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QCheck: A Web Server for Quality Check of Next Generation Sequencing Data with Standards and Guidelines

June 2019
Journal of Molecular Diagnostics 21

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The Journal of Molecular Diagnostics, Vol. 21, Supplement, May 2019

Abstracts of the 2nd Global Congress on Molecular Pathology:

Evidence-based Precision Medicine

The Association for Molecular Pathology

May 16 - 18, 2019

Hong Kong

___________________________________________________________________________________________________

ORAL PRESENTATIONS

Genetics/Inherited Conditions

OR01. An International Interlaboratory Study of

Complex Variant Detection by NGS

S. Lincoln1, S. Mahamdallie2, A. Fellowes3, S.

Chowdhury4, E. Klee5, J. Zook6, R. Truty1, R. Garlick7, M.

Salit8, N. Rahman2, S. Kingsmore4, R. Nussbaum1, M.

Ferber5, B. Shirts9

1Invitae, San Francisco, CA, United States, 2Institute of

Cancer Research, London, United Kingdom, 3Peter

MacCallum Cancer Centre, Melbourne, Australia, 4Rady

Children's Institute for Genomic Medicine, San Diego,

CA, United States, 5Mayo Clinic, Rochester, MN, United

States, 6National Institute of Standards and Technology,

Gaithersburg, MD, United States, 7SeraCare Life

Sciences, Gaithersburg, MD, United States, 8Stanford

University, Palo Alto, CA, United States, 9University of

Washington, Seattle, WA, United States

Introduction: Next-generation sequencing (NGS) is a

capable technique for detecting single nucleotide variants

(SNVs) and small insertions and deletions (indels) in

relatively accessible parts of a patient's genome.

However, conventional NGS methods have important

limitations. An analysis of over 80,000 patients, tested for

constitutional alterations using sensitive methods,

showed that variants of other, technically challenging

types comprise between 9 and 19% of the reportable

pathogenic findings, depending on clinical indication.

Approximately 50% of these variants were of challenging

types (large indels, single exon copy number variants

(CNVs), etc.), 20% were in challenging genomic regions

(homopolymers, non-unique sequences, etc.), and 15%

were in regions poorly covered by standard commercial

kits. A further 15% presented multiple challenges. This

study has since been expanded to include over 200,000

patients with diverse indications, and these newer data

lead to similar conclusions.

It can be difficult for both clinicians and laboratory

directors to evaluate the sensitivity of tests for such

challenging variants. The most recent AMP/CAP

guidelines for NGS bioinformatics [1] recommend that a

total of 59 variants of each type be included in validation

studies, a number that is difficult to achieve for complex

variants given the relative scarcity of positive control

specimens.

Methods: We developed a synthetic specimen

containing 22 challenging variants of diverse types in 7

commonly tested genes. Raw NGS data for these

synthetic variants was found to mimic that of the

endogenous variants and presented similar technical

challenges for NGS. This specimen was sequenced

using 10 different NGS tests by an international group of

collaborating laboratories. These tests employed different

sequencing platforms, library methods, and

bioinformatics pipelines.

Results: All 10 tests detected all of the relatively “easy”

SNVs and small indels present (with one exception).

However, only 10 of the 22 challenging variants were

detected by all tests, and just 3 tests detected all 22.

Limitations with large indels, homopolymer associated

variants, variants in non-unique regions, and other

challenges were observed. Many, but not all of these

limitations appeared to be bioinformatic in nature. Some

of these limitations were not previously known to the

respective laboratory directors, demonstrating the utility

of this approach.

Conclusions: We believe that both our prevalence data

and control specimens such as ours may be a valuable

asset to the global AMP community for evaluating and

optimizing clinical tests. The particular specimen

described is now available to members of the AMP

community and we are currently expanding this specimen

and others to include additional variants. We are

soliciting input from the community on additional variant

types and genes to include in new specimens.

OR02. High-Risk Cytogenetics in Multiple Myeloma:

Further Scrutiny of Deletions within the IGH Gene

Region Enhances Risk Stratification

J.N. Sanmann1, P.A. Althof1, S.C. Smith1, B.J. Dave1

1Munroe-Meyer Institute at the University of Nebraska

Medical Center, Human Genetics Laboratory, Omaha,

NE, United States

Introduction: High-risk (HR) cytogenetics in multiple

myeloma are best characterized using fluorescence in

situ hybridization (FISH), as low proliferation indices and

cryptic genetic aberrations often preclude informative

results by standard karyotyping. When combined with the

International Staging System, lactodehydrogenase, and

other clinical features, risk can be stratified and treatment

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options can be informed by the cytogenetics profile.

Certain translocations [i.e., t(4;14), t(14;16), t(14;20)]

involving disruption of IGH are in the HR category, while

the other IGH-disrupting translocations [i.e., t(11;14),

t(6;14)] confer standard-risk (SR). To enhance efficiency,

minimize cost, and preserve limited material (CD138+

fraction), laboratories typically use a breakapart probe

(BAP) to detect rearrangement of IGH, followed by dual

fusion probes to determine the IGH fusion partner.

However, there is a paucity of data describing

translocations and resultant fusion partners when the

BAP for IGH reveals a deletion of part or all of the 5'

(telomeric) or 3' (centromeric) gene region.

Methods: Our laboratory performed a retrospective

review of abnormal multiple myeloma FISH studies

between July 2012 and December 2017 to identify cases

harboring deletions of the IGH region using a BAP. We

catalogued these findings in conjunction with the dual

fusion studies performed [2012 - 2016: t(4;14), t(14;16),

t(11;14); 2017: the aforementioned probes plus t(14;20)]

to determine whether these IGH deletions were indicative

of clinically-significant translocations associated with HR

or SR.

Results: Of our 233 patient cohort exhibiting IGH

deletions, we identified 73 5' deletions, 29 3' deletions,

124 partial 5' deletions, and 7 partial 5' deletions with

concurrent 3' deletions. Subsequent dual fusion analysis

revealed a fusion partner in 19% (n=44) of cases. Of

these fusion-positive (FP) cases, 34% (n=15) exhibited

HR translocations, including 26% (n=9) of FP cases with

5' deletions, 100% (n=6) of FP cases with 3' deletions,

and 66% (n=2) of FP cases with partial 5' deletions. Of

note, patients with 5' deletions (partial or full) exhibited

both HR and SR translocations, while patients with 3'

deletions exhibited only HR translocations [i.e., t(4;14)].

Conclusions: Differentiating between IGH translocations

that confer HR versus SR is imperative for

prognostication and management. Although an IGH BAP

strategy is commonly used to screen cases, our data

suggest that subsequent analysis with dual fusion probe

strategies should not be restricted to cases with

traditional IGH BAP rearrangements. Specifically, our

data demonstrate that atypical signal patterns (e.g.,

deletions) are often indicative of clinically-significant IGH-

partner gene fusions. Thus, we recommend performing

dual fusion FISH testing among cases with deletion of

IGH, particularly for assessment of IGH translocations

associated with HR gene partners, to ensure appropriate

stratification and treatment.

OR03. The Clinical Utility of Tumour Mutational

Signatures for Identifying Hereditary Colorectal

Cancer and Polyposis Syndromes

D. Buchanan1,2, P. Georgeson1, M. Clendenning1, K.

Mahmood1, A. Ragunathan1,3, R. Walker1, J. Joo1,2, H.

Jayasekara1,4, C. Rosty1,5,6, F. Macrae7, B. Pope1, M.

Jenkins8, I. Winship3

1The University of Melbourne, Department of Clinical

Pathology, Melbourne, Australia, 2The University of

Melbourne, Centre for Cancer Research, Melbourne,

Australia, 3Royal Melbourne Hospital, Genomic Medicine

and Family Cancer Clinic, Parkville, Australia, 4Cancer

Council Victoria, Cancer Epidemiology and Intelligent

Division, Melbourne, Australia, 5Envoi Pathology,

Brisbane, Australia, 6The University of Queensland,

Brisbane, Australia, 7The Royal Melbourne Hospital,

Colorectal Medicine and Genetics, Parkville, Australia,

8The University of Melbourne, Centre for Epidemiology

and Biostatistics, Carlton, Australia

Introduction: Tumour mutational signatures are derived

from the type of nucleotide substitution and their

sequence context. Mutational signatures with known

aetiology include: A ubiquitous signature associated with

spontaneous deamination of 5-methylcytosine and

correlated with age (signature 1), and signatures related

to defective DNA mismatch repair (MMR; signatures 6,

15, 20, 26), polymerase proofreading exonuclease

domain mutations in POLE (signatures 10 and 14) and

base excision repair (BER) defects in NTHL1 (signature

30) and MUTYH (signature 18). The aim of this study

was to assess the utility of tumour mutational signatures

in colorectal cancers (CRCs) for identifying carriers of

pathogenic variants and classifying variants of uncertain

clinical significance (VUS) in genes underlying hereditary

colorectal and polyposis syndromes.

Methods: Hereditary CRC and polyposis susceptibility

gene mutation and VUS carriers were identified through

germline testing of participants from the Australasian

Colorectal Cancer Family Registry (MLH1, MUTYH,

POLE, POLD1, AXIN2 variant carriers) and from the

Genetics of Colonic Polyposis Study (NTHL1 and RNF43

variant carriers). Whole exome sequencing of formalin-

fixed, paraffin-embedded (FFPE) tumour DNA was

performed using Agilent Clinical Research Exome V2

sequenced on Illumina Novaseq. Using the mutational

signatures established by the Wellcome Trust Sanger

Institute, we calculated the optimal combination of

mutational signatures for each tumour sample based on

cosine similarity.

Results: Two CRCs from MLH1 gene mutation carriers

(Lynch syndrome) demonstrated signatures 6 and 15

correlating with defective MMR. The patterns in these two

carriers were not ostensibly different to a sporadic CRC

with biallelic somatic mutations in MLH1. Three CRCs

from homozygous carriers of the MUTYH (p.Gly396Asp)

mutation demonstrated a dominant signature 18

associated with defective BER. A CRC from a

heterozygous carrier of the MUTYH (p.Gly396Asp)

mutation and a VUS in MUTYH (p.Arg304Ser) did not

demonstrate signature 18 suggesting the VUS is not

pathogenic. The CRC from a biallelic NTHL1 carrier

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showed a dominant signature 30. A MMR-proficient CRC

from a carrier of a POLD1 VUS in the exonuclease

domain (p.Gly321AlafsTer72) showed no evidence of

hypermutation or defective DNA repair signatures. CRCs

from a truncating RNF43 (p.Arg132Ter) mutation carrier

with Serrated Polyposis Syndrome, and an AXIN2

truncating variant carrier (p.P350LfsX13) also did not

demonstrate DNA repair signatures.

Conclusions: The tumour mutational signatures

observed in CRCs from known pathogenic mutation

carriers were consistent with the underlying DNA repair

defect. The presence or absence of expected tumour

mutational DNA repair signatures in CRCs from

individuals with a VUS may help classify rare variants in

hereditary CRC and polyposis genes. Further exploration

of somatic mutation patterns in CRCs from RNF43 and

AXIN2 carriers may identify novel mutational signatures.

OR04. Prospective Study to Determine the Spectrum

of Mutations among Patients with Multiple Endocrine

Neoplasia 1 (MEN-1) and to Identify “At Risk”, First-

Degree Relatives

R. Pai1, A. Hesaraghatta2, S. Rajaratnam2, N. Thomas2,

T. Paul2, M.J. Paul3, D. Abraham3, A. Chacko4

1Christian Medical College, Pathology, Vellore, India,

2Christian Medical College, Endocrinology and

Metabolism, Vellore, India, 3Christian Medical College,

Endocrine Surgery, Vellore, India, 4Christian Medical

College, Neurosurgical Sciences, Vellore, India

Introduction: Multiple Endocrine Neoplasia (MEN-1) is

an autosomal dominant disorder characterized by the

combined occurrence of tumors of the parathyroid

glands, pancreatic islets and the anterior pituitary. About

90% of patients with familial MEN-1 disease are known to

carry mutations in the MEN1 gene. However, there is

very little information about the spectrum of mutations

seen in India.

Methods: Twenty-five clinically suspected cases of

MEN-1 were enrolled prospectively over a two-year

period. Of the 25 clinically suspected cases of MEN-1, 18

patients had definite clinical MEN-1 based on the

presence of at least 2 of the endocrine tumours

associated with MEN-1; 12 had family history of MEN-1.

Further, 16 first-degree relatives of index cases with

MEN-1 were screened for mutations that were seen in

the index cases. The MEN1 gene (exons 2-10), 3' and 5'

UTR regions of MEN1 gene, CaSR genes (exons 2-7)

and the CDKN1B gene (exons 1-2) were screened in all

index cases by PCR followed by Sanger sequencing. In

the absence of mutations, the samples were screened for

large deletions by Multiplex Ligase Dependent Probe

Amplification (MLPA) assay.

Results: Among the 25 index cases of MEN-1, 17 (68%)

had pituitary adenoma, 19 (76%) had primary

hyperparathyroidism and 17 (68%) had pancreatic

neuroendocrine tumors. Three patients had thymic

tumors, four had adrenal tumors, four had collagenomas,

one had angiofibroma and one had papillary

microcarcinoma thyroid. Of the 18 cases with definitive

MEN-1, 61% (n=11) were positive for mutations.

However, there were no hotspot of mutations, though 4 of

the 11 harboured an exon 10 mutation. None of the 7

probable cases of MEN-1 were positive for mutations.

Further, all the mutation negative cases that were

screened for large deletions by MLPA, 3' and 5' UTR

regions of MEN1 gene, CaSR and CDKN1B gene,

continued to be negative for mutations. However, certain

established polymorphisms in the MEN1 gene, CaSR

and the CDKN1B gene were seen among the mutated

and mutation negative cases. Interestingly, 7 of the 9

definitive cases of MEN-1 that were MEN1 mutation

negative were found to be positive for the p.V109G

polymorphism in the CDKN1B gene.

Sixteen first degree relatives including 9 members of a

single large family, were screened for the same

mutations as seen in the index case. Of the 9 members 6

were found to harbor the same mutations as in the index

case (c.1151_1152insGAGG; p.A384fs) while three were

negative. Among the other “at risk” families tested 3 of

the remaining 7 tested, harboured the same mutations as

in the index case.

Conclusion: The study has helped to clearly document

the spectrum of mutations seen among patients with

MEN-1. However, the absence of MEN-1 mutation in ~

39% of the cases and the presence of the p.V109G

polymorphism in the CDKN1B gene among the MEN-1

mutation-negative cases raises the question whether

such polymorphisms could be independently contributing

to pathogenesis.

OR05. Five Year Experience of Clinical Next-

Generation Sequencing for Somatic Overgrowth and

Related Syndromes: Non-PIK3CA Cumulative

Findings

Y. Cao1, M. Evenson1, M. Corliss1, M. Schroeder1, J.

Neidich1, J. Heusel1

1Washington University School of Medicine, Department

of Pathology and Immunology, St. Louis, MO, United

States

Introduction: A number of oncogenes with recurrent

mutations in cancer are now understood to drive

segmental overgrowth and related syndromes. Our

clinical laboratory has pioneered the sensitive detection

of causative mutations in cancer and applied that

expertise to mutations associated with overgrowth and

related phenotypes. Presented here are our non-PIK3CA

cumulative findings.

Methods: We use targeted hybridization capture coupled

with Next-Generation Sequencing (NGS) for deep and

comprehensive coverage. This assay has undergone

frequent modifications due to its growing knowledge

base. The current assay includes baits targeting exonic

regions and selected intronic sequences from 177 genes

recurrently mutated across all cancer types

(Comprehensive Cancer Gene Set, version 3.2). The 35

genes reported for somatic overgrowth are divided into

seven discrete orderable subsets depending on the

presenting phenotype. Paired-end 101bp sequencing

was performed on Illumina HiSeq 2500 with an average

unique on-target coverage depth of >1000x. Variants

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were called and analyzed using a combination of multiple

software programs, and Clinical Genomics Workspace

was used for data visualization and interpretation.

Accepted specimen types include fresh and formalin

fixed tissues, buccal swabs and fibroblasts. Of note, the

somatic analysis of a peripheral blood sample was

inappropriate in most cases.

Results: Clinical NGS analysis was performed on

specimens from 306 patients. The age of patients ranges

from newborn to 62 years, with an average age of 7.4

years. About half of the patients were under the age of

four and 18% were tested within their first year of life. A

total of 180/306 patients had reportable genetic variants,

85/180 had non-PIK3CA variants. Among those, 71/85

had a pathogenic (P) or likely pathogenic (LP) variant,

and 14/85 had variant of uncertain significance (VUS). A

total of 92 non-PIK3CA variants were identified in 23

genes in those 85 cases: 73/92 were P/LP and 19/92

were VUS. Half of the 92 variants were identified in six

genes: GNA11 (12/92), KRAS (8/92), PIK3R2 (7/92),

GNAS (7/92), GNAQ (6/92), and MTOR (6/92). Sanger

sequencing was performed on a secondary non-affected

specimen (peripheral blood) on 62/85 cases as a means

to confirm the origin of the causative variant. Among

those, 39/62 (63%) cases had negative results, indicating

the somatic origin of the variants, 23/62 (37%) cases had

positive results, indicating the germline or multi-tissue

mosaic origin. In correlation, 63% (37/59) variants are

with variant allele frequency (VAF) < 0.2 and 37% (22/59)

with VAF>0.2.

Conclusion: Our cumulative clinical experience

demonstrates the utility of NGS testing for segmental

overgrowth and related syndromes. With new clinical

trials for treatment that are often gene or variant specific,

somatic testing for causative variants in patients with

overgrowth syndromes has become an important part of

clinical care.

Hematopathology

OR06. Validation of Immunohistochemistry by a

Monoclonal Antibody CAL2 on Trephine Biopsy for

CALR Mutation Detection in Myeloproliferative

Neoplasms

M.K. Ng1, C.W. Lam1, C.H. Au1, B.K. Ip1, M.Y.R. To1, G.

Choy1, H.Y. Lam1, T.L. Chan1, S.K.E. Ma1

1Hong Kong Sanatorium & Hospital, Department of

Pathology, Hong Kong, Hong Kong

Introduction: Calreticulin (CALR) mutation is a major

diver genetic event in essential thrombocythemia (ET)

and primary myelofibrosis (PMF). Mutations in CALR

lead to 1+ base pair frameshifts and generate a novel

amino acid C-terminus. The monoclonal antibody CAL2

binds to the novel epitope of CALR mutant protein and

allows the recognition of CALR mutations. The objectives

of this study were to (1) validate the IHC detection of

CALR mutation detection by CAL2 in Myeloproliferative

Neoplasm (MPN) cases with known mutation status; (2)

to develop an IHC scoring system for CALR mutation

detection in the MPNs.

Methods: A total of 70 bone marrow (BM) trephines were

selected for paraffin CAL2 IHC, including 31 known

CALR mutations (22 ET and 9 PMF) and 31 known

JAK2V617F mutations (22 ET and 9 PMF). Molecular

testing for CALR mutation was performed by PCR

Sanger sequencing. JAK2V617F mutation was confirmed

by allele-specific PCR and PCR followed by restriction

enzyme digestion analysis (PCR-RFLP). Individual cases

were confirmed by fragment analysis or next-generation

sequencing as indicated. A known CALR mutated

specimen was used as an internal positive control, while

2 normal BMs and 6 cases of other myeloid neoplasms

(AML (M5), AML (AML-TMDS), MDS-SLD, MDS-MLD

and 2 CML-CP) were also used as negative control.

Mutant CALR mouse monoclonal antibody (CAL2,

Dianova, Germany) was titered at 1/100 by following

manufacturer's instructions. Slides were stained on

BOND III automated immunostainer (25 mins epitope

retrieval, 30 mins antibody incubation, and 8 mins

OptiView detection plus amplification). We developed a

scoring system calculated by megakaryocytes (MK)

signal strength (0-3+) times positive ratio of index (out of

1.0) for calling CALR mutation.

Results: Among 31 known CALR mutations cases with 9

various types of CALR mutations including 52 bp deletion

and 5 bp insertion, 22 positives with strong CAL2

cytoplasmic staining (intensity 2-3/3) and 8 positives with

weak CAL2 cytoplasmic staining (intensity 1) were

recognizable in the MK. The mean CAL2 IHC score was

1.87, 95% CI [1.56, 2.18]. There were no significant

differences between ET and PMF. 1/31 cases with

multiple clones of 2 CALR mutations in cis was negative

by CAL2 IHC. CAL2 staining was negative in 31 known

JAK2V617F cases, as well as in 2 normal BMs and 6

cases of other myeloid disorders. The CAL2 IHC results

correlated well with the molecular status.

Conclusions: The specificity and sensitivity of CAL2 IHC

were 100% and 97% respectively. CAL2 IHC is an

accurate method to identify CALR mutations in MPN.

While CAL2 antibody can recognize CALR mutations with

1+ base pair frameshift, a limitation is failure to detect

CALR mutation that results in non-canonical amino acid

sequence, albeit of uncertain pathogenicity. We have

successfully validated CAL2 IHC and the scoring scheme

is ready for clinical use on BM trephine. IHC by CAL2

antibody is a simple, quick and cheap method to identify

CALR mutation in MPN.

OR07. Comparison of a 640-Gene Next Generation

Sequencing Panel to a Virtual 41-Gene Panel for the

Diagnostic Utility in Unexplained Cytopenia

G. Zheng1, P. Chen1, L. Haley1, L. Jiang1, A.

Pallavajjalla1, M.-T. Lin1, C. Gocke1

1Johns Hopkins University, Baltimore, MD, United States

Introduction: The diagnostic utility of somatic mutations

for cytopenia is unclear: myeloid neoplasms including

myelodysplastic syndrome are clonal, but clonal

hematopoiesis can be found in healthy individuals. We

examined a large cohort of well-studied patients with

cytopenias with next generation sequencing to determine

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its diagnostic utility, and also assessed the impact of

panel size on diagnostic performance.

Methods: This cohort study comprised 207 patients with

cytopenias and a specimen (blood or marrow) submitted

to a CLIA-certified molecular pathology laboratory for

640-gene NGS from 09/2015 to 03/2018. Patients with

diagnoses of acute leukemia, lymphoma, or history of

cytotoxic therapy were excluded. Using a combination of

marrow morphology and cytogenetics as a diagnostic

“gold standard” for MDS, we assessed the diagnostic

utility of somatic mutations detected by the 640 gene

NGS panel and a virtual panel of 41 genes which are

commonly mutated in MDS and aplastic anemia.

Results: 586 somatic mutations were identified in 201

patients with definitive diagnosis: 102 of 105 MDS

patients, 20 of 34 aplastic anemia patients, and 27 of 52

patients with no evidence of a primary marrow disorder

showed somatic mutations. The sensitivity of the large

640-gene NGS panel for MDS was 98.3%. When the

variant allele frequency for one or more variants was set

at 20% the positive predictive value (PPV) for MDS was

94.9% with a specificity of 94.2%. Presence of two or

more somatic mutations at any variant allele frequency

(VAF) in a specimen showed a PPV of 76% for MDS,

which increased to 95.6% when the VAF cutoff was set to

10%. While the virtual 41 gene panel (Figure 1) showed a

mild decrease of sensitivity (96.5% versus 98.3%), a

better specificity was observed when VAF was set at ≥

20% (98.8% versus 94.2%). Remarkably, the presence of

two or more somatic mutations at VAF ≥ 10% showed

100% specificity for MDS with the 41 gene panel.

Mutation analysis can also predict high versus low grade

MDS: mutations in TP53 and RUNX1 are associated with

high-grade MDS, while SF3B1 mutations are, as

expected, associated with MDS with ring sideroblasts. In

addition PIGA mutations were present exclusively in

aplastic anemia patients.

Conclusions: The 41 gene panel showed better

disgnostic specificty than the 640 gene panel. Both

panels showed high sensitivity in excluding MDS, and the

presence of a mutation with a VAF ≥ 20%, or two or

more mutations with a VAF≥10%, was highly predictive

of MDS. Presence of PIGA mutations was highly

predictive of aplastic anemia. Such mutation analysis has

the potential to improve the diagnostic approach and

accuracy for unexplained cytopenias.

[Figure 1. Somatic Mutations Identified by a virtual 41-

gene NGS panel in 201 cytopenia patients]

OR08. T-Cell Clonality Testing by Next Generation

Sequencing Facilitates the Initial Diagnosis and

Disease Monitoring of Cutaneous T-Cell Lymphomas

J. Yao1, M. Syed1, C. Ho1, W. Yu1, L. Maciag1, K.

Petrova-Drus1, C. Moung1, A. Zehir1, M. Roshal1, A.

Dogan1, K. Nafa1, M. Arcila1

1Memorial Sloan Kettering Cancer Center, New York, NY,

United States

Introduction: The diagnosis of cutaneous T-cell

lymphomas (CTCLs) is often challenging based on

morphology and immunohistochemistry. Clonality

assessment of T cell receptor (TCR) rearrangement is a

valuable adjunct in routine clinical work up; however,

current gold standard methods by fragment analysis (FA)

have distinct limitations due to low sensitivity and

specificity. Next generation sequencing (NGS) offers

unique advantages over traditional assays as it provides

sensitive and sequence specific data. This allows more

accurate initial diagnosis of cutaneous lesions as well as

highly sensitive assessment of disease involvement

beyond skin and the possibility to monitor disease post

treatment. Here we describe our clinical implementation

of TCR gamma gene (TRG) based NGS clonality testing

in the diagnosis and monitoring of CTCLs.

Methods: Clinical samples submitted for routine TRG

clonality testing were included in this study, including

formalin fixed paraffin embedded tissue, peripheral blood

and bone marrow. Testing was performed using the

LymphoTrack (LT) TRG kits (Invivoscribe) followed by

sequencing on Illumina MiSeq instrument. Data was

analyzed by LymphoTrack MiSeq Software and in-house

developed MSK-LymphoClone bioinformatics pipelines.

Performance characteristics were evaluated by directly

comparing to FA assay (BIOMED-2 primers) and flow

cytometry (FC).

Results: A total of 47 clinical samples from 33 patients

were analyzed, including Sézary syndrome/mycosis

fungoides (22), cutaneus gamma-delta T cell lymphoma

(3), anaplastic large cell lymphoma/lymphomatoid

papulosis (2), CD4+ small/medium T-cell

lymphoproliferative disorder (2), CD8+ aggressive

epidermotropic T-cell lymphoma (1), primary cutaneus

perihernial T-cell lymphoma (1), and CTCL, unclassifiable

(2). Among 34 initial characterization samples, 11 (32%)

were diagnosed as CTCLs after morphologic and

immunophenotypic assessment and a clone was

confirmed by both FA and LT. In 23 cases (68%), a

diagnosis of atypical lymphohistiocytic infiltrate was

rendered. TRG clonal rearrangement was detected in

16/23 (70%) by FA and 22/23 (96%) by LT. Among nine

monitoring samples with corresponding FC results, 9/9

and 6/9 were positive by LT and FC, respectively.

Conclusions: TRG NGS clonality testing demonstrated

higher clonal detection rate compared to FA and FC.

Improved clonal detection at initial assessment facilitated

the diagnosis of CTCLs in 26% more cases compared to

FA. The unique ability to search for diagnostic clonal

sequences enables more sensitive and specific staging

and monitoring in the subsequent specimens.

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OR09. A Noise-Cancelling Method Based on

Information Theory for ctDNA Variant Calling

H. Chen1, F. Zhang1, M. Mao1

1Seekin Inc., Shenzhen, China

Introduction: Circulating tumor DNA (ctDNA) is

fragmented cell-free DNA (cfDNA) in plasma, originating

from tumor cells rather than normal somatic cells. Since it

carries great potential for noninvasive early diagnosis,

progression monitoring, and prognosis, ctDNA soon

arose as one of the hottest topics in cancer research now

that NextGen Sequencing makes it feasible to identify

ctDNA in clinically acceptable cost and time. However,

due to low content of ctDNA in total cfDNA, a challenge

we confronted in clinical application was how to identify

meaningful low-frequency somatic variants carried by

ctDNA from chaotic noise introduced by sample

preparation and sequencing.

Information theory has been proven powerful to handle

signal-noise problems in various disciplines. Particularly

in bioinformatics studies, it has achieved competitive

accuracies in challenging signal process problems such

as identifying differential gene expression and aligning

distantly related protein sequences. Here we implement a

method based on knowledge of information theory to

cancel the noises in ctDNA sequencing data and amplify

the true variant signal.

Methods: Data preparation: SeqMaker

(https://github.com/opengene/seqmaker.jl) was used to

generate simulated ctDNA sequencing data containing

73 single nucleotide polymorphism (SNP) variants and

their brinks of 1000bp in both upstream and downstream

with average depth of 1000x. The frequencies of these

variants varied from 0.001-0.1 and the sequencing error

rate was 0.001. Simulated data were then trimmed by

fastp and were then aligned by BWA (0.7.17-r1194-dirty).

The output was converted by Samtools into BAM and

PILEUP files.

Variant calling and performance benchmarking: The

information theory-based algorithm gave each base

position a score derived from the base frequencies in the

output PILEUP files. The detail of this score will be

provided in following publications. Then all base positions

were ordered by their score. Applying stepwise cutoffs on

these ordered positions, the true positive rate and the

false positive rate were determined. The receive operator

characteristic (ROC) figure summarizes all results.

Results: The ROC curve of our algorithm is displayed in

Figure 1. In this simulated ctDNA data, our algorithm can

achieve the true positive rate of 95% with the false

positive rate of 0.2% when calling variants. The Area

Under Curve (AUC) is 0.986.

Conclusions: Our information theory-based method can

cancel sequencing noises and accurately identify low-

frequency true variants in ctDNA samples, which could

improve diagnosis accuracy in clinical practice.

[Figure 1. ROC curve of our information theory-based

algorithm on simulated ctDNA data]

OR10. Clinical Implications of Cytogenetic

Heterogeneity in BCR-ABL1 Fusion Positive Adult B

Cell Acute Lymphoblastic Leukemia

X. Lu1, L.J. Medeiros,2

1Northwestern University Feinberg School of Medicine,

Pathology, Chicago, IL, United States, 2The University of

Texas MD Anderson Cancer Center, Hematopathology,

Houston, TX, United States

Introduction: Cytogenetic heterogeneity is frequently

observed in adults with t(9;22)(q34.1;q11.2)/BCR-ABL1

positive B cell acute lymphoblastic leukemia (Ph+B-ALL).

We retrospectively investigated the cytogenetic

heterogeneity and correlated additional chromosomal

abnormalities (ACAs) with clinical outcomes in 144 adults

with Ph+B-ALL.

Methods: There were 85 (59.1%) men and 59 (40.9%)

women with a median age of 54-years. Patients were

sorted into 6 subgroups based on the karyotypic findings

in stemline including: 32 (22.2%) with t(9;22) as the sole

aberration, 23 (15.9%) with t(9;22) plus one ACA, 26

(18.1%) with t(9;22) as part of a complex karyotype, 18

(12.5%) showed a variant t(9;22), 30 (20.8%) showed

t(9;22) as the stemline with ACAs in the sideline(s), and

15 (10.4%) had the t(9;22) and hyperdiploidy. Of these,

89 cases had one, 41 had two and 14 had ≥ three

clones, respectively.

Results: The median follow-up was 25.8 months. The

median overall survival (OS) was 25.6 months and the

median relapse-free-survival (RFS) was 20.6 months.

Patients with variant t(9;22) had a poorer OS and RFS

when compared with all other subgroups combined (P =

0.0018 and 0.0049, respectively). In addition, patients

with two or more clones had poorer outcomes than those

with one clone (P = 0.0427). Multivariate analysis

confirmed that variant t(9;22) and clone numbers are the

independent negative risk factors.

Conclusion: These data suggest that traditional

karyotypic analysis of adult Ph+B-ALL identifying

cytogenetic heterogeneity is helpful for the risk

stratification of these patients.

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Infectious Diseases

OR11. Incidental Detection of Malignancies in

Metagenomics Cell-Free DNA Testing

W. Gu1, E. Talevich2,3, A. Gopez1, S. Federman1, Y.

Sucu1, S. Arevalo1, M. Gottschall1, Z. Qi1, L. Wang1, B.

Karlon1, T. Kurtz1, J. Yu1, I. Yeh2, J. DeRisi4,5, S. Miller1,

C. Chiu1, J. Simko2

1University of California, San Francisco, Laboratory

Medicine, Menlo Park, CA, United States, 2University of

California, San Francisco, Pathology, San Francisco, CA,

United States, 3DNANexus, San Francisco, CA, United

States, 4Chan Zuckerberg Biohub, San Francisco, CA,

United States, 5UCSF, Biochemistry, San Francisco, CA,

United States

Introduction: Metagenomic next-generation sequencing

(mNGS) is an unbiased test for detecting DNA pathogens

through whole genome sequencing of cell-free DNA

(cfDNA) in plasma and body fluids. At University of

California San Francisco (UCSF) Clinical Laboratories,

we launched a clinically validated mNGS test for

undiagnosed meningitis and encephalitis using

cerebrospinal fluid and are validating a plasma mNGS

test for sepsis using cfDNA. In addition, we apply cell-

free metagenomics as a general approach to body fluids

suspected of infection. Using discarded human host data

that account for >99% of the sequencing reads, we found

genome-wide copy number variation signatures caused

by tumor derived cfDNA.

Methods: Clinical samples consisting of patient body

fluids and plasma were identified due to clinical suspicion

or laboratory confirmation of infection or malignancy. In

both blood and body fluids, we reduced the majority of

leukocyte human by centrifugation and sequenced only

the supernatant or plasma. We then created sequencing

libraries and performed whole genome sequencing. A

similar protocol was used for formalin-fixed, paraffin-

embedded (FFPE) material obtained from primary tumor

tissue. The fastq output was aligned to hg38 and binned

into segments of the human genome for read counting

and normalization.

Results: In the initial set of plasma samples from

patients newly admitted for suspected sepsis, 4/9 plasma

from metastatic cancer patients showed at least five large

(>20 Mb) copy number gains or losses. The positive

cases were validated based either on tissue copy number

variant (CNV) detection or cytogenetics for hematological

malignancies. All body fluid samples (e.g. pleural fluid,

peritoneal fluid, spinal fluid) that were confirmed positive

for malignancy on cytology also carried at least 5 large

(>20 Mb) copy number gains or losses. In addition, we

found five examples of positive tumor signatures in body

fluid specimens that had resulted 'benign' on cytology.

For a subset of the samples that had correlated tissue

blocks of the tumor available or had cytogenetic data, the

same CNV pattern was consistently found in a primary

tissue.

Conclusions: We show that incidental data from cfDNA

metagenomic testing can pick up large CNVs in cfDNA.

This is particularly useful in cases where an undiagnosed

infectious and/or malignant etiology overlaps as

possibilities on a clinical differential. The high specificity

of requiring multiple large CNVs at a high tumor fraction

(>5%) will mitigate the risk of false positives. This test

may also be useful for monitoring the vast majority of

tumors in different body sites, working especially well for

metastatic disease with high tumor burdens. Larger

studies are needed to validate this finding across a

broader range of sample types and tumors and to assess

for clinical utility.

OR12. Comparative Whole-Genome Analysis

between Pregnant Women-Colonizing and Neonate-

Infecting Group B Streptococcus Isolates

L. Wu1, F. Wang1, R.E. Nunez2, W. Qiu2, Y. Tang3, Y.

Zhu1

1Bao an Maternal and Child Health Hospital, Jinan

University, Shenzhen, China, 2City University of New

York, Hunter College, New York, NY, United States,

3Memorial Sloan Kettering Cancer Center, New York, NY,

United States

Introduction: Group B Streptococcus (GBS) is a

frequent resident of the vaginal tract in pregnant women

and a major cause of invasive infection in neonates. GBS

is readily transmitted from mother to neonate; however,

molecular mechanisms for the transmission and invasion

from mothers to their neonates remain unknown.

Methods: In this study, 48 GBS isolates were collected

for whole-genome sequencing by using the Illumina next-

generation sequencing technologies and Five GBS

genomes have been fully assembled by PacBio,

Including 26 invasive (high-virulence, HV) isolates from

bloodstream-infected neonates and/or vaginal/mother

milk from colonization mothers, and 22 colonizing (low-

virulence, LV) isolates recovered from vaginal

colonization mothers without invasive infection in

neonates.

We applied Align short reads to reference genome

(B111) by bwa, and call variants by samtools mpileup;

and annotate and extract single nucleotide

polymorphisms (SNPs) by customized BioPerl scripts.

The genome sequences between the mothers and

neonates in the four pairs were virtually identical. We

further compared the genomes between the HV and LV

isolates recovered from mothers with and without

subsequent neonate infections.

Results: In total, 34,395 variants, and 22,893 SNPs were

analyzed, all 48 GBS isolates genomes divided into five

major clades, Clade 1 consists of a significantly large

number of HV isolates (19 HV isolates included, p=6.7e-4

by Fisher's test) and other clades consist of more

balanced mixed isolates. Opportunistic infections occur in

all clades (no fixed diffs between invasive and colonizing

isolates) and no single gene or SNP was found to

correspond to all HV or LV (no fixed diffs between

invasive and colonizing isolates). Recombination (more

than mutation) has caused increase of virulence in the

HV clade (we used LDhat to run recombination hotspot

tests).

There are 2-3 loci that stand out as the most promising

virulence factor (a large number of nonsynonymous

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changes that are statistically significant), including:

LPXTG-domain surface protein (“CDH81_02355"), which

is a known virulence factor in Gram+ bacteria, and

phosphoribosylformylglycinamidine synthase

(“CDH81_07310"). This suggests that these are HV

factors that could be used as diagnostic markers and that

this is deserving of further research.

Conclusion: There are a group of GBS clones

circulating exclusively in China. Some clades are

associated with high-virulence in neonatal patients.

Multiple SNPs and genes are associated with high-

virulence, in particular the adhesion known as pullulates.

OR13. Suppressor of Cytokine Signaling (SOCS)-3

Downregulation Is Associated with Increased

Proinflammatory Responses in Diabetic Individuals

with M. tuberculosis Infection

K. Iqbal1, M. Rottenberg2, Z. Hasan1, M. Irfan1, Q.

Masood1

1The Aga Khan University, Pathology and Laboratory

Medicine, Karachi, Pakistan, 2Karolinska Institute,

Stockholm, Sweden

Introduction: Pakistan ranks 5th in high tuberculosis (TB)

burden countries and 6th in countries with high burden of

diabetes mellitus (DM). Individuals with diabetes have

been shown to exhibit dysregulation of host protective

cytokines such as, IFN-γ, TNFα and IL6. Suppressor of

cytokine signaling (SOCS) molecule 3 plays a role in

maintaining balance between pro and anti-inflammatory

cytokines in patients with TB. Given the co-incidence of

both diseases we investigated the role of SOCS3 in

modulation of host immunity in individuals infected with

the pathogen, Mycobacterium tuberculosis (MTB),

comparing latent (LTB) and active disease.

Methods: Peripheral blood mononuclear cells (PBMCs)

were isolated from healthy controls (EC, n=20), those

with LTB (n=10), DM (n=13), DM with LTB (n=15) and TB

patients (n=15). PBMCs were stimulated with PPD

(10µg/ml) for 18 hours. Cell supernatants were collected

and tested for Th1/Th2 Cytokines. RNA was extracted

from stimulated PBMCs and mRNA used for RT-PCR

based analysis of IFNγ, TNFα, IL6 and SOCS3 genes.

Results: Measurement of stimulated cell supernatants

showed increased levels of IFN-γ in DM-LTB as

compared with EC (p=0.01), LTB (p=0.03), DM (p=0.01)

and TB (p=0.03) cases. IL12 and TNFα levels were

increased in DM-LTB as compared with LTB (p value:

IL12, 0.005; TNFα, 0.006), DM (IL12, p=0.021; TNFα,

0.019). Levels of IL6 were increased in DM-LTB as

compared with EC (p=0.001) and LTB (0.021).

Coordinately, we observed increased IFN-γ (p=0.033)

and IL6 (p=0.01) mRNA expression in patients with DM-

LTB as compared with EC; also, increased TNFα mRNA

expression as compared with LTB (p=0.034) cases.

SOCS3 mRNA expression was decreased in DM-LTB as

compared with LTB. In patients with TB, IFN-γ mRNA

levels were increased as compared with EC (p=0.0457),

while SOCS3 mRNA levels were decreased as compared

with LTB (p=0.0188).

Conclusion: We observed increased pro-inflammatory

response in diabetics with latent TB to be associated with

a downregulation in SOCS3 expression. As SOCS3 is

required for mycobacterial clearance, our data suggests

that this could be a mechanism which increases

susceptibility to active TB in DM cases who are infected

with MTB.

OR14. High Prevalence of NDM-1-Producing

Enterobacter cloacae from Three Tertiary Hospitals

in China

B. Huang1, Y. Cai1, C. Chen2, M. Zhao3, X. Yu1, K. Liao1,

K. Lan2, P. Guo1, W. Zhang2, X. Ma1, Y. He1, J. Zeng2, L.

Chen4, W. Jia3, Y. Tang5

1Department of Laboratory Medicine, The First Affiliated

Hospital of Sun Yat-sen University, Grangzhou, China,

2Department of Laboratory Medicine, The Second

Affiliated Hospital of Guangzhou University of Chinese

Medicine, Grangzhou, China, 3Department of Laboratory

Medicine, Ningxia Hospital of Ningxia Medical University,

Yinchuan, China, 4Public Health Research Institute

Tuberculosis Center, New Jersey Medical School,

Rutgers University, Newark, NJ, United States,

5Department of Laboratory Medicine, Memorial Sloan

Kettering Cancer Center, New York, NY, United States

Introduction: Enterobacter cloacae has recently

emerged as one of the most common carbapenem-

resistant Enterobacteriaceae. The emergence and

spread of NDM-1-producing E. cloacae have posed an

immediate threat globally. Here, we investigated the

molecular characteristics of carbapenem-resistant

Enterobacter cloacae (CREL) isolates collected from

three tertiary hospitals in certain regions of China from

2012 to 2016.

Methods: Eighty-six nonduplicate CREL isolates were

collected from Southern (e.g. Grangdong) and

Northwestern (e.g. Ningxia) China. Species identification

and antimicrobial susceptibility testing were performed

using a VITEK-2 system. Susceptibility to carbapenems

were also confirmed using broth microdilution method.

Isolates were screened for antibiotic resistant genes by

PCR, and expression of ompC and ompF was

determined by qRT-PCR. Genetic relatedness was

performed by pulsed-field gel electrophoresis (PFGE),

and selected isolates were subjected to whole-genome

sequencing. Multilocus sequence typing (MLST) and

plasmid-typing were also mined in our study.

Results: Among the 86 CREL isolates, 50 (58.1%) were

detected as carbapenemase producers. NDM-1 was the

dominant carbapenemase (46.5%), followed by IMP-26

(4.7%) and IMP-4 (3.5%). Notably, we identified the first

NDM-1 and IMP-1 co-producing E. cloacae, carrying

plasmids of three incompatibility (Inc) groups, including

IncHI2, IncHI2A, and IncN. In addition, Most strains

showed decreased expression of ompC and/or ompF,

and contained a broad distribution of extended-spectrum

β-lactamases (ESBLs) and AmpC β-lactamase genes.

The high-throughput sequencing demonstrated that

seven blaNDM-1-harboring plasmids contained a highly

conserved gene environment around blaNDM-1 (blaNDM-1-

bleMBL-trpF-dsbD-cutA1-groES-groEL). Meanwhile, ltrA

and QacEΔ1 genes were frequently observed

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downstream blaIMP-26 and blaIMP-4. All the CREL isolates

could be divided into 60 PFGE clusters. Nineteen blaNDM-

1-positive E. cloacae isolates obtained from Ningxia had

the same PFGE pattern (Type 1) and were classified to

ST78 in clonal complex 74 (CC74) by MLST analysis.

The replicon typing indicated that IncX3 plasmids

mediated the dissemination of blaNDM-1 within these

homologous strains.

Conclusions: These findings suggested that different

molecular mechanisms, including carbapenemase,

ESBLs and AmpC β-lactamases plus the loss of porins,

have contributed to carbapenems resistance in these

CREL isolates. We first reported an outbreak of NDM-1-

producing E. cloacae ST78 with contribution of IncX3

plasmids in northwestern China. There´s an immediate

need to to heightened surveillance efforts attentively to

prevent and control the further spread of NDM-1 in

China. (Y.C., C.C., MZ, and X.Y. contributed equally to

this article)

OR15. Application of a Multiplex PCR to Detect

Intestinal Parasite Infections in Antenatal Anemia

V. Gupta1,2, P. Deshpande1,2, Q. Zahiruddin3, A.

Gaidhane4

1Datta Meghe Institute of Medical Sciences, Molecular

Biology and Epidemiology Laboratory, Wardha, India,

2Datta Meghe Institute of Medical Sciences, Pathology,

Wardha, India, 3Datta Meghe Institute of Medical

Sciences, Community Medicine, Wardah, India, 4Datta

Meghe Institute of Medical Sciences, Community

Medicine, Wardha, India

Introduction: Anemia and poor nutrition status,

especially in pregnant women, are partially attributable to

intestinal parasitic infections. The majority of the global

burden is considered due to the five main parasites:

Ascaris lumbricoides, Necator americanus, Ancylostoma

dudonale, Trichuris trichiura, Giardia duodenalis, and

Entamoeba histolytica. Diagnosis of these parasites has

always relied on the classical microscopic examination of

stool samples as it is relatively simple to perform and

does not require expensive laboratory equipment.

However, stool microscopy is highly observer dependent

and has several shortcomings that greatly affect the

efficacy of current management of intestinal parasitic

infections in pregnant women. The study aim to assess

the utility of multiplex PCR for detection of intestinal

parasites as compared to microscopy in establishing

intestinal parasitic infections in antenatal anemic.

Methods: Thirty-four antenatal anemic subjects, enrolled

from neighboring villages of Wardha district of India as

part of pilot study funded by Public Health Foundation of

India in collaboration with Department of Science and

Technology, India.

Samples were collected and stored in 5% potassium

dichromate. DNA extraction was done using conventional

protocol. Extracted DNA was subjected to multiplex real

time PCR using TaqMan probes, forward, and reverse

primers for the following species Ascaris lumbricoides,

Ancylostoma dudonale, Trichuris trichiura, Necator

americanus, Giardia duodenalis, and Entamoeba

histolytica was designed.

[Primers and Probes for multiplex real time PCR used to

detect different parasites]

PCR was performed using PikoRealTM Real time PCR

System by Thermo Fisher Scientific. Final reaction

volume was set at 20µl, with UNG incubation at 50ºC for

2 min, polymerase activation at 95ºC for 10 min, followed

by 45 cycles of denaturation (95ºC for 15 sec) and

annealing and extension (60ºC for 1 min). The results

were compared with standard microscopy.

Results: The age range of antenatal anemic patients

was 18-30 years median age 24 years. 16/34 (41%)

patients were positive for one or the other parasite.

Maximum number 11/34 (32%) had infection with Ascaris

lumbricoides, 7/34 (12%) were positive for Ancylostoma

dudonale, 1/34 was positive for Entamoeba histolytica.

4/34 had multiple infection with Ascaris lumbricoides and

Ancylostoma dudonale. On microscopy only two cases

(Ascaris lumbricoides and Entamoeba histolytica each)

were positive and none was positive for co-infection.

Conclusion: The real-time PCR-based diagnosis

outperforms microscopy and the multiplex PCR approach

was also superior in terms of detection with multiple

infections and can be used for detection and

management in antenatal anemia.

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Informatics

OR16. Identifying True Somatic Variants in Cancer

Using Machine Learning

C. Wu1, X. Zhao1, M. Welsh1, K. Costello2, K. Cao1, M.M.

Li1,3,4, M. Sarmady1,3

1Children's Hospital of Philadelphia, Division of Genomic

Diagnostics, Philadelphia, PA, United States, 2Temple

University, College of Science and Technology,

Philadelphia, PA, United States, 3Perelman School of

Medicine, University of Pennsylvania, Department of

Pathology and Laboratory Medicine, Philadelphia, PA,

United States, 4Perelman School of Medicine, University

of Pennsylvania, Department of Pediatrics, Philadelphia,

PA, United States

Introduction: Molecular profiling has become

increasingly essential for tumor risk stratification and

treatment selection. However, the diversity of somatic

events, technical artifacts and genomic complexity make

the differentiation of reportable variants from artifacts a

challenge. Variant refinement via manual inspection is

costly for clinical labs and is subjective to personal bias.

Here, we present a machine learning-based classifier

developed at Children's Hospital of Philadelphia (CHOP)

to distinguish artifacts from bona fide Single Nucleotide

Variants (SNV)s of tumor samples.

Methods: All of the SNVs used in this study were from

CHOP cancer panels and were manually reviewed and

labeled as either true positive or artifact. An array of

sequencing attributes were calculated for each SNVs,

such as coverage, strand bias and mapping quality.

Feature selection was performed to remove attributes

that provided little separation between the two classes

such as mapping quality. A random forest classifier was

trained on a training set of 3367 SNVs from 84 tumor

samples. The classifier achieved 100%

sensitivity/specificity and 0.98 F-1 score in a 10-fold

cross validation. Following this, an independent test set

of 2362 SNVs from 66 samples were used to fine-tune

the baseline model. During this process, a confidence

interval (CI) in the range of [0, 1] was assigned to each of

the variants by the model reflecting the certainty of the

classification. The range indicates confidence level of the

model for a variant being 100% an artifact 0.0 or real 1.0.

To achieve optimal performance, different weights were

assigned to the two classes to account for different

misclassification costs, i.e. a true variant being classified

as an artifact (false negative) could lead to a worse

adverse clinical consequence than a false positive

misclassification.

Results: We identified a model that yielded the optimal

performance on the test set. Additionally, a range of CI

scores was derived to label SNVs with CI scores within

[0.05, 0.9] “uncertain” to meet clinical assurance, which

would require manual inspection. The combination of the

classifier and the CI thresholds, achieved 100%

sensitivity/specificity with 11 SNVs labeled as “uncertain”

and zero misclassifications on the test set. We then

benchmarked its performance on an independent

validation set of 5634 SNVs from 141 samples. As a

result, our model achieved 100% sensitivity/specificity

with only 3% SNVs labeled as “uncertain” and zero

misclassifications.

Conclusions: We presented a computational classifier to

identify true positive SNVs from tumor sequencing. The

classifier demonstrated high sensitivity/specificity, and

utility on a wide range of tumor samples. Overall, 90% of

the SNVs detected will receive a definite label and thus

be exempt from manual review. Implementing the model

can greatly reduce the hands-on time and hence improve

the efficiency without compromising the quality of the

clinical tests.

OR17. Development and Clinical Validation of a

Bioinformatics Pipeline for CNV Detection on Cancer

NGS Panels

K. Cao1, F. Lin1, F. Chang1,2, M. Welsh1, M. Li1,2, M.

Sarmady1,2

1Children's Hospital of Philadelphia, Philadelphia, PA,

United States, 2Perelman School of Medicine, University

of Pennsylvania, Philadelphia, PA, United States

Introduction: Copy number variation (CNV) is the most

common genomic alteration in pediatric cancer that

directly impact patient care. NGS has emerged as the

method of choice to analyze a wide range of genes

known to be associated with various cancers. We have

launched multiple NGS panels targeting childhood

cancers including 13 hereditary cancer panels (128

genes), 9 bone marrow failure panels (159 genes), one

hematological cancer panel (118 genes), and one solid

tumor panel (238 genes). To optimize the detection of

CNVs based on the sequencing data, we developed an

accurate and efficient bioinformatics pipeline to dentify

and visualize CNVs allowing simultaneous detection of

SNVs, indels and CNVs.

Methods: To overcome the limitation of targeted panel

sequencing that lacks continuous coverage required for

the detections of CNV and to meet the needs of

identifying ploidy changes in cancers, thousands of single

nucleotide polymorphism (SNP) probes relatively evenly

covering the entire genomic regions of targeted genes

were added to each panel as the “backbones”. FastQ

files generated by HiSeq sequencer are first aligned to

GRCh37 assembly. CNVs are called by VarScan2 on the

data from patients and controls sequenced using the

same set of baits. B-allele frequency was generated

based on SNP calls from the backbone and the regions

of interest of the panel for each patient and compared to

that of the controls. CNVs were annotated with gene

symbols and chromosome locations. CNVs and B-alleles

were then post-processed and visualized by home-brew

software using R Shiny. The pipeline were designed for

analyzing multiple samples in parallel to increase analytic

efficiency and accuracy.

Results: To assess the performance of the pipeline, we

validated it on 30 different clinical samples with known

CNVs. The results showed 100% concordance to that of

our previously validated commercial software yet with

only 20% of the processing time. Analyzing multiple

cases in paralell also improves the accuracy of the CNV

calls by eliminating batch effect. In addition to detecting

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simple CNVs, the pipeline successfully detected complex

genomes with complicated del/dup/amp and ploidy

changes in neuroblastoma, rhabdomyosarcoma, and

osteosarcoma exemplified by the identification of a

pseudo-hyperdiploid genome in a near-haploid ALL

patient which would be indistinguishable from true

hyperdiploid ALL by cytogenetics or histology/flow

cytometry (figure 1).

Conclusions: We have developed a clinical

bioinformatics pipeline for CNV calling, annotation and

visualization for targeted NGS panels. The validation

results demonstrate 100% accuracy in identifying cancer

associated CNVs with increased efficiency and accuracy.

[figure 1 for caok g-amp 2019 abstract]

OR18. Pan-Cancer Repository of Validated Natural

and Cryptic mRNA Splicing Mutations

P. Rogan1,2, E. Mucaki1, B. Shirley2

1University of Western Ontario, Biochemistry, London,

ON, Canada, 2Cytognomix, London, ON, Canada

Introduction: Next generation sequencing continues to

reveal large numbers of novel variants whose impact

cannot be interpreted from curated variant databases or

through reviews of peer-reviewed biomedical literature.

This has created a largely, unmet need for unequivocal

sources of information regarding the molecular

phenotypes and potential pathology of variants of

unknown significance (VUS); in cancer genomes, such

sources are critically needed to assist in distinguishing

driver mutations from overwhelming numbers of

bystander mutations.VUS classification criteria highlight

the limitations in genome interpretation due to ambiguous

variant interpretation.

Methods: We present a major public resource of mRNA

splicing mutations validated according to multiple lines of

evidence of abnormal gene expression. Likely mutations

present in all tumor types reported in the Cancer

Genome Atlas (TCGA) were identified based on the

comparative strengths of splice sites in tumor versus

normal genomes and then validated by respectively

comparing counts of splice junction spanning and

abundance of transcript reads in RNA-Seq data from

matched tissues and tumors lacking these mutations.

Results: The comprehensive resource features 351,423

of these validated mutations, the majority of which

(69.1%) are not present in the Single Nucleotide

Polymorphism Database (dbSNP 150). There are

117,951 unique mutations which weaken or abolish

natural splice sites, and 244,415 mutations which

strengthen cryptic splice sites (10,943 affect both

simultaneously). 27,803 novel or rare flagged variants

(with < 1% population frequency in dbSNP) were

observed in multiple tumor tissue types. Single variants

or chromosome ranges can be queried using a Global

Alliance for Genomics and Health (GA4GH)-compliant,

web-based Beacon “Validated Splicing Mutations” either

separately or in aggregate alongside other beacons

through the public Beacon Network (http://www.beacon-

network.org/#/search?beacon=cytognomix), as well as

through our website

(https://validsplicemut.cytognomix.com/).

Conclusions: The Validated Splicing Mutation resource

should substantially contribute to reducing the number of

outstanding VUS in tumor (and possibly some germline)

genomes, and substantially increases the number of

functional variants with previously unappreciated

consequences to mRNA splicing, in particular, activation

of cryptic splice sites We previously (doi:

10.1038/srep07063) identified 988 TCGA breast cancer

mutations that significantly altered normal splicing (19%

of total). This database greatly expands the size of the

repository. Here, a higher ratio of rare or novel mutations

have been validated (24% of total were flagged). The

higher yield could be related to the same mutation being

present in multiple samples from the same tumor type

and other tumor tissues, which would be expected to

increase the probability of observing abnormally

expressed splice forms for the mutation.

OR19. Error Rate Normalization to Establish Position-

Specific Limits of Detection in Next Generation

Sequencing Assays

J. D'Angelo1, S. Ferguson2, Z. Xie1, L. Chamberlain1, A.

Carson1, J. Miller1

1Invivoscribe Technologies, San Diego, CA, United

States, 2LabPMM, San Diego, CA, United States

Introduction: Next-generation sequencing (NGS)

continues to be the method of choice for high throughput

genetic assays. Many assays employ a uniform assay-

specific limit of detection (LoD) that tends to be overly

conservative due to “worst-case” error and artifact rates

(generated by PCR, sequencing and alignment errors,

etc.). The process of differentiating sequencing artifacts

from real, low-frequency variants is extremely important

for clinical utility of NGS-based assays, especially as

clinicians push for tracking minimal residual disease

(MRD). The motivation for this study was to identify a

strategy for finding the inherent error-rate, at single base

resolution, for our MyMRD assay. This strategy improved

the specificity and sensitivity of our MyMRD assay and

can be implemented in any NGS-based assay.

Methods: Our study focused on Invivoscribe's MyMRD

assay panel and investigated the observed error rates at

all 53,308 targeted bases. We looked at each of these

positions independently to evaluate the observed

frequency of every possible single nucleotide variation

(SNV) and every observed insertion/deletion (indel) in

213 samples (36 samples from reference cell line

NA12878 and 177 contrived/clinical samples).

S12 jmd.amjpathol.org ● The Journal of Molecular Diagnostics

We calculated various inter and intra-sample metrics

(e.g. minimums, maximums, average error frequencies)

independently for each SNV and indel to understand

overall assay trends as well as sample-specific trends at

single base resolution.

Results:

Sample

Set

Mini

mum

Obse

rved

SNV

Freq

uenc

Maxi

mum

Obse

rved

SNV

Freq

uenc

Aver

age

Obse

rved

SNV

Freq

uenc

Mini

mum

Obse

rved

Indel

Freq

uenc

Maxi

mum

Obse

rved

Indel

Freq

uenc

Aver

age

Obse

rved

Indel

Freq

uenc

High

est

Freq

uenc

SNV

Type

NA12878

0% 0.30

%**

4.4x1

0^-

0% 0.33

%**

0.022

C--

Contrive

d/Clinical 0% 0.49

4.8x1

0^-

0% 0.10

0.002

C--

[Table 1]

Table1: The analysis showed that error-rates widely vary

across different positions and across different variant

types (even within a single targeted base).

**Known variants (in NA12878) and high probability

germline variants and artifacts were filtered from these

analyses to focus on the background error-rate at

homozygous reference positions.

Conclusions: We were able to increase the sensitivity

and specificity of the MyMRD panel by creating non-

uniform LoDs that take into account variant specific error

rates at every targeted position. We improved the

sensitivity of the assay by decreasing the LoD (often

dropping from 0.5% to lower than 0.1%) and at >99% of

the targeted sites. However, we also identified potential

artifacts where the LoD was increased to avoid calling

false positives which would have arisen if a uniform LoD

was used. By increasing the LoD at these positions, we

were also able to improve the assay's specificity. Overall,

performing this analysis makes it easier to differentiate

sequencing artifacts from true low-frequency variants,

which is particularly important for MRD tracking

applications.

Solid Tumors

OR20. Clinical Implication of ctDNA Analysis in

Advanced Lung Carcinoma Patients Using Different

Technologies: Real-Time PCR and MALDI-TOF

P.-J. Lamy1, N. Lozano1, C. Becht2, L. Gambier1, N.

Pourel3, C. Alix-Panabieres4

1Inovie, Institut d'Analyse Génomique, Imagenome,

Montpellier, France, 2Centre de Cancérologie du Grand

Montpellier, Thoracic Oncology, Montpellier, France,

3Institut Sainte Catherine, Onco-Thoracic Unit, Avignon,

France, 4University Medical Centre of Montpellier,

Laboratory of Rare Human Circulating Cells, Montpellier,

France

Introduction: With liquid biopsy (LB) entering the routine

in personalized cancer treatment it is important to

understand the differences with regards to technical and

clinical sensitivity and mutation coverage inherent to

technologies for analyzing circulating tumor DNA (ctDNA)

and its implication for the patient care.

Methods: Circulating cell free DNA (cfDNA) extracted

from blood plasma (QiaAMP ctDNA, Qiagen) of 100 lung

carcinoma patients were assessed for DNA quality and

quantity using DNA 3K Assays on the LabChip GX Touch

24 (PerkinElmer). Each 10 ng cfDNA were analyzed for

somatic mutations detection in ctDNA using two

technologies: Real-time PCR test Cobas EGFR Mutation

Test v2 (Roche) that identifies 42 mutations in the EGFR

gene (63 patients tested), and UltraSEEK Lung Panel on

MALDI-TOF based MassARRAY (Agena Bioscience) that

covers 67 mutations in 5 oncogenes: EGFR (43), BRAF

(4), ERBB2 (2), KRAS (14) and PIK3CA (4) with a

sensitivity down to 0.1% (100 patients tested). All

samples were also analyzed with the UltraSEEK EGFR

Panel (Agena Bioscience) that surveys the 3 most

frequent activating mutations in EGFR and the resistance

markers EGFR T790M and EGFR C797S. A set of

controls was added to each run: 0.5% positive control

(EGFR cfDNA Multiplex), EGFR wild-type control

(Horizon Discovery) and no-template control.

Results: All cfDNA samples showed the expected

fragment-size distribution on LabChip and no significant

abundance of genomic DNA.

Four of the 63 (6%) samples tested on Cobas EGFR

Mutation Test v2 were invalid for analysis. Concordant

results were achieved for 85% of patients (50/59) for

overlapping markers in EGFR. With the UltraSEEK Lung

Panel 10 additional EGFR mutations (4*Exon 19 deletion,

3*T790M, 1*Exon 20 insertion, 1*L861Q/R, 1*G917A)

were detected in 8 of 59 patients (14%). 1 EGFR Exon

19 deletion was detected by Cobas only (2%). EGFR

T790M mutation was detected in 7 samples with Cobas

and in 10 samples with UltraSEEK (70% concordance).

UltraSEEK Lung Panel identified 7 non-EGFR mutations

(6*KRAS, BRAF) in 6 patients (10%). Across all 100

patient samples in EGFR 32*Exon 19 deletions,

18*T90M, 17*L858R, 5*G719X, 3*L861X and 1*S768I

could be identified with UltraSEEK Lung Panel as well as

9*KRAS and 2*BRAF V600E. The UltraSEEK EGFR

Panel confirmed all overlapping mutations in EGFR also

assessed with the UltraSEEK Lung Panel.

S13 jmd.amjpathol.org ● The Journal of Molecular Diagnostics

Conclusion: This study showed that the UltraSEEK

Lung Panel improved sensitivity for ctDNA analysis in

comparison to Cobas EGFR Mutation Test v2, a method

widely used in clinical routine. The UltraSEEK Panel

could detect more EGFR primary or secondary

resistance mutations. With the use of a panel that covers

mutations in multiple genes, additional actionable

mutations to EGFR (KRAS, BRAF) could be also

detected in LB and used as a guidance for therapy.

These new data may lead to improve clinical sensitivity of

LB and its clinical utility in the management of lung

cancer patients.

OR21. Adjusting for Variation in Tumor Mutation

Burden Using a Cancer-Specific Threshold in Whole

Exome Sequencing

W. Song1, E. Fernadez1, K. Eng1, O. Elemento1, M.

Shah1, A. Sboner1

1Weill Cornell Medical College, New York, NY, United

States

Introduction: Tumor mutational burden (TMB) has

recently been shown to be an improved biomarker for

predicting response to immunotherapy, with high TMB

patients responding better to immunotherapy than low

TMB patients. Defining a patient as TMB high or TMB low

has not been standardized and often uses a pan-cancer

threshold. On the other hand, almost all previous

publications on TMB cut-off have used comprehensive

panel which covers a small portion of the whole

genome/exome, and no systematic whole exome

sequencing data has been analyzed to analyze the TMB

distribution among different cancer types. Hereby, we

aim to understand the differences between using a pan-

cancer threshold compared to a cancer-specific

threshold, as well as understand the clinical context of

TMB.

Method: Using whole exome sequencing (WES) data

from primary tumors in The Cancer Genome Atlas

(TCGA) (n=3886) and advanced Weill Cornell Medicine

(WCM) samples (n=979), TMB status was determined by

using both a pan-cancer and cancer-specific threshold.

Survival curves and number of TMB high classifications

were used together to evaluate the differences between

the thresholds. Cox regression was performed to

understand other clinical variables that may influence the

use of TMB as a prognostic biomarker.

Result: The distribution of TMB varied between cancer

types. The cancer-specific threshold was able to adjust

for the different TMB distributions, while the pan-cancer

threshold was often too stringent. The dynamic nature of

the cancer-specific threshold resulted in more TMB high

classifications compared to the static pan-cancer

threshold. Additionally, the survival curves between the

cancer-specific and pan-cancer threshold were similar for

both TMB high and TMB low groups.

Conclusion: TMB is relative to the context of cancer

type, metastatic state, and disease stage. A cancer-

specific threshold results in more patients being classified

as TMB high while maintaining clinical outcome

comparable to pan-cancer threshold

[Distribution of TMB Among Different Cancer Types]

[Comparison of TMB-H Samples by WCM versus

Chalmers' Cut-off]

S14 jmd.amjpathol.org ● The Journal of Molecular Diagnostics

OR22. Integrated Genomic Profiling of 289 Pediatric

Brain Tumors Uncovers Genomic Signatures

Significantly Impacting Patient Care

F. Lin1, L. Surrey1, A. Resnick2, P. Storm2,3, M. Luo1, D.

Gallo1, K. Cao1, X. Zhao1, S. MacFarland2, M. Santi1, A.

Waanders2, M. Li1,2

1Children's Hospital of Philadelphia, Department of

Pathology and Laboratory Medicine, Philadelphia, PA,

United States, 2Children's Hospital of Philadelphia,

Center for Childhood Cancer Research, Philadelphia, PA,

United States, 3Children's Hospital of Philadelphia,

Department of Pediatrics, Division of Neurosurgery,

Philadelphia, PA, United States

Introduction: Genomic mutation profiling plays a

significant role in brain tumor diagnosis, prognosis, and

treatment. Large Next-Generation Sequencing (NGS)

panels are rapidly replacing traditional single-gene or

focused multigene testing to provide more

comprehensive information about the tumor genome.

Here we describe genomic profiles of a large cohort of

pediatric brain tumors tested in a single clinical laboratory

using a custom-designed NGS panel, CHOP OncoMark

Solid, and their impact on patient care.

Methods: Brain tumor samples from patients 0-21 years

old over a 33-month period were subjected to NGS

testing. Genomic alterations were assessed using the

CHOP OncoMark Solid panel to interrogate 238 cancer

genes for single nucleotide variants (SNVs), indels, and

copy number variants (CNVs), and 110 fusion gene

partners for over 600 known and novel fusions. Identified

variants were categorized and reported according to the

AMP/ASCO/CAP guidelines. The clinical impact of the

results were evaluated based on their significance on

diagnosis, prognosis and treatment.

Results: 289 consecutive tumors were tested, the most

common of which were pilocytic astrocytoma (87/289),

medulloblastoma (24/289) and diffuse midline glioma

(21/289). Clinically significant genomic alterations were

identified in 93% of the tumors. 191 clinically significant

SNVs/indels in 73 genes were reported 323 times in 164

cases. The most frequent mutations were BRAF V600E

(32/323) in multiple tumor types and H3F3A K28M

exclusively in diffuse midline glioma. TP53 and NF1 were

second and fourth most mutated genes in this cohort.

More than 1500 CNVs were reported in 175 cases with

gain of 1q and 7q the most prevalent. 32 fusions were

detected in 97 cases with KIAA1549-BRAF the most

common. These results provided diagnostic evidence in

73.3% of patients including 9 patients whose diagnoses

were changed and 41 patients whose diagnoses were

further defined; prognostic evidence in 34.6% of patients,

and evidence for targeted therapy in 18% of patients.

Distinctive genomic profiles for specific tumor subtypes

were observed: 64% of pilocytic astrocytomas harbored

KIAA1549-BRAF, all diffuse midline gliomas were H3F3A

K28M positive with 52% of them showing H3F3A gain,

isochromosome 17q was highly enriched in non-

WNT/non-SHH medulloblastomas, and loss/cnLOH of

chr22 was observed in 100% of ATRT cases, 42% of

which displayed additional SMARCB1 mutations. 71

patients were suspected of carrying germline mutations,

of which 25 were evaluated and 11 confirmed. The

average tumor mutation burden (TMB) was 3.88

mutations/Mbp and all 5 cases with high TMB (>10

mutations/Mbp) harbored a germline mutation in MSH2

or MSH6.

Conclusion: Integrated genomic profiling of pediatric

brain tumors demonstrates significant impact on clinical

care in 78.2% of patients in our institution. Broad-base

genomic profiling should be implemented as part of the

routine patient care for pediatric brain tumors.

OR23. Biomarker Analysis on Limited Biopsy Lung

Cancer Samples in Tertiary Care Cancer Centre in

South India Basavatarakam Indo American Cancer

Hospital, Hyderabad, India

S. Sudha Murthy1, L. Nambaru2, S.J. Rajappa3, G.

Sandhya Devi1, S.A. Sahithi1, K.M. Mallavarapu3, F.

Daphne1, T.S. Rao4, C. Veeraiah5

1Basavatarakam Indo American Cancer Hospital,

Pathology & Laboratory Medicine, Hyderabad, India,

2Basavatarakam Indo American Cancer Hospital,

Laboratory Medicine, Hyderabad, India, 3Basavatarakam

Indo American Cancer Hospital, Medical Oncology,

Hyderabad, India, 4Basavatarakam Indo American

Cancer Hospital, Surgical Oncology, Hyderabad, India,

5Basavatarakam Indo American Cancer Hospital,

Radiology, Hyderabad, India

Introduction: Lung cancer diagnosis and management

has undergone a paradigm shift in the last decade with

multiple actionable mutations and genetic alterations

being detected. Biomarker studies in lung cancers from

India are limited owing to the different tissue handling

processes and lack of standardisation in the preanalytic

phase. We present the data of prevalence of EGFR

(Epidermal growth factor), ALKD5F3 (Anaplastic

lymphoma kinase; clone D5F3) and ROS1 in a cohort

from a tertiary care centre in south India seen over the

last two years.

Methods: EGFR testing was done by RT-PCR (Qiagen

Rotorgene) using Therascreen EGFR RGQ PCR kit,

ALKD5F3 testing was done by immunohistochemistry

(IHC) on Ventana Benchmark XT automated

immunohistochemistry platform and ROS1 testing by

Flouroscence in situ hybridisation (FISH) using Zytolight

break-apart probe.

Results: Of a total 515 lung biopsies performed. EGFR

mutation testing was done in 269 (52.2%) cases.

Morphology included 406 (78.8%) adenocarcinomas,

51(9.9%) adenosquamous carcinomas, 35 (6.7%)

squamous cell carcinomas and 23 (4.4%) of poorly

differentiated carcinomas. Overall EGFR mutation was

detected in 141(52.4%) patients. The predominant

mutations identified were Del 19 in 77 (28.6%) cases,

L858R in 52 (19.3%) cases and 102 (37.9%) cases were

of wild type. Dual mutations Del19+L858R were noted in

two cases, Del19+L861Q in one case, L858R+T790M in

one case and T790M+Del19 in two cases. The

biomarkers were mutually exclusive in majority of the

cases, however one case showed dual ALKD5F3

expression by IHC and EGFR mutation (insertion). One

S15 jmd.amjpathol.org ● The Journal of Molecular Diagnostics

other case harboured Del19 mutation and ALKD5F3

positivity. 200 patients underwent ALKD5F3 testing by

IHC, of which 36 (18%) patients expressed ALK

positivity. 195 patients underwent ROS1 testing by FISH

of which five (2.56%) patients harboured ROS1 gene

rearrangement.

T790M testing was done at the time of progression to test

for secondary resistance by RTPCR on biopsy samples

in 48 patients. Of these 26 patients (54%) harboured

T790M mutations indicating secondary resistance

pattern. Of these 26 patients, 17(65.3%) harboured Del

19 mutaion, six (23%) harboured L858R, two had a

report of mutation (done elsewhere) and one was wild

type (done elsewhere).

Conclusion: Triaging of tissue samples and coordination

between multidisciplinary teams is the key to successful

biomarker analysis and patient management in lung

cancers.

OR24. Oncologists' Review of ctDNA EGFR Mutation

Testing Reports

Z. Deans1, G. Holland1, N. Iley1, F. Blackhall2, A.

Greystoke3, D. Talbot4, R. Butler5

1GenQA/UK NEQAS, Edinburgh, United Kingdom, 2The

Christie NHS Foundation Trust, Manchester, United

Kingdom, 3Newcastle upon Tyne Hospitals NHS

Foundation Trust, Newcastle upon Tyne, United

Kingdom, 4Oxford University Hospitals NHS Foundation

Trust, Oxford, United Kingdom, 5All Wales Genetic

Laboratory, Cardiff, United Kingdom

Introduction: GenQA is an External Quality Assessment

(EQA) Provider for Genomics which assesses laboratory

tests and interpretative content of reports. GenQA

provided an EQA for the testing of circulating tumour

DNA (ctDNA) for EGFR mutations in lung cancer

patients. The submitted reports were anonymously

reviewed by a panel of Consultant Oncologists to identify

the elements which were clear and unambiguous, and

those misleading and may cause patient harm if

misinterpreted. The findings were shared with the

laboratories to improve the reporting standard of liquid

biopsy test results.

Methods: Nine laboratories perform ctDNA testing for

EGFR mutations in lung cancer patients in the United

Kingdom, and all took part in this EQA and report review.

The EQA reports submitted for two clinical cases were

anonymised and sent to the reviewers to be rated for

ease of finding the result, understanding the test

methodology and clarity of the interpretation. Text which

the reviewer found comprehensive, and content which

was confusing or inaccurate were highlighted. Individual

laboratory reviews were provided to the laboratories.

Results: The review identified elements deemed to be

essential on the report; a clear statement of the mutation

testing result in a format which cannot be misinterpreted,

understandable test information and associated

limitations, the use of HGVS nomenclature cross

referenced to the common names e.g. T790M, a

condensed one page report and generic reference to

targeted treatment rather than naming individual drugs.

The reports submitted for a patient progressing on first

line EGFR TKI therapy with the primary EGFR mutation

and a resistance mutation detected in the plasma

sample, raised comments about reporting allelic

frequency and queried the clinical utility for this on the

report. The clinicians wished reassurance that ctDNA had

been tested as the primary EGFR mutation had been

detected.

The second report set was related to a patient relapsing

on EGFR TKI therapy with no EGFR mutations detected

in the plasma sample. The reviewers' responses were

focussed on the risk of a false negative result and the

ways the laboratories reported this varied, if reported at

all, and the recommendations on appropriate further

testing. The reviewers expressed a wish for a more

standardised approach to interpreting such results and to

emphasise the importance on highlighting that the

primary EGFR mutation had not been detected.

Conclusions: Laboratories report ctDNA EGFR mutation

testing in lung cancer patients using a multitude of

formats and terminology. The lack of standardisation

increases the risk of misinterpreting the result with the

potential to harm patients through inappropriate clinical

management or treatment. The external oncologist

review of the content of reports has provided laboratories

with feedback as to the positive and negative elements

within their reports and will aid in the improvement in

genomic test result reporting.

POSTER PRESENTATIONS

Genetics/Inherited Conditions

P001. The UHN NGS Panel for Hematologic

Malignancies: Use for Standard of Care Testing of

Acute Myeloid Leukemia in Ontario/Canada

J.M. Capo-chichi1,2,3, S. Grenier1, C. Badduke2, K. Chin1,

A. Swindell1, N. Stickle1, G. Downs1, B. Nwachukwu1, T.

Zhang2, S. Kamel-Reid1,2,3, T. Stockley1,2,3

1Genome Diagnostics, Department of Clinical Laboratory

Genetics, Laboratory Medicine Program, University

Health Network, Toronto, ON, Canada, 2Advanced

Molecular Diagnostics Laboratory, Princess Margaret

Cancer Centre, University Health Network, Toronto, ON,

Canada, 3Department of Laboratory Medicine and

Pathobiology, University of Toronto, Toronto, ON,

Canada

Introduction: Hematologic malignancies including

myeloproliferative neoplasms, myelodysplastic

syndromes and acute leukemia are characterized by

great clinical and genetic heterogeneity. Genomic

profiling is an important component for diagnosis,

prognosis and management of patients with these

hematopoietic malignancies in the updated 2016 WHO

classification of myeloproliferative neoplasms and acute

leukemia. AMP recently recommended molecular testing

of 34 critical genes as standard of care in chronic myeloid

neoplasms. Our previous work on 1,600 patients with

various hematopoietic malignancies (Princess Margaret

Cancer Center - Advanced Genomics in Leukemia study)

S16 jmd.amjpathol.org ● The Journal of Molecular Diagnostics

proved that comprehensive Next-Generation Sequencing

(NGS) was an effective aid for disease diagnosis and

clinical management of these individuals.

Methods: We developed a custom hybrid-capture NGS

panel that targets 28 hotspot gene regions and 21 entire

consensus DNA coding sequences of 49 clinically

relevant genes in hematologic malignancies.

Results: This clinical test is currently offered as part of a

newly funded program for patients with acute leukemia in

Ontario/Canada. To date, we have completed molecular

profiling on over 500 Acute Myeloid Leukemia (AML)

patients. Specific approaches were taken to enable

capture of complex gene regions and to identify

challenging variants including larger insertions, deletions

and duplications in genes such as CEBPA, CALR, FLT3

and KMT2A. We have reported over 1,000 clinically

relevant variants (preliminary data on 300 patients) using

this NGS assay: minimum: 0, maximum: 13, average 4

per case. NGS detected unsuspected variants in genes

(BRAF, NOTCH1) indicating complex clinical

presentation of disease; comprehensive NGS testing was

also useful in confirmation of diagnosis of AML and in

identification of AML patients that were further confirmed

to have familial predisposition to myeloid neoplasms

(DDX41, RUNX1, MPL mutations).

Conclusions: Molecular profiling by NGS is an effective

strategy for proper diagnosis and clinical management of

patients with hematologic malignancies. Important

considerations for NGS panel testing development and

validation in a clinical setting include relevance of

hematologic malignancy genes and/or gene regions to be

tested, complete gene coverage and uniform coverage of

targeted gene regions. Bioinformatics support is required

to improve assay performance and analytical sensitivity

particularly for detection of large insertions, deletions and

duplications.

P002. Real-World Diagnostic Value of Targeted Panel

Sequencing in Korean Hearing Loss Patients

J.H. Rim1, J. Jung1, H.Y. Gee1, S.-T. Lee1, J.Y. Choi1,

J.R. Choi1

1Yonsei University College of Medicine, Seoul, Republic

of Korea

Introduction: As more genes have been identified as

linked to hearing loss and more public databases

become available, a substantial proportion of patients

with hearing loss are diagnosed for genetic causes using

next generation sequencing (NGS) results. In this study,

we present clinical and diagnostic utility of a targeted

NGS panel in Korean patients with hearing loss, along

with several strategies to maximize the diagnostic yield.

Methods: A total of 39 unrelated Korean pediatric

patients with moderate to severe nonsyndromic hearing

loss (NSHL) at Severance Hospital were tested using a

customized NGS panel (Celemics, Korea) targeting all

coding exons and exon/intron boundaries of 182

deafness-related genes. After target enrichment,

sequencing was performed on the MiSeq System

(Illumina, USA) according to the manufacturer's

instructions. Our customized bioinformatics pipeline with

segregation studies, extensive literature review, and well-

curated genotype-phenotype correlation were applied to

improve the diagnostic yield. The classification of variants

followed the NSHL-optimized American College of

Medical Genetics and Genomics (ACMG) guidelines.

Results: Among 39 patients, 11 patients (28.2%) had

pathogenic or likely pathogenic mutations in 12 genes

including TECTA, POU3F4, POU4F3, NLRP3,

CATSPER2, SLC26A4, USH2A, TMPRSS3, OTOG,

MYO7A, GJB2 and KCNQ4. Types of pathogenic

variants were variable including missense (n=9),

frameshift (n=3), nonsense (n=1), splice site (n=1), and

deletion (n=1). Interestingly, a 22-year old female patient

had dual pathogenic mutations in GJB2 and KCNQ4,

highlighting the clinical utility of NGS. In addition, whole

gene deletions including CATSPER2 with varying sizes

confirmed by Multiplex Ligation-dependent Probe

Amplification were identified in three patients, including

possibly two carrier individuals. Clinical genotype-based

phenotype review confirmed that the identified

pathogenic variants were strongly matched in most

patients according to NSHL-optimized ACMG guideline

interpretation.

Conclusions: Our results demonstrate that this targeted

NGS panel has an excellent diagnostic performance and

great utility in patients with hearing loss. Deep

understanding of the genetic architecture of hearing loss

was also of utmost importance, and thus the targeted

NGS panel could be used in routine clinical diagnostics

with careful interpretation and optimal strategies.

(Keywords: Hearing loss, Next generation sequencing,

Targeted gene panel, Deafness)

P003. Distinct Craniofacial and Skeletal Features,

Keratosis, Hearing Loss, Short Stature, and Complex

Congenital Heart Disease with Vasculopathy:

Expanding the Phenotype of BRD4-Related

Conditions

L. Starr1, A. Yetman1

1University of Nebraska Medical Center, Children's

Hospital, Pediatrics, Omaha, NE, United States

Introduction: Patients with point mutations and deletions

encompassing BRD4 have been described.

Haploinsufficiency of this gene can cause a Cornelia de

Lange-like (CdL) syndrome with short stature,

microcephaly, characteristic facial features, congenital

heart defects, hirsutism, and neurodevelopmental delays.

Herein is a detailed description of a new patient with

distinct physical characteristics. An updated genetics

consultation was requested for a 29 year old male that

resulted in a gestalt of a hybrid CdL and Rubenstein

Taybi phenotype.

Methods: An exome analysis with trio reference was

performed which resulted in a single genetic mutation of

interest. A missense mutation in BRD4 (NM_058243.2,

c.860G>T (p.Glyc287Val)) was classified as that of

uncertain significance. This de novo mutation had a

minor allele frequency of zero, was highly conserved, and

was predicted in most models to be deleterious.

Results: The distinct physical characteristics included a

S17 jmd.amjpathol.org ● The Journal of Molecular Diagnostics

craniofacial appearance reminiscent of the

aforementioned syndromes. He had microcephaly,

micrognathia, poor dentition, thick and course hair with

multiple whorls and hirsutism with atypical hair placement

and density. He had a very narrow chest wall. There was

a single testicle s/p orchiopexy, small hands and feet,

hyperkeratosis of the palms, scleroderma-like tight skin,

and an angiofibromatous persistent rash-like appearance

on the face. He had short stature and hearing loss,

presumed to be secondary to cholesteatomas. He had a

small adrenal mass of no known consequence. He had

intellectual disability with a pleasant personality.

Cardiovascular complications included tetralogy of Fallot

type double outlet right ventricle, progressive dilation of

the ascending aorta and moderate dilation of the celiac

artery.

Conclusions: This patient had features of CdL similar to

previously reported patients with BRD4

haploinsufficiency. The three individuals previously

reported with missense mutations were in a cohort

chosen for their CdL phenotype, which was qualified as

mild. In contrast, our patient had more distinct features.

While his moderate aortic dilation may be at least in part

due to his congenital cardiac defect, the celiac artery

dilation would support a more systemic vasculopathy.

This patient may represent the severe-end of the BRD4-

related disorder spectrum and expands the phenotype.

P004. Somatic Mosaic Truncating Mutations in Exon

6 of PPM1D in Leukocytes Are Likely to Be Related to

the Effects of Chemotherapy

D. Won1, B. Kim1, S.T. Lee1, J.R. Choi1

1Yonsei University College of Medicine, Department of

Laboratory Medicine, Seoul, Republic of Korea

Introduction: PPM1D (Protein phosphatase magnesium-

dependent 1δ) plays a key role in the negative regulation

of p53 and modulates the DNA damage response

pathway. Gain-of-function mutations of the PPM1D gene

are associated with a wide variety of cancers including

breast cancer and ovarian cancer while over suppressing

the function of p53. Reportedly, truncating mutations in

exon 6 of PPM1D can cause overexpression of PPM1D

and the mutations of mosaic form in peripheral blood are

more common in the ovarian cancer or breast cancer

group than in the control group. On the other hand, it is

reported that PPM1D mosaic truncating mutations in

leukocytes are associated with chemotherapy rather than

predisposition of solid cancers.

Methods: We examined the PPM1D mutations in next-

generation sequencing (NGS) results of patients with

suspected hereditary solid tumors. The NGS tests were

conducted on hereditary cancer panel with peripheral

blood between July 2016 and August 2018. Detected

variants were further examined by visual verification

using the Integrative Genomic Viewer (IGV). The

pathogenicity of variants is classified according to the

ACMG criteria. We also examined the mutations in blood

and cancer specimen by sanger sequencing in three

cases.

Results: A total of 1195 patients were included in the

study. Among 1,195 patients, the proportion of patients

with breast and/or ovarian cancer (80.3%) was the

highest. Truncating mutations in PPM1D were detected

in 4 (4/965, 0.41%) of the patients with breast cancer

and/or ovarian cancer. All truncating mutations were in

exon 6, in mosaic form. The mean percentage of mutant

reads was 11.15% (range= 5.4%-15.4%). All four had a

history of chemotherapy. Of the total, 395 were treated

with chemotherapy and odds ratio of chemotherapy

about PPM1D truncating mutations in exon 6 was 18.4

(95% CI: 0.99-342.67, p=0.051). In three cases, A low

percentage of mutations in peripheral blood by NGS were

confirmed by Sanger sequencing but no corresponding

mutations in the tumor tissues were observed.

Conclusions: In our NGS analysis, PPM1D mosaic

truncating mutations in peripheral blood were observed in

0.41% of breast and/or cancer patients, which was

similar to that reported previously. However, the

mutations were somewhat significant in the group treated

with chemotherapy. Clonal hematopoiesis which is

related to age cause hematologic cancers according to

acquisition of somatic mutations due to chemotherapy.

Patients with truncating PPM1D mutations in our study

may be subjects with a so-called clonal hematopoiesis of

indeterminate potential (CHIP). Nevertheless, it is unclear

whether the mosaic truncating mutations of PPM1D in

the blood are the cause acting as a driver of solid cancer

or the result of chemotherapy. We need to study more

samples and make sure that the mutations are in normal

tissues.

P005. Molecular Diagnosis of Huntington's Disease in

Suspected Cases of Indian Origin

P. Chheda1, T. Dama1, D. Goradia1, S. Pande1, S.

Vinarkar1

1Metropolis Healthcare Ltd., Molecular Pathology,

Mumbai, India

Introduction: Huntington's disease (HD), an autosomal-

dominant, progressive, neurodegenerative genetic

disorder is caused by an increase in the number of CAG

repeats in the huntingtin (HTT) gene. Genetic tests that

accurately determine the number of CAG repeats are

performed for confirmation of diagnosis. The aim of our

study was to evaluate utility of triplet-primed polymerase

chain reaction (TP-PCR) for routine diagnosis of HD in

suspected cases from India.

Methods: We ran a combination of CAG flanking PCR

and triplet-primed PCR for estimation of CAG repeats in

732 cases with clinical suspicion of HD. Age of onset

versus the number of CAG repeats were studied.

Results: There were 363 cases (49.6%) that showed the

presence of expanded alleles, with 349 (47.67%) being

fully penetrant alleles and 13 (1.77%) in the reduced

penetrance category. The remaining suspected cases

(N=369) showed CAG repeats in normal range, thereby

excluding the diagnosis of HD and these cases should be

further evaluated for Huntington's disease-like (HDL)

syndromes. There were 13 juvenile cases with an age of

onset lower than 20 years, with the longest allele

comprising 106 CAG repeats found in an 8-year-old male

S18 jmd.amjpathol.org ● The Journal of Molecular Diagnostics

patient. The results demonstrated an inverse (R = - 0.66)

relationship between CAG length and age at clinical

onset.

Conclusion: Our study on pan-Indian cases is one of the

largest studies reported so far in India and focuses on the

most accurate and comprehensive molecular diagnostic

evaluation of HD.

P006. Development of a Clinical Next Generation

Sequencing Panel for Diagnosis of Cystic Lung

Diseases: Evaluation of Diagnostic Yield and

Optimization of Bioinformatics Pipelines

J. Oh1, B. Kim1, Y. Kim1, K.-A. Lee1

1Gangnam Severance Hospital, Yonsei College of

Medicine, Laboratory Medicine, Seoul, Republic of Korea

Introduction: Multiple cystic lung disease (CLD)

represents a diverse group of uncommon disorders that

can present a diagnostic challenge due to the increasing

number of diseases associated with this presentation.

Among CLD, several diseases have well-defined

causative mutations in the relevant genes; e.g.

lymphangioleiomyomatosis (LAM), Birt-Hogg-Dube

syndrome (BHD), tuberous sclerosis complex (TSC) and

cystic fibrosis (CF). Thus far, the molecular diagnosis of

CLD is mainly based on Sanger sequencing. As Sanger

sequencing of all the candidate genes substantially

increase the cost, genetic testing usually starts with the

most commonly involved genes and proceeds to less

likely genes only when clinical suspicion is very high. In

recent years, targeted next-generation sequencing (NGS)

platform has been further developed, allowing us to focus

specifically on genomic regions of interest for cheaper

multiplexed sequencing of more cases. However,

inaccuracy in detecting the length of homopolymers

repeats and complexity in detecting structural variation

became a critical barrier against accurate detection of

genomic variations. Herein we seek to evaluate

diagnostic yield of customized CLD panel using NGS

platform.

Methods: 49 patients with multiple lung cysts was

enrolled. Mutations in FLCN gene were characterized

through Sanger sequencing, Multiplex Ligation-

dependent Probe Amplification (MLPA) and quantitative

PCR (qPCR). Evaluation of diagnostic yield of the CLD

NGS panel was done using Ion torrent S5 NGS platform.

Genomic DNA reference materials obtained from Coriell

cell repository and results of Sanger sequencing-

confirmed mutations in FLCN was used to validate

analytical performance of the CLD panel. Two

bioinformatics(BI) pipeline for processing NGS data were

used; the Torrent Suite(TSS) with a plug-in Torrent

Variant caller (Thermo Fisher Scientific, MA, USA) and

NextGENe(NG) software (Softgenetics, PA, USA).

Results: Out of 49 patients with multiple lung cysts, 18

FLCN pathogenic variants and 1 FLCN large deletion

were observed confirmed by Sanger sequencing and

MLPA respectively. Using CLD NGS panel test, 1 extra

pathogenic variant and 3 VOUs were newly observed. In

analytical performance evaluation, analytical sensitivity of

TSS and NG was 77.1% (59.4-89.0) and 94.3% (79.5-

99.0). Specificity of TSS and NG was both 100%. After

adapting additional hotspot bed file to TSS, sensitivity

went up to 100%. And after adapting IGV visual

inspection of the pathogenic variant hotspot list,

sensitivity of NG was 100%.

Conclusions: Diagnostic yield using NGS went up from

38.7% to 46.9% compared with FLCN Sanger

sequencing alone. Even though the pathogenic hotspot

of FLCN is mostly filtered out using TSS BI pipeline due

to 8 homopolymers repeat sequences, adjusting BI can

dramatically improve the overall performance.

Optimization of the BI pipeline is essential when

designing difficult NGS panel.

P007. DNA Methylation and Its Correlation with

Breast Cancer Pathological Prognostic Staging

V. Gupta1,2, P. Deshpande3, A. Bhake4

1Jawaharlal Nehru Medical College, Datta Meghe

Institute of Medical Sciences, Pathology, Wardha, India,

2Datta Meghe Institute of Medical Sciences, Molecular

Biology and Epidemiology Laboratory, Wardha, India,

3University of Dayton, Ohio, OH, United States,

4Jawaharlal Nehru Medical College, Pathology, Wardha,

India

Introduction: Breast cancer is the most common form of

malignant disease in women worldwide and is the major

cause of mortality among women. It is a heterogeneous

disease with histopathological, genetic and epigenetic

characteristics. Epigenetic alterations such as in DNA

methylation, characterized by addition of a methyl group

at the carbon-5 position of cytosine residue within CpG

dinucleotide, are considered an early event in tumor

development. However, the changes in DNA methylation

and its correlation with the progression of breast cancer

is not well studied. The study aims to assess the

relationship of DNA methylation in breast cancers

presenting at different pathological prognostic stages, for

understanding the progressive behavior.

Methods: Cross sectional study on surgically resected

mastectomy specimens from breast carcinomas,

diagnosed as invasive ductal carcinoma and matched

adjacent normal tissues from 63 breast cancer patients

were included in the study, conducted in Department of

Pathology and Molecular Biology and Epidemiology

Laboratory at Jawaharlal Nehru Medical College, Datta

Meghe Institute of Medical Sciences, Wardha, India.

Clinico-pathological data was obtained from the record in

the surgical pathological section. The specimens were

collected after pathological examination in phosphate

buffer saline.

DNA extraction and methylation: A conventional

phenol/chloroform extraction procedure was followed.

DNA methylation at CpG islands was studied using

ELISA by Epigentek kit.

Statistical analysis: performed using SPSS 23.0. Fisher's

exact tests was used to calculate the agreement from

different sources (tumor, and adjacent tissues) among

cases.

Results: Sixty-three, breast cancer patients were

enrolled in the study. Median age 50 years, range (26 -

S19 jmd.amjpathol.org ● The Journal of Molecular Diagnostics

77). Molecular subtyping was as follows: Luminal A&B,

18(29%); HER2 enriched 27(42%); triple negative,

18(29%) cases respectively. Maximum number of

patients were in stage III i.e., 36 (57%) followed by stage

II: 18 (29%) and stage I: 9 (14%), as categorized in

Pathological Prognostic Staging system.

The median levels of CpG island methylation for invasive

ductal carcinoma tumor samples was 64% and that of

normal samples was 18%. There was a significant rise in

CpG island methylation from stage I to stage II i.e., 26%

to 97%. This rise could be due to increase in number of

CpG islands in tumor suppressor genes with the

increasing stage of breast cancer. This rise was also

significant after controlling the age of patients (p-value <

0.05). No significant association was found between

different molecular subtypes and levels of CpG island

methylations in DNA.

Conclusion: Aberrant DNA methylation is associated

with increased stage of breast cancer and can be used to

assess the biological behavior of tumor progression.

Status of DNA methylation at CpG islands can also be

used as a prognostic marker.

P008. Evaluation of Developmental Delay and Related

Disorders in Pediatric Patients Using Targeted Next

Generation Sequencing

J.-Y. Han1, K.-S. Woo1, I.-H. Jeong1, R.-Y. Goh1, J.-H.

Yoo2

1Dong-A University College of Medicine, Laboratory

Medicine, Busan, Republic of Korea, 2Dong-A University

College of Medicine, Pediatrics, Busan, Republic of

Korea

Introduction: The etiology of developmental delay (DD)

and related disorders is heterogeneous and is therefore

still posing diagnostic challenges. With the introduction of

next generation sequencing (NGS) techniques, diagnosis

and discovery of causative genes have increased

tremendously in the past several years. In this

presentation, we conducted targeted NGS using various

panels for diagnosis and classification of pediatric DD

and related conditions.

Methods: The patients were evaluated first by clinical

examination at Dong-A University hospital and further

enrolled for NGS analysis in the form of targeted

sequencing. The panels employed were hypothyroidism

panel, proportionate short stature panel, rasopathy panel,

skeletal dysplasia panel, congenital adrenal hyperplasia

(CAH) panel, and inborn error of metabolism panel,

based on the primary clinical and laboratory features of

the patients.

Results: Molecular diagnosis was performed for 25

pediatric patients. Eight proportionate short stature

panels, 7 hypothyroidism panels, 3 each of CAH and

rasopathy panels and 2 each of inborn error of

metabolism and skeletal dysplasia panels were used. Six

pathogenic variants related to the phenotype of patients

were identified by mostly hypothyroidism panel in 5

patients. The other patient showed a pathogenic variant

of FGFR3 gene with skeletal dysplasia panel. Most

common variants were obtained with DUOX2 gene.

Interestingly 13 patients showed variants of uncertain

significance (VUS).

Conclusions: DD patients show variable phenotypes

along with genetic heterogeneity. NGS analysis could be

the best choice for detection of molecular defects.

However, reevaluation of genotype-phenotype data with

literature review is still recommended to revise and

update the mutation profile especially in patients with

VUS.

P009. Impact of CYP4F2 rs2108622 Variant on

Warfarin Dose Requirement in Indian Patients

S. Shah1, M. Paradkar1, S. Natarajan1, C. Ponde2, R.

Rajani2, F. Jijina3, R. Gursahani4, T. Ashavaid5

1P. D. Hinduja Hospital & MRC, Research Laboratories,

Mumbai, India, 2P. D. Hinduja Hospital & MRC,

Department of Cardiology, Mumbai, India, 3P. D. Hinduja

Hospital & MRC, Department of Clinical Hematology,

Mumbai, India, 4P. D. Hinduja Hospital & MRC,

Department of Neurology, Mumbai, India, 5P. D. Hinduja

Hospital & MRC, Department of Laboratory Medicine,

Mumbai, India

Introduction: Warfarin dose management is challenging

due to its narrow therapeutic index and large inter-patient

variability. CYP2C9, VKORC1 variants along with known

non-genetic factors accounts for ~50% of warfarin dose

variability. Additionally a CYP4F2 variant has shown to

influence warfarin dose and therefore, due to absence of

data from the western India, the present study was aimed

to determine the allele frequency as well as to determine

the impact of CYP4F2 rs2108622 variant in warfarin dose

management in Indian patients.

Methods: The present ongoing study included a total of

101 patients undergoing warfarin therapy and 30 healthy

controls. Their mean daily warfarin dose, international

normalized ratio (INR) and demographics were recorded.

CYP2C9 and VKORC1 allele frequency data was

available from our previously published data. CYP4F2

variant genotyping was performed by allele-specific PCR

method and validated by Sanger sequencing. CYP4F2

allele frequencies and genotype frequencies were

calculated and tested for Hardy-Weinberg equilibrium.

Krushal-Wallis and Chi-square test were performed for

comparison of warfarin dose and INR with CYP4F2

mutants using Graphpad Prism (v7.02). A p value < 0.05

was considered significant.

Results: The CYP4F2 rs2108622 minor allele frequency

in the patient and controls were found to be 40% and

42% respectively, which was observed to be higher than

that reported in the Caucasian population (30%). The

mean warfarin doses as well as the mean INR were not

statistically significant with CYP4F2 genotypes. However,

on subgroup analysis, it was revealed that the CYP4F2

variant allele was significantly (p< 0.05) associated in

patients with supra and sub-therapeutic INR (>3 and < 2)

as compared to patients with therapeutic INR (2-3). Most

importantly, only 14% of supra and sub-therapeutic INR

was explained by CYP2C9 and VKORC1 genotyping,

which was significantly increased to 79% with inclusion of

CYP4F2 genotyping. Further in the above subgroup, 68%

S20 jmd.amjpathol.org ● The Journal of Molecular Diagnostics

of patients with CYP2C9+VKORC1 wild-type showed the

presence of CYP4F2 variant. Similar association

between CYP4F2 variant and supra and sub-therapeutic

warfarin dose was observed.

Conclusions: The present study, a first from Western

India showed the CYP4F2 rs2108622 variant to be

clinically relevant and would impact warfarin dose

management. Further validation on a large Indian cohort

will be required in order to establish the true clinical utility

of CYP4F2 variant in management of patients on

Warfarin therapy.

P010. Frequency of the Moyamoya-Related RNF213

p.Arg4810Lys Variant in Patients with Moyamoya

Disease

M.-N. Lee1, E.H. Lee2

1Green Cross Labs, Molecular Unit, Yongin-si, Republic

of Korea, 2Green Cross Labs, Yongin-si, Republic of

Korea

Introduction: Moyamoya disease (MMD) is a rare,

genetically heterogeneous cerebrovascular disease

resulting from progressive steno-occlusion of the distal

internal carotid arteries accompanied by abnormal

collateral vessels. RNF213 was identified as a

susceptibility gene for MMD and a variant in the Ring

Finger 213 gene (RNF213), altering arginine to lysine at

the codon of 4810 (c.14429G>A, p.R4810K)

(rs112735431) is associated with MMD in Asian

populations. Interestingly, many studies have reported

that a certain proportion of the general population in

Japan, Korea, and China also has this variant. Patients

with the homozygote of the variant were also reported to

present more severe form of Moyamoya disease. In this

study, we investigated the frequency of the variant

including heterozygous and homozygous frequency in

Korean MMD patients.

Methods: During January 2017 to October 2018, a total

of 460 MMD patients were included in this study. MMD

was diagnosed as either definite (bilateral) or probable

(unilateral) according to published guidelines. We

sequenced RNF213 exon 60 (encoding p.R4810K) to

determine the genotype of the variant. After obtaining

written informed consent from all subjects, peripheral

blood samples were obtained. Genomic DNA was

extracted from peripheral blood leukocytes by using

Chemagic DNA blood kit (PerkinElmer, Baesweiler,

Germany), according to the manufacturer´s instructions.

Exons of the RNF213 and their ﬂanking introns were

amplified using primer sets designed by the authors

(available upon request). The PCR was performed with a

thermal cycler (model 2720, Applied Biosystems, Foster

City, CA, USA). Direct sequencing was performed with

the BigDye Terminator Cycle Sequencing Ready

Reaction kit (Applied Biosystems) on the AB 3730 DNA

analyzer (Applied Biosystems).

Results: The c.14429G>A (p.R4810K) variant was

identified in 44.3% (204/460) of patients with definite or

probable MMD. A heterozygote of the variant was

observed in 43.0% (198/460) patients and homozygote

was identified in 1.3% (6/460) patients. Homozygotes

had a slightly earlier age at diagnosis compared with

heterozygotes or wild types (median age at Diagnosis 43,

49 and 52 yrs, respectively), which did not achieve the

statistical significance. However, the proportion of

childhood onset (age under 15 yrs) were significantly

higher in patients with homozygotes compared with

patients with heterozygotes.

Conclusions: In summary, our results confirm that

alteration in RNF213 predisposes patents of Korean

MMD patients and the homozygous c.14429G>A

(p.R4810K) variant in RNF213 could be a useful

molecular marker for predicting the severe type of MMD,

for which proper medical intervention is recommended,

and may provide a better monitoring and prevention

strategy. This study will provide the frequency of

c.14429G>A (p.R4810K) variant in RNF213, and an

effective strategy for the molecular diagnosis of MMD in

Koreans.

P011. Thiopurine S-Methyltransferase (TPMT) Allele

Frequencies in Hong Kong Population

S.K.F. Cheung1, J.C.Y. Ho1, Y.S. Chan1, I. Tang1, J.

Kwok1

1Queen Mary Hospital, Division of Transplantation and

Immunogenetics, Department of Pathology, Hong Kong,

Hong Kong

Introduction: Thiopurine drugs, such as Azathioprine, 6-

Mercaptopurine and 6-Thioguanine, are effective

immunosuppressive agents in the management of

inflammatory bowel disease, autoimmune diseases and

organ transplantation. It is also used in anti-leukemia

therapy. Two modes of action have been proposed for

thiopurine. Firstly, the thiopurine metabolite, 6-methyl

Thioinosine monophosphate, inhibits the cell proliferation

of fast growing lymphocytes by suppressing de novo

purine synthesis. In addition, 6-thioguanine nucleotide,

another thiopurine metabolites, exerts a pro-apoptotic

effect to activated T cells [1]. Thiopurine S-

methyltransferase (TPMT) is the key enzyme responsible

for catalytic conversion of the thiopurine metabolites to

non-cytotoxic products. TPMT variants with reduced

enzyme activity may cause accumulation of these

cytotoxic active thiopurine metabolites which can lead to

myelosuppression [2]. Studies have shown that the

TPMT gene is more polymorphic in the Western

populations than in the Chinese populations [3-5]. In this

study, we investigated the allele frequencies of the most

common TPMT variants (TPMT*2, *3A, *3B and *3C) in

the Hong Kong population.

Methods: Genomic DNA was extracted from whole blood

of 300 Hong Kong healthy blood donors. The TPMT*2,

*3B and *3C variant alleles were first detected using a

modified Amplification-Refractory Mutation System

(ARMS) PCR method [6]. Beta-2-microglobulin was used

as the internal control. All samples with variants detected

were further confirmed by Sanger sequencing of the

TPMT exon V, VII and X. Rare TPMT alleles were not

determined.

Results: TPMT*1/*3C genotype was detected and

confirmed in 9 out of 300 healthy subjects. TPMT*2 and

S21 jmd.amjpathol.org ● The Journal of Molecular Diagnostics

TPMT*3B alleles were not detected. The allele frequency

of TPMT*3C allele in the Hong Kong population is 1.5%.

Conclusions: This study showed the allele frequency of

the most common TPMT variants in Hong Kong. Hong

Kong as well as the Chinese population has a lower

frequency of TPMT*3C variant as compared with the

Western populations and only heterozygous TPMT*1/*3C

genotype (3%) was found. It was estimated that 3% of

the Hong Kong population has an intermediate TPMT

enzyme activity. TPMT genotype could be performed for

patients requiring thiopurine therapy to guide the

reduction of drug dose to minimize the risk of

myelosuppression.

P012. Characterization and Distribution of Dystrophin

Gene Mutations Observed in Patients Suspected of

Duchenne Muscular Dystrophy in Pakistan

Z. Ahmed1, A. Nasir1

1Aga Khan University, Pathology and Laboratory

Medicine, Karachi, Pakistan

Introduction: Duchenne muscular dystrophy (DMD) is

an inherited X-linked recessive genetic disorder caused

by a defect in the dystrophin gene. This results in

quantitative and/or qualitative abnormality in the

dystrophin protein causing progressive muscular damage

and weakness. The objective of this study was to

investigate the spectrum of deletions and duplications in

the dystrophin gene of patients suspected of DMD or of

being DMD carriers.

Methods: A retrospective review of DMD testing

performed from March 2017 to June 2018 was conducted

at Aga Khan University Hospital, Karachi. MLPA was

used for identification of deletion/duplication in 79 exons

of the dystrophin gene.

Results: In total 92 cases, 85 males and 7 Females were

examined. In males, deletions and duplication were

identified in 44 (52%) of the 85 cases, deletions were

found in 38 cases (45%) and duplications in 6 cases

(7%). In 7 cases referred for females, 2 of the 7 females

(30%) were found to have a heterozygous deletion,

suggestive of carrier status.

Conclusions: This study identifies deletions in

dystrophin exon 42-52 to be the most common while

duplications were mostly found in exons 3-7. This

information can facilitate the use of new treatments with

exon-skipping drugs, which are mutation specific for

DMD.

P013. Association of Vitamin D Receptor (VDR) Start

Codon FokI Polymorphism with Acute Myelogenous

Leukemia

M. Faiz1, A. Shahid1

1Institute of Nuclear Medicine and Oncology, Lahore,

Pakistan

Introduction: The role of chromosomal translocations,

gene mutations, and polymorphisms in the pathogenesis

of leukemia/lymphoma have been extensively studied.

Recently, the role of vitamin D and vitamin D receptor

(VDR) polymorphisms in hematologic malignancies has

been considered. Vitamin D start codon polymorphism

has been suggested to be associated with an increased

risk of developing different hematological malignancies

including AML. The objective of this study was to

investigate the association between VDR start codon F/f

polymorphism and the risk of AML among Pakistani

patients.

Methods: A total of 70 Pakistani patients with acute

myelogenous leukemia were enrolled in this hospital-

based case-control study. In addition, 70 age and sex

matched healthy volunteers were included as a control

group. Five milliliters (ml) of venous blood were collected

and genomic DNA was extracted from all samples using

standard methods. This was followed by the detection of

VDR Fok-I polymorphism by polymerase chain reaction

and restriction fragment length polymorphism.

Results: A 260 bp fragment of the VDR gene was

amplified by PCR and digested by restriction enzyme

(FokI) to detect the polymorphism. The genotype FF was

the most frequent (76%) in patients with AML, followed

by the genotype Ff (20%) and genotype ff(4%). Similarly

in the control group the genotype F/F also was the most

frequent (57%) followed by the genotype f/f (39%) and F/f

genotype (4%). There was statistically significant

association between AML and each of the genotypes FF

and Ff (p=0.0001). No statistically significant correlation

between the VDR Fok-I polymorphism and sex/age

(P=0.611) was found.

Conclusion: Both FF and Ff genotypes are associated

with increased risk of AML among Pakistani patients.

P014. Human Genome Reference Assembly for Blood

Group Systems

D.-H. Ko1, J.J. Yang1, H. Kim2, S.-H. Hwang1, H.-B. Oh1

1Asan Medical Center, Department of Laboratory

Medicine, Seoul, Republic of Korea, 2Seoul National

University Hospital, Department of Laboratory Medicine,

Seoul, Republic of Korea

Introduction: Clinical applications of next-generation

sequencing (NGS) are continuously expanding. Human

reference genome build comparison is a commonly used

method for variant detection but such an approach is

limited for polymorphic regions encoding blood group

antigens. In this study, we analyzed and typed the

sequences of two human reference genomes to provide

a reference blood group for application of NGS.

Methods: Two human reference genome builds,

GRCh37 and GRCh38 were analyzed. Representative

reference allele sequences were selected for clinical

important blood groups: ABO, Rhesus, Kidd, and Kell

blood groups (AJ536122.1 for ABO gene; X63094.1 and

L08429.1 for RHD gene; DQ322275.1 for RHCE gene;

NM_015865.7 for SLC14A1 gene; and M64934 for KEL

gene). The reference alleles were ABO*A1.01, RHD*01,

RHCE*01, JK*02, and KEL*02, respectively. Sequences

from the two genome builds were compared to the

reference alleles to detect the presence of variants in

coding regions.

Results: Compared with the reference ABO allele, both

reference genome builds had c.261delG, representative

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of the O allele. In addition, GRCh37 assembly showed

additional variants in coding regions (c.106C>T,

c.188G>A, c.189C>T, and c.220C>T). Multiple intronic

variants were found in both reference genome builds. For

RHD, there was no difference between the two reference

genome builds. On the other hand, c.636C>A and

c.1036T>C variants were detected when compared to the

L08429.1 and X63094.1, respectively. These findings are

possibly sequencing artifacts within the reference alleles

and require further analysis. There was no difference

between the reference alleles and the referenced

genome builds for RHCE and SLC14A1. Several intronic

differences were found between the two reference builds

for KEL.

Conclusions: Several differences between the reference

genome build and reference alleles were found. Two

reference genome builds also had differences. Findings

from our study suggest that caution is necessary when

using reference genome builds for polymorphic regions

such as blood group antigens.

P015. An Artificial Intelligence Engine for High-

Throughput Matching of Genetic Variants to Their

ACMG/AMP Classification for Inherited Disease Gene

Panels

E. Frise1, S. Nohzadeh-Malakshah1, M. Falcioni1, C.

Bellavia1, F.M. De La Vega1,2, E. Kiruluta1

1Fabric Genomics Inc., Oakland, CA, United States,

2Stanford University School of Medicine, Dept of

Biomedical Data Science, Stanford, CA, United States

Introduction: The ACMG and AMP defined a set of

evidence-based guidelines to support variant

pathogenicity assessment and reporting in inherited

disease diagnostics. The guidelines define several

criteria, each assessing particular supporting evidence

information that is checked independently for each

variant. Criteria are grouped by different levels of

evidence and a set of rules combines the evaluated

criteria and classifies a variant accordingly as pathogenic

(P), likely pathogenic (LP), benign (B), likely benign (LB),

or uncertain significance (VUS). Although widely adopted

in clinical interpretation of inherited disease variants, this

process has remained a largely manual and time-

consuming process in clinical labs that is not scalable to

the volume of NGS testing data. The rich evidence

needed to properly classify variants is difficult to

effectively incorporate in manual classification schemes,

resulting in discrepancies of interpretation, excessive

specialized reviewing labor, and higher costs. Informatics

tools have been proposed to ease the application of the

guidelines in clinical practice, such as the web-based

ClinGen Pathogenicity Calculator. However, such tools

do not solve the issue of interpreting a large set of

variants, since they do not automate the entire

classification process.

Methods: For these reasons, we developed

VariantMatch, an artificial intelligence engine to

automatically infer the classification of variants that

utilizes a forward-chaining inference engine at its core to

implement the ACMG-AMP criteria returning a predicted

classification for each variant. The engine is able to

incorporate criteria and rule refinements for specific

genes and related diseases by separating the rule

specification from the code that executes them. A critical

input is careful curation of the gene-disease relationship

attributes and computed features including variant

deleteriousness scores such as VVP. Moreover, a natural

language generation module provides a rationale of the

classification reached for reference by clinical geneticists

when reviewing and reporting cases. To evaluate the

performance of our method, we analyzed a set of 2,978

of BRCA1 variants of diverse prior classifications

compiled from ClinVar, VariSNP, JapanData, and the

Color Database.

Results: Our preliminary results show that VariantMatch

was able to correctly classify automatically 96% of 2 stars

or more ClinVar variants and was able to reclassify 69%

and 76% of Color and JapanData VUS variants. The

output provided explanatory text indicating the key rules

matched that led to a classification and was easily

understandable by clinicians.

Conclusion: We plan to integrate VariantMatch into

Fabric Enterprise, a cloud-based software platform for

clinical genomic analysis and reporting, to enable high-

throughput genetic labs to score variants rapidly,

reproducibly, cost-effectively and at scale.

P016. Development of a NGS-based Neotelomere

Mapping Method for Detection of Terminal Deletions

B. Zhang1, A. Iqbal1, W. Crowe1, P. Elliott1, A. Campbell1,

A. Perkins1, P. Rothberg1

1University of Rochester Medical Center, Pathology,

Rochester, NY, United States

Introduction: Human telomeres are composed of

repetitive (TTAGGG)n sequences. Broken chromosomes,

when not repaired by homologous recombination or non-

homologous end-joining, can be stabilized by telomere

capture or by the telomerase-mediated direct addition of

telomeric repeats to nontelomeric DNA ends, forming a

neotelomere (Flint J et al., 1994 PMID:7521575). This

end-repair process is called telomere healing, which

results in terminal deletions. Neotelomeres are found in

human terminal deletion syndromes, which occurs in at

least 1/5,000 individuals (Shaffer LG et al., 2000

PMID:11092830; Knight SJL et al., 1999

PMID:10568569). However, it is not easily detected by

current assay designs and informatics pipelines, and has

not been characterized in a systematic manner.

Methods: We developed a novel NextGen-sequencing

(NGS) based method and an informatics algorithm to

map neotelomeres attributable to telomere healing. This

method uses one telomere-anchored primer and one

ligated adaptor primer together for library generation

followed by deep sequencing. We applied this assay in a

pilot study to human samples with known terminal

deletions, and successfully identified telomere junctions

for most chromosomes in the initial 4 specimens as well

as disease-causing terminal deletions.

Results: The clinical performance and utility of the assay

to screen for fetal chromosomal terminal deletions in cell-

S23 jmd.amjpathol.org ● The Journal of Molecular Diagnostics

free DNA from maternal plasma are being assessed, and

Conclusion: We are also developing a cellular model to

study the molecular pathways regulating neotelomere-

mediated end repair.

P017. Determining Genetic Predispositions Using

Ultrafast Amplicon-Based NGS Hereditary Cancer

Panel

L. Lee1, Y.L. Liu1, K. Pendleton1, L. Lin1, G. Liu1, J. Liu1,

Z. Liu1

1Paragon Genomics, Inc., R&D, Hayward, CA, United

States

Introduction: As genetic testing becomes more widely

accepted, medical professionals are increasingly

interested to use it as a means to screen for hereditary

conditions. While hybrid capture-based target enrichment

methods are mainstream for surveying large number of

targets, they are time-consuming and cumbersome to

perform requiring highly trained operators with

specialized equipment.

Methods: We have developed a multiplex PCR-based

CleanPlex technology to make it possible to produce

high-quality coverage of genomic targets using a fast and

sample target enrichment and NGS library construction

workflow. Here we present an NGS panel targeting

hereditary mutations that covers the full exons of 37

genes using 1445 pairs of PCR primers. In addition, the

panel also detects two clinically relevant intronic

mutations. This panel paired with the CleanPlex workflow

can enable the quick analysis of genes associated with

an increased risk of developing hereditary cancers using

small amounts of genomic DNA.

The CleanPlex 3-step workflow includes a multiplex PCR

step with targeted primers, a background cleaning step to

remove by-products, and a final PCR to add Illumina

adapter sequences and sample indexes. NGS libraries

were made using 10 ng of genomic DNA per pool (40 ng

total). The CleanPlex Hereditary Cancer Panel contains

primers divided into four pools for the multiplex PCR.

Samples were sequenced at ~2,500 read depth on an

Illumina NextSeq. Sequenced reads were demultiplexed.

Mapping rate and on-target rates were calculated, and

variants were identified using Paragon Genomics' variant

calling algorithm.

Results: The samples exhibit >96% uniformity at 0.2X

mean, and all exons were sufficiently covered to provide

accurate variant calling information. The assay is highly

reproducible and sensitive, exhibiting an R2 value of 0.95

between independently prepared replicates, and a

detection rate of >99% for single nucleotide variants.

Conclusion:The Paragon Genomics CleanPlex

Hereditary Cancer Panel can be used for rapid profiling

of cancer risk by using small quantities of genomic DNA.

With the easy workflow and quick turnaround, this panel

can make genetic monitoring easy, efficient, and

economical.

P018. Understanding Next Generation Sequencing

for Autosomal Dominant Polycystic Kidney Disease

(ADPKD)

H. Rennert1, A. Tan1, J. Blumenfeld1

1Weill Cornell Medical College, New York, NY, United

States

Introduction: ADPKD is a genetically heterogeneous

disorder caused by mutations in PKD1 and PKD2,

accounting for ~85% and 15% of cases, respectively.

PKD1 consists of 46 exons spanning ~52 Kb of genomic

DNA, a large part of which is duplicated in six homologue

genes (i.e. pseudogenes) showing ~98% homology with

PKD1 exons 1-33. PKD2 spans 15 exons and a coding

sequence of ~3Kb. Gene-based mutation testing

currently is the predominant method of ADPKD

genotyping. However, the marked allelic heterogeneity of

the disease-associated mutations, the vast majority of

which are private, and the duplicated structure of PKD1

together with its large size, make mutation screening a

significant technical challenge.

Methods: The key step in ADPKD genotyping

procedures is selective amplification of PKD1 sequences,

while excluding the pseudogenes. This was traditionally

achieved by utilizing long-range PCR (LR-PCR) with

primers located to the rare mismatch sites that

distinguish PKD1 and the pseudogenes, while the single-

copy regions of PKD1 and PKD2 are directly amplified

from gDNA. Amplicons are then analyzed by Sanger

sequencing. Introduction of next generation sequencing

(NGS) has revolutionized the field of molecular genetics

and was recently applied to PKD genetic testing. In this

method, LR-PCR is coupled with NGS. Bar-coded LR-

PCR libraries from individual patients are pooled together

and analyzed using paired-end NGS in a single

sequencer flow-cell (Illumina). Sequencing results are

then sorted according to the sample barcode, aligned

against the reference PKD genes and mutations are

called using a computational bioinformatics analysis

pipeline.

Results: This approach has a sensitivity of 100%

compared with Sanger sequencing, while retaining high

specificity, enabling identification of single nucleotide

variants (SNVs), indel and copy number variation (CNV).

Moreover, the high read-depth and average coverage

(>400x) obtained over exonic regions significantly

increases testing sensitivity compared to Sanger

sequencing, allowing detection of low-level mutations

(~3% VAF), especially important for the detection of

genetic mosaicism. Recently, both WES and WGS have

been applied to PKD gene testing, eliminating the need

for LR-PCR. However, the sensitivity of these methods

varied significantly between the duplicated and single-

copy PKD1 compared to LR-PCR based sequencing

(30%~80% versus 100% sensitivity), primarily due to low

read-depth particularly in GC-rich regions and mapping of

reads from duplicated regions.

Conclusion: NGS has the potential to dramatically

improve PKD genetic testing by simplifying workflow and

allowing simultaneous detection of SNV/indels and CNV

S24 jmd.amjpathol.org ● The Journal of Molecular Diagnostics

in a single test, increasing mutation detection rate and

significantly reducing costs and turn-around time.

However, careful evaluation of these novel methods and

the corresponding computational pipelines is required to

ensure high quality clinical testing.

P019. A New Scalable and Automatable Method for

the Extraction of cfDNA

J. Doh1

1Beckman Coulter Life Sciences, Genomic Reagents,

Indianapolis, IN, United States

Introduction: Cell free DNA (cfDNA) consists of small

(150 - 500 bp) DNA fragments that circulate in the blood.

cfDNA levels are generally low in healthy, non-pregnant

patients, and increase in patients with cancer, pregnancy,

or extensive damage to tissue. cfDNA is believe to derive

mostly from apoptotic cells, and biomarkers for a variety

of diseases have been found in cfDNA, increasing

interest in its use as a less invasive way to monitor

disease progression.

Methods: In this study, cfDNA was extracted from

horizon control and cancer patient samples. The

mechanism of DNA extraction was optimized to maximize

yield of cfDNA. The DNA extraction is scalable, and able

to work with low amounts of plasma (~200 µL) up to

several milliliters of plasma. The DNA extraction can also

be run on a variety of automation, and was run on both

Kingfisher and Biomek machines. The efficacy of this

extraction method was measured by looking at detection

of known mutations in horizon control samples and the

effect of input amount on detection levels was

determined. Cancer patient samples were also extracted

and analyzed for associated mutations.

Hematopathology

P020. Assessment of Minimum Residual Disease

Detection in Multiple Myeloma Samples Using Next

Generation Sequencing Based LymphoTrack Assays

and Flow Cytometry

Y. Huang1, A. Jacobsen1, J. Panganiban1, E. Vigil1, K.

Hutt1, F.W. Martinez1, M. Blankfard1, A. Medina2, C.

Jiménez2, R. García-Sanz2, J.E. Miller1

1Invivoscribe, San Diego, CA, United States, 2Hospital

Universitario de Salamanca-IBSAL, Salamanca, Spain

Introduction: Multiple myeloma (MM), characterized by

the presence of >10% clonal plasma cells in bone

morrow (BM), represents the second most common

hematological malignancy. Multiparameter flow cytometry

(MFC) is a standard tool used to detect and monitor MM

patients. Next generation sequencing (NGS) based

methods have demonstrated advantages with improved

sensitivity, and international organizations (NCCN, IMWG

and ESMO) have recently included NGS for minimum

residual disease (MRD) assessment in MM. Here we

report the results of a pilot study comparing the

performance of NGS-based LymphoTrack Assays and

MFC by testing 101 MRD MM samples.

Methods: 101 paired BM samples from MM patients

were tested in this study. The MFC methods utilize the 8-

color direct immunofluorescence technique and test 1-5

million cells. Our NGS based MRD assessment tests only

~1/10 as many cell equivalents of genomic DNA (0.7µg,

~107,000 cell equivalents) from the collected specimens

which were blinded prior to testing with four

LymphoTrack Assays (IGH FR1, FR2, FR3 and IGK) on

a MiSeq. LymphoTrack Software was used to sort data

by target and index. The clonal rearrangement

(clonotype) identified by LymphoTrack FR1 or FR2 Assay

was then tracked to test the 91 subsequent samples.

LymphoQuant Internal Control was added to each PCR

reaction at 100 cell equivalents to estimate the cell

equivalents within each sample. LymphoTrack Software -

MiSeq and LymphoTrack MRD software were used to

analyze the sequencing results from baseline and follow

up samples, respectively.

Results: Despite the limited DNA available for testing,

out of 101 baseline samples, 84 (83%), 80 (79%), 63

(62%) and 87 (86%) samples were detected as clonal

positive by IGH FR1, FR2, FR3 and IGK, respectively.

When combing FR1 and FR2, 94% (95/101) clonal

positivity was achieved. When combining all 4 targets,

100% (101/101) clonal positivity was achieved.

Using LymphoTrack MRD Software to identify clonotype

sequences, we performed MRD assessment on 91

samples that were positive for IGH FR1 or FR2. 47 (52%)

were positive, 43 (47%) were negative, and 1 (1%) was

invalid, due to insufficient reads. MRD results by MFC on

the same 91 patients showed 45 (49%) positive, 37

(41%) negative, and 9 (10%) invalid samples. Excluding

samples with DNA less than 700 ng (~107,000 cell

equivalents), concordance was 85.4%. Three samples

were detected by MCF but not by NGS while four

samples were detected by NGS but not by MCF.

Conclusions: LymphoTrack assays were shown to

detect clonal sequences in 100% of MM baseline

samples and, despite testing approximately 1/10 the

number of cell equivalents, were able to achieve 85.4%

agreement with MFC in detecting MRD. This suggests

that LymphoTrack assays are a useful tool in identifying

and tracking disease status in MM samples. Unlike MFC,

the LymphoTrack assays and accompanying

bioinformatics software can be submitted for approval to

regulatory authorities worldwide.

S25 jmd.amjpathol.org ● The Journal of Molecular Diagnostics

P021. Comparative Analysis of Molecular

Approaches Used for Targeted Detection of

Mutations in FLT3 and NPM1 from DNA and RNA

Templates

C. Wei1,2,3, J.P. Nadeau1, S. Grenier1, B. Nwachukwu1,

J.M. Capo-Chichi1,2,3, S. Kamel-Reid1,2,3, T. Stockley1,2,3

1Genome Diagnostics, Department of Clinical Laboratory

Genetics, Laboratory Medicine Program, University

Health Network, Toronto, ON, Canada, 2Advanced

Molecular Diagnostics Laboratory, Princess Margaret

Cancer Centre, University Health Network, Toronto, ON,

Canada, 3Department of Laboratory Medicine and

Pathobiology, University of Toronto, Toronto, ON,

Canada

Introduction: FLT3 and NPM1 are key biomarkers in

diagnosis, prognosis and disease management of Acute

Myeloid Leukemia (AML). WHO recommends molecular

profiling of Internal Tandem Duplications in FLT3 (FLT3-

ITD), hotspot missense variants in the Tyrosine Kinase

Domain of FLT3 (FLT3-TKD) and insertions in NPM1 for

standard of care testing in AML. Molecular approaches

used to characterize FLT3-ITD, FLT3-TKD and NPM1

mutations differ in performance and analytical sensitivity;

there is no consensus on whether levels of FLT3-ITD

should be assessed from DNA or RNA templates.

Methods: We developed fluorescent PCR based

fragment analysis for targeted detection of FLT3-ITDs,

FLT3-TKDs and NPM1 insertions from DNA and RNA; in

parallel, we used a Next-Generation Sequencing (NGS)

panel for comprehensive analysis of 49 genes altered in

hematologic malignancies, which include FLT3 and

NPM1. We compared the performance of NGS, DNA and

RNA-based fragment analyses approaches on peripheral

blood or bone marrow from 100 AML patients. We

assessed the diagnostic yield of these different

methodologies; variant allelic fraction from DNA versus

RNA, length of FLT3-ITD captured, assay sensitivity.

Assay limitations were also evaluated in this study.

Results: Preliminary data on 50 patients shows that

genotyping calls for FLT3-ITD, FLT3-TKD and NPM1

insertions are concordant in 47/50 of cases analyzed.

Discordances were seen for variant with allelic fraction

detected below 5% by fragment analysis and missed by

NGS. Allelic fraction of variants, particularly FLT3-ITDs

differ between DNA and RNA templates; allelic fractions

were also inconsistent when comparing NGS and

fragment analysis results performed on DNA. NGS was

effective in resolving the nature of in-frame and out of

frame insertions/duplications; amplicon sizing using

fragment analysis could not differentiate these types of

variants occurring in FLT3.

Conclusion: NGS and fragment analysis are

complementary strategies for detection of FLT3-ITDs,

FLT3-TKDs and NPM1 insertions. Fragment analysis is

suitable for critical clinical decision making in AML

patients due to faster turnaround time; NGS

comprehensive molecular profiling aids with diagnosis,

prognosis and management of AML. Recent reports are

in favor of measurements of FLT3-ITDs by RNA; further

investigations and correlation of FLT3-ITDs levels with

clinical outcome of patients will be useful in determining

the optimal nucleic acid template (DNA or RNA) to use

for better stratification of AML patients.

P022. Clinical Application of Next-Generation

Sequencing in Myeloproliferative Neoplasms:

Genetic Profiles and Prognostic Significance for

Personalized Medicine

J.M. Lee1, E. Han1, K. Han1, M. Kim1, Y. Kim1

1Catholic University of Korea, Laboratory Medicine,

Seoul, Republic of Korea

Introduction: The mutational landscape of

myeloproliferative neoplasms (MPNs) has been

discovered including three diagnostic driver mutations,

JAK2, MPL, and CALR. Nowadays, next-generation

sequencing (NGS), especially targeted panel

sequencing, is widely applied in clinical laboratory to

identify mutational profile in hematologic malignancies.

Herein, we evaluated genetic profiles in MPNs using

NGS and explored the clinical application in the single

center cohort.

Methods: A total of 432 patients were included; 196

essential thrombocythemia (45.37%), 112 polycythemia

vera (25.9%), 72 primary myelofibrosis (16.7%), 14 other

MPNs (3.2%) and 38 myelodysplastic/myeloproliferative

neoplasms (MDS/MPN, 8.8%). JAK2 V617F, CALR and

MPL 515 mutations were firstly analyzed using allele-

specific PCR and fragment analysis. Targeted panel

sequencing including 86 genes was performed. We

analyzed relationships between genetic proflies and

clinical outcomes including acute transformation, bone

marrow fibrosis and death.

Results: Three driver mutations were detected in 82.4%

of patients; JAK2 V617F in 64.8%, CALR in 15.7% and

MPL in 1.9%. We also detected mutations in other genes

through NGS which included ASXL1 (27.0%), TET2

(7.6%), KMT2C (3.2%), SRSF2 (2.9%), RUNX1 (2.9%),

CSF3R (2.5%), ATM (2.2%), SETBP1 (1.8%), DNMT3A

(1.8%), TP53 (1.8%), U2AF2 (1.8%), KMT2D (1.8%) and

PTPN11 (1.8%). Among the three phenotypic subtypes,

PMF showed shorter event free survival (EFS) than the

others (hazard ratio: PMF-ET 2.62, PMF-PV and 2.97). In

the aspect of genetic profile, TP53 mutation showed

worst outcome followed by chromatin or spliceosome

mutation (Median EFS: 77 and 99 months, 95%

Confidential interval: 5.0-264 and 72-226, respectively).

Conclusions: We demonstrated that NGS effectively

identified mutations that were more relevant to the

prognosis in MPN patients. Genetic profiles can improve

a risk stratification to make efficient therapeutic plan

including targeted therapy in MPN patients.

S26 jmd.amjpathol.org ● The Journal of Molecular Diagnostics

P023. Potential Role of JAK2V617F and ASXL1

Mutations as Biomarkers for Disease Outcomes

Amongst Thai Patients with Philadelphia-Negative

Myeloproliferative Neoplasms

P. Arnutti1, D. Apipongrat2, T. Numbenjapon2, T.

Nimmanon1, P. Jindatanmanusan3

1Phramongkutklao College of Medicine, Pathology,

Bangkok, Thailand, 2Phramongkutklao College of

Medicine, Medicine, Bangkok, Thailand,

3Phramongkutklao College of Medicine, Pediatric,

Bangkok, Thailand

Introduction: Polycythemiavera (PV), essential

thrombocythemia (ET), and primary myelofibrosis (PMF)

are three main disorders constituting Philadelphia-

negative myeloproliferative neoplasms (Ph-negative

MPNs). The JAK2V617F mutation is present in the

majority of these patients and has been included in

diagnostic criteria for these conditions. Furthermore,

ASXL1 mutations have been demonstrated as prognostic

markers for risk stratification of MPNs. This study

therefore aimed to determine the prevalence of

JAK2V617F and ASXL1 mutations and their relationship

with disease outcomes in Thai Ph-negative MPNs.

Methods: A total of 139 cases (78 male and 61 female)

of Ph-negative MPNs, consisting of 51 PV cases, 67 ET

cases, 18 PMF cases, and 3 MPN-U cases, were

enrolled. All cases were investigated for JAK2V617F and

ASXL1 mutations, utilising allele-specific PCR (AS-PCR)

and Sanger sequencing, respectively.

Results: The JAK2V617F mutation was detected in

64.8% of Ph-negative MPN cases (72.6% of PVcases,

61.2% of ET cases, 50.0% of PMF cases, and 100.0% of

MPN-U cases), whereas ASXL1 mutations were detected

in 10.8% of Ph-negative MPN cases (22.2% of PMF

cases, 11.8% of PV cases, 7.5% of ET cases, and 0.0%

of MPN-U cases). ASXL1 mutations were demonstrated

in 7 out of 90 JAK2V617F-negative cases (7.8%) and 8

out of 49 JAK2V617F-positive cases (16.3%).

Interestingly, JAK2V617-negative ASXL1-positive

patients were shown to have distinctive clinical profiles in

all groups of patients. However, no difference in

thrombosis-free survival was seen amongst MPN

patients with different mutations.

Conclusions: JAK2V617F and ASXL1 mutations might

become potential biomarkers for disease outcomes in

Thai patients with Ph-negative MPNs.

P024. Accurate Detection of Low AF Variants

Relevant to AML by Anchored Multiplex PCR and

Next Generation Sequencing

V. Johnson1, K. Moore1, L. Griffin1, A. Berlin1, A. Licon1,

R. Walters1

1ArcherDX, Boulder, CO, United States

Introduction: Acute Myeloid Leukemia (AML) is clinically

and biologically heterogeneous, requiring the detection of

multiple mutations for characterization. For instance,

FLT3-ITDs and CEBPA mutations represent important

markers in AML, however they are difficult to detect by

NGS due to the highly variable nature of ITDs, the high

GC content of CEBPA, and the difficulty in mapping

repeated sequences to a wild-type reference. Tracking

low frequency mutations is also of growing importance.

The ability to accurately detect variants at low allele

fractions (AFs) using a single test can be used to assess

treatment efficacy and potential relapse.

Methods: We developed Archer VariantPlex myeloid

assays based on Anchored Multiplex PCR (AMP) to

detect important mutations in myeloid malignancies. AMP

is a target enrichment strategy that uses molecular-

barcoded adapters and gene-specific primers for

amplification, permitting open-ended capture of DNA

fragments from a single end. This approach enables

flexible and strand-specific primer design to provide

better coverage of ITD-containing and GC-rich regions.

We also developed a method to assess SNV sensitivity

taking into account both unique coverage depth and

noise for single base substitutions. This strategy enables

utilization of position-specific detection thresholds and

maximizes sensitivity and specificity. We tested this

approach using the VariantPlex Core Myeloid panel, by

titrating reference inputs into background normal samples

to examine detection of low AF variants.

Results: Our assay enables calling of a 30bp FLT3-ITD

down to sub-0.05% allele frequencies. Using optimized

low AF conditions improves coverage depth, consistency

of low AF FLT3-ITD detection, and sensitivity (98.5% of

bases are powered to call a true variant at an allele

frequency of 3.0% with 1M reads and 200ng of input).

We show >1000X unique molecule coverage across the

coding region of CEBPA and use this challenging region

to visualize the minimum detectable AF (MDAF) at which

a variant has a >95% probability of being detected above

the noise (95MDAF). Finally, we show consistent single

nucleotide variant (SNV), insertion and deletion (indel),

and ITD calling at sub-0.5% allele frequencies, and

demonstrate the utility of reporting variant-specific

MDAFs and normal dataset P-values when analyzing low

AF variants.

Conclusion: AMP provides NGS-based detection of

complex mutation types that are relevant in AML. We

demonstrate robust calling of FLT3-ITDs and other

variants at low AFs. We also demonstrate full coverage

of CEBPA with high depth and low noise such that the

95MDAF predicts confident variant calling at low AFs.

This approach is accurate and scalable, enabling

simultaneous detection of multiple mutation types across

multiple target genes in a single assay.

S27 jmd.amjpathol.org ● The Journal of Molecular Diagnostics

P025. Downregulation of RAG1 Expression Inhibits

the Leukemic IK6 Isoform Formation in B-Cell

Lymphoblastic Leukemia Cells

L. Lyu1,2, W. Zhao1, T. Huang1,2, H. Tu3, P. Ru3, X. Pen3,

D. Jones3

1The Ohio State University College of Medicine,

Pathology, Columbus, OH, United States, 2Wuhan No.1

Hospital, Tongji Medical College, Urology, Wuhan, China,

3The Ohio State University College of Medicine, James

Molecular Laboratory at Polaris, Columbus, OH, United

States

Introduction: The IKZF1 gene isoform, IK6 is a common

pathogenic finding in acute lymphoblastic leukemia (ALL)

with or without BCR-ABL1 fusion gene. The formation of

IK6, however, might be caused by either exon deletion or

aberrant RNA splicing. RAG1, one of the key elements

for DNA V(D)J recombination, was predicted to be the

key player in a previous study ( Harvey et al. Blood 2010)

but no direct evidence was given. In this study, we

examined if down regulation of RAG1 would affect the

IK6 formation and what are the other effects of RAG1 in

the biology of ALL.

Methods: The RAG1 gene was specifically knocked

down by small interference RNA (SiRAG1) or

nonspecifically down regulated by givinostat, a type II

Histone Deacetylase and potent apoptosis inducer for

ALL (Li and Yao et al., 2016 and 2017). The presence

IK6 isoform was measured by quantitative RT-PCR. In

addition, a next generation sequencing (NGS) was

performed to study whole transcription analysis to

investigate the differentially expressed whole RNA in the

down-regulated RAG1 ALL.

Results: The IK6 isoform was only detected in RNA but

not in genomic DNA from this cell line confirmed to be

resulted aberrant RNA splicing. The RAG1 was

significantly knocked down by both by 200 nM SiRAG1

and 25 uM Givinostat, 65% and 90%, respectively, (P<

0.001). Meanwhile, IK6 was co-inhibited, 60% and 75%,

respectively (P < 0.001) compared to the controls while

wild-type IKZF1 was not significantly affected. Further

WTS study showed that knockdown RAG1 only

upregulate SOX11 and CCND2 genes and downregulate

most CDK genes associated with cell cycle regulation.

Finally, the analysis of 150 upregulated genes by

SiRAG1 were positively correlated to givinostat treated

specimen (P=0.016), indicating these genes might be

involved in givinostat induced antineoplastic effect

including the apoptosis.

Conclusion: The role of RAG1 in RNA splicing

regulation has not been studied and our results in this

study were the first to support this hypothesis. In addition,

our results provided the evidence that the formation of

IK6 in B-cell ALL can be regulated by RAG1 or

expression of RAG1 with deacetylation inhibitor,

givinostat. Our finding not only revealed the molecular

mechanism in IK6 isoform formation but provided

evidence of clinical investigation if RAG1 might be a

target for personalized medicine in treatment of acute

lymphoblastic leukemia.

P026. Panel-Based Next-Generation Sequencing of

Chinese Diffuse Large B-Cell Lymphoma

B. Cao1,2, C. Sun1,2, R. Bi1,2, Z. Liu1,2, Y. Jia1,2, W. Cui1,2,

M. Sun1,2, X. Zhou1,2

1Fudan University Shanghai Cancer Center, Department

of Pathology, Shanghai, China, 2Shanghai Medical

College, Fudan University, Department of Oncology,

Shanghai, China

Introduction: Diffuse large B-cell lymphoma (DLBCL),

an aggressive and heterogeneous malignancy, remains a

challenging clinical problem, as up to one-third of patients

are not cured with initial therapy. Recently, next-

generation sequencing (NGS) studies in DLBCL have

uncovered recurrent gene mutations. We designed a

NGS multi-gene panel to identify genetic characteristics

of Chinese DLBCL patients and provide relevant

information for panel-based NGS tests to clinical

laboratories.

Methods: A panel of 116 DLBCL genes was designed,

based on literature, the Catalogue of Somatic Mutations

in Cancer (COSMIC) database, and FoundationOne

Heme. We studied 96 Chinese DLBCL biopsy samples

using targeted sequencing.

Results: We identified the mutation frequency of SPEN

(17%) and DDX3X (6%) in our study was higher than

Western studies. Despite functional similarities between

NOTCH1 and NOTCH2, there was no overlap in cases of

NOTCH1 and NOTCH2 mutations, suggesting a distinct

pathogenesis of N1 (NOTCH1 mutations) subtype and

N2 (NOTCH2 mutations) subtype. KMT2D pathogenic

mutations in total patients and patients treated with R-

CHOP-like therapy were associated with significantly

favorable prognosis. MYD88 L265P mutation, MYD88

pathogenic mutations, TP53 and BCL2 pathogenic

mutations were important influence factors of poor

prognosis in DLBCL.

Conclusions: This study underlines the genetic

heterogeneity of DLBCL and demonstrates the

contribution of panel-based NGS to molecular targeted

therapy in DLBCL.

P027. Low-Frequency Variant Detection in Cell-Free

DNA with Ultrafast Amplicon-Based Targeted

Sequencing Using Unique Molecular Identifiers

Y.L. Liu1, K. Pendleton1, L. Lee1, L. Lin1, G. Liu1, J. Liu1,

Z. Liu1

1Paragon Genomics, Inc., R&D, Hayward, CA, United

States

Introduction: The use of cell-free DNA (cfDNA)

applications has seen tremendous growth in recent years

with liquid biopsy being a noninvasive and easily

obtainable sample type with several diagnostics and

prognostic values. While liquid biopsy can potentially

enable applications such as early cancer detection,

treatment monitoring, and drug resistance screening, it

also presents new challenges to variant detection due to

the low fraction of mutant DNA in cfDNA and artifacts

from background noise resulting from PCR and

sequencing errors. While some success has been made

S28 jmd.amjpathol.org ● The Journal of Molecular Diagnostics

by using adapters carrying unique molecular identifiers

(UMIs) in hybrid capture-based methods and performing

deep sequencing, such approaches suffer from long,

tricky, and tedious workflows and disappointingly low on-

target rates.

Methods: We have developed the CleanPlex UMI

technology to provide a fast, simple, and reliable NGS

solution to low-frequency variant detection. The

technology features three simple steps to generate

molecular-barcoded and target-enriched NGS libraries in

3.5 hours. This amplicon-based method consists of first,

a multiplex PCR reaction for molecular barcoding and

target amplification, then followed by a proprietary

background removal step, and finally a second round of

PCR to add platform-specific adapter sequences and

sample indexes. We validated this technology with an

NGS panel optimized for cfDNA to target hotspots in 23

genes frequently mutated in lung cancer. NGS libraries

were prepared from commercial reference cfDNA at

minor allele frequencies (mAF) ranging from 0.1% to

0.5% and sequenced on an Illumina NextSeq to evaluate

the performance.

Results: Overall, the detection sensitivity for low-

frequency alleles is high even at low DNA inputs. We

observe significant reductions in the number of false

positive calls when analyzing the samples using UMIs.

Nearly all known mutations in the reference cfDNA are

detected at 0.1% mAF. At an mAF of 0.25%, all known

mutants are detected with 100% PPV using 50ng of

DNA. Detection of all mutants at mAFs of 0.5% and PPV

of 100% require only 20ng or less of DNA. We are also

able to accurately quantify the detected allele frequencies

due to the high numbers of reference and mutant UMIs

identified for each genetic target.

Conclusion:The CleanPlex UMI technology

demonstrates high sensitivity for the detection of low-

frequency alleles with low false positive detection rates.

P028. Targeted Sequencing Reveals Pathogenic

Genes in EBV-associated T/Natural Killer-Cell

Lymphoproliferative Disorders

L.-M. Gao1, W.-P. Liu1

1West China Hospital of Sichuan University, Pathology,

Chengdu, China

Introduction: EBV associated T/NK cell

lymphoproliferative disorders are characterized by clonal

proliferation of EBV positive T or NK cells. Extranodal

NK/T cell lymphoma-nasal type (ENKTL) is the most

common type of this diseases and it can occur both in the

upper aerodigestive track (nasal-ENKTL) and extranasal

sites (extranasal-ENKTL). However, diverse biological

behaviors and prognosis were observed for ENKTL

involving different sites. The mechanism underlying such

heterogeneity remains elusive. Aggressive natural killer

cell leukemia (ANKL) and EBV positive T cell

lymphoproliferative disorders (EBV+T-LPD) are relatively

rare. There are some overlapping clinicopathologic

features among ENKTL, ANKL and EBV+T-LPD.

Methods: A total of 169 cases of EBV associated T/NK

cell lymphoproliferative disorders were collected. This

included 123 ENKTL cases (87 nasal-ENKTL and 36

extra-nasal ENKTL), 12 ANKL cases and 34 EBV+T-

LPD cases. The targeted sequencing gene panel covers

all exons and elected introns of 64 genes involving in the

development of lymphoma. The mutation profiles of

ENKTL, ANKL and EBV+T-LPD were compared.

ENKTL is the most common disease of the three

EBV+T/NK-LPD, we further done the analysis of gene

mutation of ENKTL cases, Comparison of mutated genes

between nasal and extra-nasal ENKTL, protein express

detection of KMT2D, TET2, EP300 and NOTCH1,

relationship analysis of protein loss expression and gene

mutation and prognosis in 123 target sequencing ENKTL

cases.

Results: STAT3, KMT2D, DDX3X, NOTCH1 and TET2

showed high mutation rates in these three EBV+T/NK-

LPD. The comparison of mutated genes of these three

disorders showed that the exclusive mutated genes of

ENKTL are PRDM1 et al., the ANKL are ITPKB et al., the

EBV+T-LPD is MFHAS1. The mutated rates of KMT2D

and NOTCH2 in extra-nasal ENKTL are greater than

those in nasal-ENKTL.

Further study in ENKTL showed that the loss of KMT2D

and TET2 protein were associated with the mutation of

KMT2D and TET2 gene respectively. Prognostic analysis

showed that the mutation of KMT2D or TET2 gene, the

loss of expression of KMT2D or TET2 protein all

significantly correlated with inferior overall survival of

ENKTL cases.

Conclusion: We conclude that the exclusive mutated

genes of ENKTL, ANKL and EBV+T-LPD are different，

the mutation of KMT2D and TET2 may play an important

role in the development of ENKTL.

P029. Longitudinal Monitoring of AML Tumors with

High-Throughput Single-Cell DNA Sequencing

Reveals Rare Clones Prognostic for Disease

Progression and Therapy Response

D. Eastburn1, C. McMahon2, R. Durruthy-Durruthy1, M.

Carroll2, C. Smith3, A. Perl2

1Mission Bio, Inc., South San Francisco, CA, United

States, 2University of Pennsylvania, Division of

Hematology-Oncology, Philadelphia, PA, United States,

3University of California, San Francisco, Division of

Hematology-Oncology, San Francisco, CA, United States

Introduction: AML (acute myeloid leukemia) is

increasingly being treated with precision medicine. To

better inform treatment, the mutational content of patient

samples must be accurately determined. However,

current tumor sequencing paradigms are inadequate to

fully characterize many instances of the disease. A major

challenge has been the unambiguous identification of

potentially rare and genetically heterogeneous neoplastic

cell populations, capable of critically impacting tumor

evolution and the acquisition of therapeutic resistance.

Standard bulk population sequencing is unable to identify

QCheck: A Web Server for Quality Check of Next Generation Sequencing Data with Standards and Guidelines

Figures