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Integrative characterization of intraductal tubulopapillary neoplasm (ITPN) of the pancreas and associated invasive adenocarcinoma

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Pancreatic intraductal tubulopapillary neoplasm (ITPN) is a recently recognized intraductal neoplasm. This study aimed to clarify the clinicopathologic and molecular features of this entity, based on a multi-institutional cohort of 16 pancreatic ITPNs and associated adenocarcinomas. The genomic profiles were analyzed using histology-driven multi-regional sequencing to provide insight on tumor heterogeneity and evolution. Furthermore, an exploratory transcriptomic characterization was performed on eight invasive adenocarcinomas. The clinicopathologic parameters and molecular alterations were further analyzed based on survival indices. The main findings were as follows: 1) the concomitant adenocarcinomas, present in 75% of cases, were always molecularly associated with the intraductal components. These data definitively establish ITPN as origin of invasive pancreatic adenocarcinoma; 2) alterations restricted to infiltrative components included mutations in chromatin remodeling genes ARID2 , ASXL1 , and PBRM1 , and ERBB2 - P3H4 fusion; 3) pancreatic ITPN can arise in the context of genetic syndromes, such as BRCA -germline and Peutz–Jeghers syndrome; 4) mutational profile: mutations in the classical PDAC drivers are present, but less frequently, in pancreatic ITPN; 5) novel genomic alterations were observed, including amplification of the Cyclin and NOTCH family genes and ERBB2 , fusions involving RET and ERBB2 , and RB1 disruptive variation; 6) chromosomal alterations: the most common was 1q gain (75% of cases); 7) by transcriptome analysis, ITPN-associated adenocarcinomas clustered into three subtypes that correlate with the activation of signaling mechanism pathways and tumor microenvironment, displaying squamous features in their majority; and 8) TP53 mutational status is a marker for adverse prognosis. ITPNs are precursor lesions of pancreatic cancer with a high malignant transformation risk. A personalized approach for patients with ITPN should recognize that such neoplasms could arise in the context of genetic syndromes. BRCA alterations, ERBB2 and RET fusions, and ERBB2 amplification are novel targets in precision oncology. The TP53 mutation status can be used as a prognostic biomarker.
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Integrative characterization of intraductal tubulopapillary
neoplasm (ITPN) of the pancreas and associated invasive
Andrea Mafcini
, Michele Simbolo
, Tatsuhiro Shibata
, Seung-Mo Hong
, Antonio Pea
, Lodewijk A. Brosens
Liang Cheng
, Davide Antonello
, Concetta Sciammarella
, Cinzia Cantù
, Paola Mattiolo
, Sergio V. Taormina
Giuseppe Malleo
, Giovanni Marchegiani
, Elisabetta Sereni
, Vincenzo Corbo
, Gaetano Paolino
, Chiara Ciaparrone
Nobuyoshi Hiraoka
, Daniel Pallaoro
, Casper Jansen
, Michele Milella
, Roberto Salvia
, Rita T. Lawlor
, Volkan Adsay
Aldo Scarpa
and Claudio Luchini
© The Author(s) 2022
Pancreatic intraductal tubulopapillary neoplasm (ITPN) is a recently recognized intraductal neoplasm. This study aimed to clarify the
clinicopathologic and molecular features of this entity, based on a multi-institutional cohort of 16 pancreatic ITPNs and associated
adenocarcinomas. The genomic proles were analyzed using histology-driven multi-regional sequencing to provide insight on
tumor heterogeneity and evolution. Furthermore, an exploratory transcriptomic characterization was performed on eight invasive
adenocarcinomas. The clinicopathologic parameters and molecular alterations were further analyzed based on survival indices. The
main ndings were as follows: 1) the concomitant adenocarcinomas, present in 75% of cases, were always molecularly associated
with the intraductal components. These data denitively establish ITPN as origin of invasive pancreatic adenocarcinoma; 2)
alterations restricted to inltrative components included mutations in chromatin remodeling genes ARID2,ASXL1, and PBRM1, and
ERBB2-P3H4 fusion; 3) pancreatic ITPN can arise in the context of genetic syndromes, such as BRCA-germline and PeutzJeghers
syndrome; 4) mutational prole: mutations in the classical PDAC drivers are present, but less frequently, in pancreatic ITPN; 5) novel
genomic alterations were observed, including amplication of the Cyclin and NOTCH family genes and ERBB2, fusions involving RET
and ERBB2, and RB1 disruptive variation; 6) chromosomal alterations: the most common was 1q gain (75% of cases); 7) by
transcriptome analysis, ITPN-associated adenocarcinomas clustered into three subtypes that correlate with the activation of
signaling mechanism pathways and tumor microenvironment, displaying squamous features in their majority; and 8) TP53
mutational status is a marker for adverse prognosis. ITPNs are precursor lesions of pancreatic cancer with a high malignant
transformation risk. A personalized approach for patients with ITPN should recognize that such neoplasms could arise in the context
of genetic syndromes. BRCA alterations, ERBB2 and RET fusions, and ERBB2 amplication are novel targets in precision oncology. The
TP53 mutation status can be used as a prognostic biomarker.
Modern Pathology;
Pancreatic intraductal tubulopapillary neoplasm (ITPN) is recog-
nized as a subtype of pancreatic neoplasms that form a
heterogeneous group of intraductal lesions, which also includes
intraductal papillary mucinous neoplasm (IPMN) and intraductal
oncocytic papillary neoplasm (IOPN)
. ITPN accounts for up to
35% of all intraductal pancreatic neoplasms
Similar to IPMN, ITPN shows various intraductal growth
degrees. However, compared to IPMN, ITPN is less frequently
cystic, forming instead eshy and solid masses in the involved
. Histologically, ITPNs are hypercellular tumors compris-
ing nodules of back-to-back tubular glands with absent or very
scant mucin formation
. The tubular areas are predominant,
whereas papillary components are limited. In addition to
architectural complexity, ITPN displays uniform high-grade
cytological atypia with numerous mitotic gures and frequent
foci of necrosis. Intra-cytoplasmic and extra-cellular mucins are
consistently absent
Received: 7 April 2022 Revised: 18 July 2022 Accepted: 18 July 2022
Department of Diagnostics and Public Health, Section of Pathology, University and Hospital Trust of Verona, Verona, Italy.
ARC-Net Research Center, University of Verona,
Verona, Italy.
Division of Cancer Genomics, National Cancer Center Research Institute, and Laboratory of Molecular Medicine, The Institute of Medical Sciences, The University of
Tokyo, Tokyo, Japan.
Department of Pathology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, South Korea.
Department of General and Pancreatic
Surgery - The Pancreas Institute, University and Hospital Trust of Verona, Verona, Italy.
Department of Pathology, University Medical Center, Utrecht, The Netherlands.
Department of Pathology and Laboratory Medicine, Warren Alpert Medical School of Brown University and Lifespan Academic Medical Center, Providence, RI, USA.
Division of
Pathology and Clinical Laboratories, National Cancer Center Hospital, Tokyo, Japan.
Laboratory for Pathology Eastern Nertherlands, Hengelo, The Netherlands.
Department of
Medicine, Section of Oncology, University and Hospital Trust of Verona, Verona, Italy.
Department of Pathology, Koç University Hospital and Koç University Research Center for
Translational Medicine (KUTTAM), Istanbul, Turkey.
These authors contributed equally: Andrea Mafcini, Michele Simbolo. email:;
Pancreatic ITPN is a presumed precursor of invasive ductal
adenocarcinoma, although denitive evidence is still lacking.
Concomitant adenocarcinomas have been reported in up to 70%
of cases at diagnosis
. Despite the high-grade cytological and
architectural features and the frequent association with concomi-
tant invasive cancer, ITPN usually has a more favorable prognosis
than conventional pancreatic ductal adenocarcinoma (PDAC),
even when associated inltrative lesions are present. However, a
small subset of patients presents with locally advanced or
metastatic disease at diagnosis or will develop local recurrence
or distant metastases after surgical resection; thus, better
comprehension of this lesion type is warranted.
ITPN has a distinct mucin immunohistochemical prole,
rendering immunohistochemistry (IHC) an important supportive
tool in the ITPN diagnosis. ITPNs are usually characterized by the
expression of MUC1 and MUC6 and generally lack expression of
the MUC5AC and MUC2 proteins
. Moreover, pancreatic ITPN is
molecularly distinct from IPMN and conventional ductal adeno-
carcinoma, showing rare (but not absent) mutations in the KRAS
and TP53 genes and more common PI3KCA mutations and FGFR2
In the present study, we performed a multi-institutional analysis
of the molecular prole of different ITPN components (tubular and
papillary areas) and concomitant invasive cancers through
histology-driven multi-regional sequencing. This study aimed to
clarify the genomic features of pancreatic ITPN, including tumor
heterogeneity and the molecular progression to invasive cancers.
Based on the results of our analyses, we provide specic insights
into molecular markers with clinical impact and suggest possible
novel targets for precision oncology.
Case selection and clinicopathologic analysis
The following electronic databases were searched for pancreatic ITPN
cases: Verona University and Hospital Trust (Verona, Italy), National Cancer
Center Research Institute (Tokyo, Japan), Asan Medical Center (Seoul, South
Korea), University Medical Center (Utrecht, The Netherlands), and Indiana
University (Indianapolis, IN, USA). Cases with material available for
molecular analysis were selected. Our cohort comprised 16 cases, which
were subsequently conrmed by histology performed by two pancreatic
pathologists. All cases were negative for BCL10, chromogranin A, and
synaptophysin. Medical records and electronic databases were used to
obtain supplementary clinicopathologic data, including prognostic out-
comes. Cases were staged using the American Joint Committee on Cancer
staging, 8
Multi-regional massive parallel DNA sequencing
To understand better tumor heterogeneity and evolution, a multi-regional
sequencing approach for genomic analysis was adopted. The most
representative inclusion from each case were selected for analysis. The
tubular area and the papillary region for the 16 ITPNs were then selected.
Co-occurring adenocarcinomas, when present, were also analyzed.
Genomic DNA was obtained from formalin-xed, parafn-embedded
tissues after enrichment for neoplastic cellularity, using manual micro-
dissection. DNA was extracted and quantied as previously described
using the GeneRead DNA FFPE kit (Qiagen - Hilden, Germany) according to
the manufacturers instructions.
DNA sequencing was performed for both tubular and papillary tumor
components, following the previously described SureSelectXT HS CD
Glasgow Cancer Core assay (, hereafter referred to as
. The CORE panel spans 1.8 Mb of the genome and searches 174
genes for somatic mutations, copy number alterations, and structural
rearrangements. The details of the targeted genes are reported in
Supplementary Table 1. Sequencing was performed on a NextSeq 500
(Illumina, San Diego, CA, USA) loaded with two captured library pools using
a high-output ow cell and 2 × 75 bp paired-end sequencing.
CORE panel analysis started with demultiplexing performed with FASTQ
Generation v1.0.0 on the BaseSpace Sequence Hub (https://, last access 11/16/2021). Forward and reverse
reads from each demultiplexed sample were aligned to the human
reference genome (version hg38/GRCh38) using Burrows-Wheeler Aligner
version 0.7.17-r1188
. Mapped reads were subjected to PCR duplication
removal and indexed, using biobambam2 v2.0.146 (
german.tischler/biobambam2.git; last access 11/16/2021)
. Coverage
statistics were calculated using the same software
. Single nucleotide
variants were identied using shearwater
. Small (<200 bp) insertions and
deletions were identied using Pindel version 0.2.5b8
. All candidate
mutations were manually reviewed using the Integrative Genomics Viewer
version 2.4 to exclude sequencing artifacts
Microsatellite instability was calculated using the method described by
Papke et al.
. Copy number alterations of targeted genes were detected
using the GeneCN software (; last access
06/30/2021). Structural rearrangements were detected using the BRASS
, and visually reviewed using the Integrative Genomics Viewer,
version 2.4
Tumor variants were classied as benign (class 1), likely benign (class 2),
variant of uncertain signicance (class 3), likely pathogenic (class 4), or
pathogenic (class 5), according to the guidelines of the American College
of Medical Genetics and Genomics and the Association for Molecular
Transcriptome analysis
Gene-expression analysis of 20,815 human genes was performed on the
co-occurring adenocarcinomas to obtain their transcriptomic prole,
according to previously described methods
.Briey, libraries were
prepared using the Ampliseq Transcriptome Human Gene Expression Kit
(Thermo Fisher Scientic, Waltham, MA, USA) with 1 µg of retro-
transcribed RNA for each multiplex PCR amplication. The AmpliSeqRNA
plugin generated each samples expression data (counts per transcript).
Counts were normalized and transformed using the DESeq2 package for
. Visualization and clustering were performed using the Complex-
Heatmap package for R
. The NbClust package was adopted to estimate
the best number of clusters. Then, a hybrid hierarchical k-means
approach was used to perform principal component analysis and to
design a dendrogram showing the relationships between samples. To
verify the resulting associations between samples, unsupervised
consensus clustering was performed using ConsensusClusterPlus. For
tumor classication, pancreatic cancer signatures were retrieved from
studies performed by Bailey et al.
, Collisson et al.
,andMoftt et al.
and cluster-specic enriched gene sets were determined using the
normalized count matrix. We applied gene set enrichment analysis
(GSEA) using the GAGE-R package between clusters to obtain signicant
pairwise up- and down-regulated pathways
. We performed z-score
normalization of pathway scores in each cluster.
Chromogenic multiplex IHC and additional IHC
Adenocarcinoma gene expression proling related to immune microenvir-
onment composition was cross-validated using chromogenic multiplex IHC
analysis as previously described
. Based on the results of the transcrip-
tome analysis, two T-lymphocyte markers, CD4 (labeled in red) and CD8
(DAB), and the class 2 macrophage marker CD163 (green) were selected for
this study. Cells were considered positivewhen the cell membrane was
stained. The expression of these markers was evaluated as previously
reported, using a semi-quantitative (05) scoring system: 0 =negative (no
stained cells), 1 =rare (110 positive cells per high-power eld, HPF; 400×
magnication), 2 =low (1120 positive cells per HPF), 3 =moderate
(2130 positive cells per HPF), 4 =high (3150 positive cells per HPF), and
5=very high (>50 positive cells per HPF)
In the case of ERBB2 amplication, a specic IHC analysis for Her2
(Hercep test, Dako, Germany) was performed. Finally, all cases were tested
for p53 with IHC (clone: DO-7, 1:50 dilution, Novocastra, UK).
Survival analysis
Univariate and multivariate Cox regression analyses were performed to
investigate any association between clinicopathologic and molecular data,
and survival outcomes. The outcomes considered were overall survival,
cancer-specic survival, disease-free survival, and composite outcome.
Multivariable analysis was planned using the factors signicantly
associated with the survival outcomes of interest with a p-value < 0.10 in
the univariate analyses. Data from the Cox regression analyses were
graphically reported using KaplanMeier curves. The results were
presented as hazard ratios with a 95% condence interval. Statistical
analyses were performed using SPSS version 20.0 (Chicago, IL, USA).
A. Mafcini et al.
Modern Pathology
Clinicopathologic analysis
The crucial clinicopathological features of the 16 cases are
summarized in Table 1. Five patients were men (31.2%) and 11
were women (68.8%), with an average age at diagnosis of 63.2
years (range 4776). Three cases (18.8%) were incidentally
diagnosed in asymptomatic individuals; of these, two were
diagnosed during routine follow-up for genetic syndromes, such
as hereditary breast and ovarian cancer syndrome (HBOC) and
PeutzJeghers syndrome.
At diagnosis, co-occurring invasive adenocarcinoma was pre-
sent in 12 cases (75%), represented by glandular/tubular
adenocarcinoma. Regarding tumor stage, four cases (25%) were
resected at stage 0 (i.e., non-invasive), four (25%) at stage I, four
(25%) at stage II, three (18.8%) at stage III, and one case (6.2%) at
stage IV due to the presence of a single liver metastasis.
Follow-up data were available for 15/16 patients. The majority
(10, 62.5%) were alive and disease-free at the last follow-up
(average follow-up time: 27.9 months). Two pancreatic lesions
were analyzed in one patient; an initial lesion during surgical
resection for a non-invasive ITPN (case #7a), and a later lesion
during local adenocarcinoma tumor relapse (case #7b), observed
38 months after the surgical resection. After surgical re-
intervention for relapse, the patient remains alive and disease-
free at the most recent follow-up (8 months after re-intervention).
Molecular analysis
Multiregional massive parallel sequencing. All cases were investi-
gated using multi-regional sequencing to assess their genomic
proles. In the single metastatic case, we investigated the invasive
pancreatic adenocarcinoma and the liver metastasis in addition to
the papillary and tubular intraductal components. Thus, we
provided the molecular characterization of tubular and of papillary
intraductal components of six cases, whereas in the remaining 10
cases, the co-occurring adenocarcinoma was also investigated
(Fig. 1). For two cases (#1 and #3), the invasive component was not
suitable for molecular analysis. The mutational proles and copy
number variations are summarized in Table 2and structural
alterations are shown in Table 3.
Sequencing revealed recurrent mutations in the classical PDAC
drivers: KRAS mutations in four cases (25%), in both the ITPN and
the concomitant inltrating adenocarcinoma; TP53 mutations in
four cases (25%), three of which had a co-occurring adenocarci-
noma; SMAD4 mutations in two cases (12.5%), restricted to the
tubular area and not altered in either the papillary or the
adenocarcinoma, the latter present in only one case; BRAF was
mutated in two cases (12.5%), both displaying the same V600E
mutation; RNF43 was mutated only in the papillary component of
a noninvasive case; and no mutations were detected in CDKN2A or
GNAS in any of the cases. None of the ITPN samples showed
microsatellite instability. The four cases harboring TP53 mutations
showed aberrant staining pattern in p53 IHC, with strong and
diffuse nuclear positivity of >90% tumor cells (Supplementary
Fig. 1). Other cases were interpreted as wild type.
Two ITPNs associated with concomitant adenocarcinoma (case
#9 and #14) were detected as part of the spectrum of familial
cancer syndromes. One case was diagnosed in a patient with
PeutzJeghers syndrome; the germline variation was associated
with LOH of STK11. The other case was detected in a patient with
HBOC syndrome carrying a BRCA2 germline variation coupled with
LOH on chromosome 13.
Copy number variations of the Cyclin family genes were noted
in three cases (18.75% of cases), in particular, CCNE1 was amplied
in two cases (12.5%) and CCND3 in one case (6.25%). Gene gain/
amplication is frequently observed in the NOTCH and FGFR
families in ITPN. Here, alterations in FGFR involved two cases with
gene gain (12.5%) and one with amplication (6.25%), and in
NOTCH, two cases with gain (12.5%) and two with amplication
(12.5%). We also observed NTRK1 amplication in all the
components in two cases (12.5%); one was the relapsing case
and the alteration was maintained in the recurrent neoplasm.
Finally, ERBB2 was amplied in all the components of one ITPN
sample with concomitant adenocarcinoma. In IHC staining, Her2
expression showed a heterogeneous pattern, from a weak to a
focally strong positivity (although there are no specic guidelines
for assessing Her2 in pancreatic tumors; Supplementary Fig. 2).
Six cases harbored structural genomic alterations (Table 3).
Among these, ve showed gene fusions and one showed
translocation. RET was fused with C14orf93 or TRIM24,FGFR2 with
STCP1 or HSD17B4, and ERBB2 with P3H4. Translocation by
asymmetric breakdown and repair of chromosome 1 in LMNA
(1q22) and chromosome 13 in RB1 (13q14.2) resulted in dicentric
and acentric chromosomes, respectively, containing the distal
parts of the q arms welded together. This generated truncated
proteins with 3-3and 5-5junctions, resulting in loss of function.
Altogether, the genomic proling data, including mutations,
variants of unknown signicance (Supplementary Table 2), copy
number variants, and gene fusion, claried that the co-occurring
adenocarcinomas were derived from the intraductal precursors
and shared with them the majority of somatic alterations. Three
cases harbored additional alterations restricted to the invasive
components, such as mutations affecting the chromatin remodel-
ing genes ARID2,ASXL1, and PBRM1, observed in three cases, and
the ERBB2-P3H4 fusion in the case with ASXL1 mutation.
Chromosomal alterations were observed in all samples (Fig. 2).
Chromosomal gains were detected in 15 cases (93.7%), whereas
chromosomal loss was observed in all cases. The most common
alterations were 1q gain, detected in 12 cases (75%), and 1p, 6q, or
18q losses found in 8 (50%), 9 (56.2%), and 9 (56.2%) cases,
respectively. Loss of heterozygosity (LOH) followed by reduplica-
tion, leading to copy-neutral LOH or LOH with additional gain, was
observed in 9 (56.2%) cases.
Transcriptome analysis. Overall, eight ITPN-concomitant adeno-
carcinomas were investigated using transcriptome analyses (Fig. 3).
A hybrid hierarchical k-means approach (k=3) was used to
perform principal component analysis and design a dendrogram
showing the relationships between samples. The resulting
consensus matrix obtained from the unsupervised consensus
clustering conrmed the associations obtained by the principal
component analysis and the dendrogram.
The three identied clusters, A, B, and C (Fig. 2), included four
and three samples, and one sample, respectively. Pairwise
differential expression analysis was performed for all identied
clusters. Cluster A showed 21 differentially expressed (DE) genes
(Supplementary Table 3), in which no genes with cluster-based
statistical signicance were identied in clusters B and C.
Comparison with the mutational analysis results showed that
cluster A was enriched with 3 cases (75%) containing KRAS
mutations and ARID2/PBMR1 chromatin remodeling. Cluster B
included one case (33%) with a BRAF mutation. The single case in
cluster C showed a BRCA alteration (germline BRCA2 mutation
coupled with LOH).
The comparison between the expression proles of each cluster
and current PDAC classiers highlighted that cluster A showed
squamous-like signatures, similar to Moftts basal-like and active
stroma proles and Collissons quasi-mesenchymal prole. In
contrast, cluster C showed features of the classical pancreatic
subtype similar to the exocrine prole. No statistically signicant
associations were identied for cluster B; nonetheless, we noted a
trend for similarity with Moftts basal-like subgroup (squamous-
like prole) (Fig. 4A). Furthermore, using the GSEA-based
approach, we identied differential biological processes among
the three clusters. Based on the z-score, cluster A showed
enrichment for induction of the epithelial-to-mesenchymal (EMT)
pathway and KRAS signaling. In contrast, cluster B presented
A. Mafcini et al.
Modern Pathology
Table 1. Summary of the most important clinicopathologic data of pancreatic ITPNs and concomitant invasive adenocarcinomas.
N case Sex, age Site in
Associated cancer Size of the
whole lesion
(T=size of
the invasive
Tumor stage VI PNI R Involved ducts Relevant clinical
history and
1 M, 76 Head-
Yes 100 mm
(25 mm)
pT2N1M0 IIB Yes No R0 Main +branch Abdominal pain Solid-cystic
AF (65)
2 F, 71 Head No 20 mm pTisN0M0 0 n/a n/a R0 Main Incidental
AF (62)
3 M, 68 Head Yes 50 mm
(36 mm)
pT2N1M0 IIB Yes No R0 Main +branch Jaundice Solid-cystic
AF (31)
4 F, 75 Head,
body, tail
Yes 130 mm
(60 mm)
pT3N0M0 IIA no No R1 Main +branch Abdominal pain Solid-cystic
DO (0)
5 M, 72 Head No 12 mm pTisN0M0 0 n/a n/a R0 Main Jaundice Solid-cystic
AF (15)
6 F, 50 Head-
No 36 mm pTisN0M0 0 n/a n/a R0 Main +branch Epigastric pain,
weight loss
of the
gland, Solid-
AF (54)
M, 57 Tail No 16 mm pTisN0M0 0 n/a n/a R0 Main Acute
Cystic lesion AD (38)
M, 59 Head-
Yes 25 mm
(12 mm)
pT1cN0M0 IA Yes Yes R0 Main +branch Diagnosed on
follow-up for
Solid-cystic AF (8)
8 F, 64 Body-tail Yes 70 mm
(23 mm)
pT2N1M0 IIB Yes Yes R0 Main +branch Abdominal pain Solid-cystic AF (9)
9 M, 60 Body-tail Yes 60 mm
(31 mm)
pT2N0M0 IB Yes Yes R0 Main +branch PeutzJeghers
Diagnosed on
Solid-cystic AF (27)
10 F, 47 Body Yes 25 mm
(3 mm)
pT1aN0M0 IA No No R0 Main n/a Solid lesion AF (3)
11 F, 63 Head Yes 35 mm
(22 mm)
pT2N2M0 III Yes Yes R0 Main Jaundice,
Solid lesion AD (54); liver
metastasis (9)
12 F, 60 Body-tail Yes 90 mm
(45 mm)
pT3N2M0 III Yes Yes R0 Main Fatigue, weight
loss, fever,
abdominal pain
Solid lesion DD (2);
recurrence (1)
13 F, 72 Head Yes 25 mm
(7 mm)
pT1bN2M0 III Yes Yes R0 Main Epigastric pain,
weight loss,
Solid lesion n/a
14 F, 59 Head Yes 25 mm
(9 mm)
pT1bN0M0 IA Yes Yes R0 Main Previous ovarian
Solid lesion DO (21)
15 F, 64 Head-
Yes 66 mm
(4 mm)
pT1aN0M0 IA Yes Yes R0 Main +branch Abdominal pain Solid-cystic DO (13)
16 F, 54 Body-tail Yes 35 mm
(30 mm)
pT2N2M1 IV Yes Yes R1 n/a D yspepsia,
abdominal pain
Solid-cystic AF (4)
Ffemale, Mmale, pTNM pathologic TNM staging, VI vascular invasion, PNI perineural invasion, Rsurgical margin status (R0 negative; R1 positive), AF alive free of disease, AD alive with disease, DO dead of other
causes, DD death of disease, ITPN intraductal tubulopapillary neoplasm, n/a not available.
Patient n. 7a experienced a tumor relapse (7b); therefore, both cases (7a and 7b) are reported.
A. Mafcini et al.
Modern Pathology
enrichment for the phosphatase and tensin homolog (PTEN)
regulation pathways, whereas NOTCH signaling was enriched in
cluster C (Fig. 4B).
Using deconvolution analysis of the different clusters, statisti-
cally signicant differences in immune cell populations were
identied. Cluster A showed enrichment in CD8+T-cells, M1-class
macrophages, and cancer-associated broblasts (CAFs). Cluster B
was enriched in CD4+T-cells and M2-class macrophages, while
cluster C was enriched in inammatory cells implicated in the
innate immune response (Fig. 4C).
Chromogenic multiplex IHC for CD4, CD8, and CD163 conrmed
these ndings, showing predominant CD8+T-lymphocyte inltra-
tion in cluster A (mean scores: CD8 =3.8; CD4 =1.6; CD163 =1.2),
predominance of class 2 macrophages and CD4+T-lymphocytes
in cluster B (mean scores: CD8 =1.2; CD4 =3.4; CD163 =4.2), and
low presence of cells positive for these markers in cluster C (mean
scores: CD8 =1.2; CD4 =1.2; macrophages =0.8).
Integrative multi-regional genomic and transcriptome analysis of
ITPN, adenocarcinoma, and liver metastasis. Integrative genomic
and transcriptome analyses were performed in the case of
metastatic ITPN; in particular, on the tubular and papillary
components of ITPN, concomitant pancreatic adenocarcinoma,
and liver metastasis. The genomic analysis showed the presence of
a truncating mutation in PBRM1 in the pancreatic adenocarcinoma.
In the transcriptome analysis, statistical analysis showed no DE
genes between the tubular and papillary components. By
comparison, up-regulation of 15 genes and down-regulation of 8
Fig. 1 Summarizing gure of histology-based genomic analysis. Histological images of the tumor areas selected for multi-regional
sequencing: (A) tubular component; (B) papillary component; (C) adenocarcinoma. Hematoxylin-eosin staining at 10× magnication for
observation of structures. The graphs show the results of the genomic analysis of all patients at diagnosis, represented per tumor component.
Each case is identied with a number followed by an acronym, indicating the specic tumor region (TUB tubular, PAP papillary, AC
A. Mafcini et al.
Modern Pathology
Table 2. Pathogenic/likely pathogenic mutations and gene copy-number variations identied in pancreatic ITPN and associated invasive carcinoma.
ID case Histology TMB MSI Clinically relevant SNV CNV
Gene Variation Mutation type Freq (%) Class Gene Variation # of copies
1 Tubular 5.6 MSS none FGFR1 Gain 3.0
RAD50 LOH 1.0
Papillary 5.6 MSS RNF43 c.571+1G>A Substitution splice site 13 4 FGFR1 Gain 3.4
RAD50 LOH 1.0
2 Tubular 3.9 MSS SMAD4 p.D415fs*20 Deletion frameshift 27 5 NOTCH1 Gain 3.6
CCNE1 Ampl 5.0
Papillary 2.8 MSS none FGFR4 Gain 3.5
CCNE1 Ampl 5.5
3 Tubular 11.7 MSS EP300 c.3591-1G>A Splice site variation 48 4 none
Papillary 13.9 MSS EP300 c.3591-1G>A Splice site variation 25 4 none
4 Tubular 10.0 MSS none NTRK1 Ampl 5.0
Papillary 10.0 MSS none NTRK1 Ampl 5.0
5 Tubular 7.8 MSS TP53 p.Y220C Substitution missense 48 5 TP53 LOH 1.0
NOTCH3 Ampl 5.0
CCNE1 Ampl 29.0
Papillary 7.2 MSS TP53 p.Y220C Substitution missense 24 5 TP53 LOH 1.0
NOTCH3 Gain 4.0
CCNE1 Ampl 29.0
6 Tubular 6.6 MSS none None
Papillary 6.1 MSS none None
Tubular 11.7 MSS BRAF p.V600E Substitution missense 35 5 NTRK1 Ampl 6.0
APC p.E1544* Substitution nonsense 5 5 STK11 LOH 1.0
STK11 p.Q159* Substitution nonsense 51 5
Papillary 11.7 MSS BRAF p.V600E Substitution missense 35 5 NTRK1 Ampl 8.0
APC p.E1544* Substitution nonsense 45 5 STK11 LOH 1.0
STK11 p.Q159* Substitution nonsense 47 5
AC 11.4 MSS BRAF p.V600E Substitution missense 21 5 NTRK1 Ampl 13.1
STK11 p.Q159* Substitution nonsense 20 5 STK11 LOH 0.9
8 Tubular 11.6 MSS none MDM2 Gain 4.0
AC 11.0 MSS none MDM2 Gain 4.0
9 Tubular 5.6 MSS KRAS p.G12S Substitution missense 11 5 STK11 LOH +gain 3.1
RUNX1 p.S318Ffs*282 Deletion frameshift 12 5
STK11 c.921-1G>C Substitution splice site 68 5
Papillary 6.1 MSS KRAS p.G12S Substitution missense 14 5 STK11 LOH +gain 3.0
RUNX1 p.S318Ffs*282 Deletion frameshift 3 5
STK11 c.921-1G>C Substitution splice site 57 5
AC 6.7 MSS KRAS p.G12S Substitution missense 15 5 STK11 LOH +gain 3.0
A. Mafcini et al.
Modern Pathology
Table 2. continued
ID case Histology TMB MSI Clinically relevant SNV CNV
Gene Variation Mutation type Freq (%) Class Gene Variation # of copies
STK11 c.921-1G>C Substitution splice site 66 5
ARID2 p.S1476Cfs*26 Deletion frameshift 3 4
10 Tubular 3.9 MSS POLE c.4149+2dupT Insertion splice site 40 4
Papillary 3.9 MSS POLE c.4149+2dupT Insertion splice site 42 4
AC 3.3 MSS POLE c.4149+2dupT Insertion splice site 44 4
11 Tubular 7.2 MSS KRAS p.G12D Substitution missense 34 5 FGFR3 Ampl 6.5
TP53 p.R248W Substitution missense 55 5 APC LOH 1.0
SMAD4 p.R445* Substitution nonsense 55 5 ERBB2 Ampl 10.3
STAT3 Ampl 8.2
AKT2 Ampl 14.6
Papillary 8.3 MSS KRAS p.G12D Substitution missense 35 5 FGFR3 Ampl 6.5
TP53 p.R248W Substitution missense 65 5 APC LOH 1.0
ERBB2 Ampl 9.6
STAT3 Ampl 7.3
AKT2 Ampl 9.9
AC 9.4 MSS KRAS p.G12D Substitution missense 41 5 FGFR3 Gain 4.0
TP53 p.R248W Substitution missense 47 5 APC LOH 1.0
ASXL1 p.Q1074* Substitution nonsense 5 4 ERBB2 Ampl 8.6
STAT3 Ampl 6.5
AKT2 Ampl 7.6
12 Tubular 12.7 MSS KRAS p.G12V Substitution missense 24 5 None
TP53 p.L194H Substitution missense 59 5
PALB2 p.E13K Substitution missense 22 4
RB1 c.138-1G>T Substitution splice site 70 4
Papillary 10.1 MSS KRAS p.G12V Substitution missense 11 5 NOTCH1 Gain 4.0
TP53 p.L194H Substitution missense 60 5
PALB2 p.E13K Substitution missense 21 4
RB1 c.138-1G>T Substitution splice site 45 4
AC 11.1 MSS KRAS p.G12V Substitution missense 20 5 None
TP53 p.L194H Substitution missense 35 5
PALB2 p.E13K Substitution missense 28 4
RB1 c.138-1G>T Substitution splice site 52 4
13 Tubular 6.6 MSS KRAS p.G12D Substitution missense 34 5 CCND3 Ampl 9.0
TP53 p.Y220C Substitution missense 35 5 NOTCH4 Ampl 9.0
MDM2 Ampl 5.0
Papillary 6.6 MSS KRAS p.G12D Substitution missense 44 5 CCND3 Ampl 9.0
TP53 p.Y220C Substitution missense 42 5 NOTCH4 Ampl 9.0
A. Mafcini et al.
Modern Pathology
Table 2. continued
ID case Histology TMB MSI Clinically relevant SNV CNV
Gene Variation Mutation type Freq (%) Class Gene Variation # of copies
MDM2 Ampl 5.0
AC 5 MSS KRAS p.G12D Substitution missense 10 5 CCND3 Ampl 9.0
TP53 p.Y220C Substitution missense 10 5 NOTCH4 Ampl 9.0
MDM2 Ampl 5.0
14 Tubular 6.5 MSS BRCA2 p.Q2960* Stop gain 85 5 MAX Hom del 0.0
Papillary 8.1 MSS BRCA2 p.Q2960* Stop gain 93 5 MAX Hom del 0.0
AC 6.7 MSS BRCA2 p.Q2960* Stop gain 54 5 NA
15 Tubular 8.3 MSS BRAF p.V600E Substitution missense 32 5 STK11 LOH 1.0
JAK3 Gain 3.0
AC 7.8 MSS BRAF p.V600E Substitution missense 32 5 STK11 LOH 1.0
JAK3 Gain 3.0
16 Tubular 4.3 MSS none None
Papillary 4.3 MSS none None
AC 5.4 MSS PBRM1 p.D554fs*4 Deletion frameshift 13 4 None
Liver met. 4.3 MSS none None
TMB tumor mutational burden, MSI microsatellite instability, MSS microsatellite stable, SNV single nucleotide variants, CNV copy number variations, AC adenocarcinoma, N/A not available, LOH loss of
heterozygosity (1 copy), Gain >2 copies, Ampl amplication (>4 copies), met metastasis, Class clinical Impact class according to AMGP/AMP guidelines (5: pathogenic; 4: likely pathogenic; Richards et al. Genet
Med 2015).
7a pancreatic resection for ITPN; 7b: local relapse as adenocarcinoma.
Neoplastic cellularity was too low for CNV analysis.
A. Mafcini et al.
Modern Pathology
genes were detected in the adenocarcinoma (Supplementary
Table 3). The case with the liver metastasis had an even more
variable prole: 135 DE genes (113 up-regulated and 22 down-
regulated) between the metastatic and intraductal areas and 156
DE genes (103 up-regulated and 53 down-regulated) between the
primary and metastatic adenocarcinoma were detected (Supple-
mentary Table 3). On the basis of the highest and statistically
signicant values of correlation to the current PDAC signatures
(Supplementary Fig. 3), the tubular and papillary components were
very similar and showed a classical pancreatic prole (Collissons
. In contrast, the adenocarcinoma showed features of
the squamous prole, with positive enrichment for Collissons
quasi-mesenchymal subtype
. By comparison, the transcriptomic
prole of liver metastasis showed classical pancreatic features, with
positive correlation with Baileys immunogenic prole
Survival analysis
In the survival analysis, the only parameter that showed
statistically signicant association with prognostic outcomes was
the TP53 mutation, associated with an increased risk of death or
recurrence (hazard ratio =10.359, 95% condence interval
1.911117.776, p=0.039; Fig. 5). The statistical signicance of
the TP53 mutation was maintained in the cases with concomitant
adenocarcinoma (hazard ratio =9.569, 95% condence interval
1.861106.371, p=0.046).
In this study, we performed a comprehensive characterization of
pancreatic ITPN and concomitant invasive adenocarcinoma in 16
cases. Below, we summarize our major ndings. 1) Clinicopatho-
logic features: concomitant adenocarcinoma was present in 75% of
cases, represented by glandular/tubular adenocarcinomas; 2) ITPN
as a precursor of pancreatic cancer: at the molecular level, the co-
occurring adenocarcinoma was always associated with pancreatic
intraductal components, establishing ITPN as a denitive precursor
of pancreatic cancer; 3) tumor progression: mutations of chromatin
remodeling genes represented a late event during ITPN oncogen-
esis. Indeed, mutations affecting such genes have been detected
only in the invasive component of three different cases; 4) clinical
genetics: ITPN can arise in the context of genetic syndromes, such
as HBOC and PeutzJeghers, with direct implications for screening,
therapy and genetic counseling; 5) mutational prole: mutations in
the classical PDAC drivers are less frequent in pancreatic ITPN; 6)
copy number variation: recurrent amplications were observed for
the Cyclin (3/16 cases, 18.75%) and NOTCH family genes (2/16
cases, 12.5%), whereas ERBB2, a potential target for molecular-
based therapies, was amplied in one case; 7) chromosomal
alterations: the most commonly observed were 1q gain (75% of
cases) and 1p, 6q or 18q loss (approximately 50% of cases); 8)
structural variations: common fusions involved the recently
identied RET and FGFR2; 9) transcriptome analysis of ITPN-
associated adenocarcinoma: three different clusters were identi-
ed, with the majority of cases displaying squamous-like features,
differential activation of EMT, KRAS-signaling, and PTEN pathways,
and variable immune microenvironment composition; and 10)
survival analysis: the TP53 mutational status emerged as a hallmark
of adverse prognosis.
At the molecular level, a critical nding emerged from the
comparative analysis between intraductal components and
concomitant adenocarcinoma: invasive cancers were present in
75% of cases and were always molecularly associated with
intraductal components. Indeed, in our case series, all ITPN and
co-occurring adenocarcinomas shared most of the genomic
alterations. These data provide denitive evidence of ITPN as
origin of invasive pancreatic adenocarcinoma. By contrast, a
previous study found that co-occurring IPMN and adenocarcino-
mas were independent (i.e., not molecularly associated) in
approximately 20% of cases
. Interestingly, we found that
acquisition of the invasive phenotype in ITPN was always
accompanied by alterations in the inltrative lesion.
Mutations in the chromatin remodeling ARID2,ASXL1 or PBRM1
were observed only in the invasive component of three different
cases. Alterations in the same class of genes have also been
reported in the biliary counterpart of these neoplasms
present and previous ndings suggest a potential role of this gene
class in tumor progression and invasion. Alterations in chromatin
remodeling genes have also been reported in the most compre-
hensive report published to-date on the molecular landscape of
pancreatic ITPN
. Chromosome 1p loss and 1q gain in the majority
of cases are additional common ndings. However, some
differences between the two studies are evident. First, Basturk
Table 3. Structural alterations of pancreatic ITPN and associated invasive carcinoma.
ID case Histology Involved genes Type of alteration
Gene 1 (region) Gene 2 (region)
1 Tubular FGFR2 (exon 17) HSD17B4 (exon 13) Fusion
Papillary FGFR2 (exon 17) HSD17B4 (exon 13) Fusion
3 Tubular RET (exon 12) C14orf93 (exon 3) Fusion
Papillary ––
4 Tubular TRIM24 (exon 9) RET (exon 12) Fusion
Papillary TRIM24 (exon 9) RET (exon 12) Fusion
5 Tubular LMNA RB1 Translocation
Papillary LMNA RB1 Translocation
8 Tubular FGFR2 (exon 17) SYCP1 (exon 24) Fusion
AC FGFR2 (exon 17) SYCP1 (exon 24) Fusion
11 Tubular ––
Papillary ––
AC ERBB2 (exon 24) P3H4 (exon 2) Fusion
ITPN intraductal tubulopapillary neoplasm.
This is a translocation by asymmetric breakdown and repair of chromosomes 1 (1q22, LMNA gene) and 13 (13q14.2, RB1 gene), resulting in a dicentric and an
acentric chromosome that contains the distal parts of the q arms, welded together. In particular, regarding LMNA and RB1, there is the 3-3 and 5-5junction of
the truncates, resulting in the loss of genes.
A. Mafcini et al.
Modern Pathology
et al.
found chromatin remodeling genes with mutations or
amplications in a substantial subset of their cases (3040% of
cases); alterations were relatively rare in our study (approximately
20%). Second, in the earlier study, alterations were commonly
detected in MLL; no such alterations were observed in our material.
Nonetheless, the picture that emerges from these studies conrms
that chromosomal alterations and mutations in chromatin remo-
deling genes are important components in the ITPN molecular
landscape, with a potential role in acquiring invasiveness.
This study is the rsttoreportthatpancreaticITPNcanariseaspart
of the spectrum of genetic syndromes, a nding conrmed by
molecular analysis. In our cases, neoplasms arose in the context of
HBOC syndrome due to BRCA2 alteration and in the context of
PeutzJeghers syndrome. Both neoplasms had inltrative compo-
nents. These ndings have immediate implications for tumor
screening and genetic counseling for patients with pancreatic ITPN
and may inuence clinical management (e.g., platinum-based
chemotherapy and PARP-inhibitors for BRCA-tumors)
. These results
emphasize the importance of a thorough anamnesis, including family
history of cancer, of all patients presenting with pancreatic ITPN.
The present study conrmed the genomic distinctiveness of
ITPN by showing that typical PDAC drivers, including KRAS,TP53,
SMAD4,andCDKN2A, are less frequently altered in this lesion in
comparison with conventional PDAC. Alterations in PDAC drivers,
at similar or lower frequency, have already been reported in
previous studies of pancreatic ITPNs
. The relative paucity of PDAC
alterations in this case series highlights the molecular differences
with conventional PDAC, but it should be acknowledged that KRAS
alterations are still present in a not-negligible subset of cases (4/16
cases in this series, 25%). This indicates that pancreatic ITPN cannot
be considered as a KRAS-independent entity, also taking into
account that MAPK-pathway can be activated in this tumor type
also through BRAF alterations (case #7).
Although the genomic landscape of pancreatic ITPN appears
largely heterogeneous, notable common events are represented
by gene amplication and fusion. Recurrent amplications were
found in genes of the Cyclin and NOTCH families. Amplication
in ERBB2 in these neoplasms represents a novel nding and a
potential target for precision oncology. It should be noted that
ERBB2 is considered one of the most important targets for
tailored treatments in breast and gastric cancer
ndings suggest new promising perspectives in treatment
strategies for pancreatic cancer. Gene fusions commonly
involved RET and FGFR2. Fusions involving FGFR2 have already
Fig. 2 Summarizing gure of the chromosomal alterations identied in all cases. In this gure, chromosomes are presented in increasing
order. Note: for chromosomal alterations, the adenocarcinoma of case #14 was not analyzed due to the low cellularity of the sample.
A. Mafcini et al.
Modern Pathology
been reported in two pancreatic ITPNs
but with different
partners from those reported here. Conversely, fusions involving
RET represent a novel ndinginpancreaticITPN,detectedhere
in two cases. Furthermore, we reported a novel ERBB2-P3H4
fusion and a newly established translocation involving LMNA
and RB1, resulting in gene loss. All these detected rearrange-
ments should be considered in molecular-based therapies,
already approved for other cancer types
. The new molecular
targets merit particular consideration as potential therapy
targets in patients with ITPN-associated pancreatic cancer,
especially in the metastatic setting.
Unsupervised clustering of DE genes in ITPN-associated
adenocarcinomas identied three different clusters; however, the
analysis at this stage should be considered exploratory due to the
small sample size. Cluster A showed activation of KRAS signaling
and EMT, and displayed squamous features, and enrichment in
CD8+T-cells, M1-class macrophages, and CAFs. Cluster B showed
positive correlation with PTEN regulation, similar features to the
PDAC squamous-like subgroup, and was enriched with CD4+
T-cells and M2-class macrophages. Cluster C showed activation of
NOTCH signaling and a transcriptomic prole toward classical-
pancreatic features. Although most cases displayed squamous
Fig. 3 Transcriptome analysis and matched genomic proles of ITPN-related adenocarcinomas. Upper panel: gene expression heatmap
stratied by the three consensus clusters (A, B, and C) derived from the transcriptome analysis of the cohort adenocarcinomas. Annotations
for clinicopathologic variables are also provided. Lower panel: genomic alterations for essential tumor-related genes found in all three clusters.
A. Mafcini et al.
Modern Pathology
features, the tumor microenvironment and biological processes
activated in the tumors showed substantial differences. These
aspects highlight the heterogeneity of tumor microenvironment
in pancreatic ITPN and should be considered in future studies to
indicate personalized therapeutic approaches
. Along these lines,
a recent study demonstrated that concurrent loss of Arid1a and
Pten in adult pancreatic ductal cells induced ITPN and ITPN-
derived PDAC in mice
. In our cohort, the majority of cases
studied by transcriptome analysis did show enrichment in the
activation of EMT and PTEN-regulation pathway. Moreover, an
ARID-gene mutation was detected in the invasive component in
one case. Overall, our study extend results from animal studies to
human disease and conrms the role of PTEN and ARID in
pancreatic ITPN and associated cancers.
Interestingly, the analysis of a primary ITPN coupled with
invasive and metastatic sites highlighted that the pancreatic
transcriptional program can be plastic across different tumor
stages. Despite genomic relatedness, the intraductal components
featured the classical pancreatic subtype, whereas squamous-like
characteristics were presented in the invasive adenocarcinoma
and classical-pancreatic features in distant metastasis. This nding
can be best appreciated in view of recent pioneering studies that
found evidence of subtype switching during tumor progres-
. Although the mechanism in PDAC is still not fully
understood, our initial analysis of a pancreatic ITPN case and the
associated primary and metastatic adenocarcinoma suggests that
subtype switching may be necessary for intraductal lesions to
acquire inltrating and further metastatic capability
Finally, a nding that merits attention is the role of TP53
mutational status in adverse prognosis; importantly, the TP53
mutational status was maintained in the multivariable analysis
that comprised cases with invasive adenocarcinoma. Association
of TP53 mutations with an adverse prognosis is commonly
encountered in different cancer types, including colorectal and
p = 0.02
p = 0.005 p = 0.019
p = 0.025
Fig. 4 Cluster-based representation of transcriptome analysis. A Heatmap showing similar statistically signicant transcriptomic proles
among the clusters identied in the current study with the existing molecular subgroups of pancreatic ductal adenocarcinoma (Mofts,
Collissons, and Baileys subgroups); BActivation of different biological mechanisms in the three clusters. Statistically signicant values are
shown. CHeatmap of the immune subpopulations inferred by gene expression of immune-related metagenes signicantly enriched in any of
the three clusters.
A. Mafcini et al.
Modern Pathology
ampullary adenocarcinomas in the gastrointestinal tract
. This
nding may help stratify patients with ITPN at diagnosis. The TP53
mutational status could, thus, be adopted as a potential
prognostic biomarker to identify high-risk lesions requiring
aggressive therapeutic and surgical strategies. As demonstrated
here, IHC is a valuable supportive tool for detecting TP53-mutated
cases; potential applications of IHC in detecting this biomarker
during routine diagnostic activity could be adopted.
It is important to acknowledge that this study has some
limitations. First, the genomic analysis did not investigate the
whole genome of the lesions; thus, potentially signicant
molecular events could have been missed. Nonetheless, the CORE
panel we adopted was based on previously reported whole-
genome sequencing focused on clinically relevant alterations.
Furthermore, although the results of the transcriptomic analysis
represent a novelty in the ITPN-context, they are based on eight
cases and should be considered as exploratory rather than
conclusive. We must also acknowledge that, despite the relatively
small sample size, the multicenter design of the current study is a
concrete answer to the difculties of collecting large case series of
rare neoplasms.
In conclusion, in this study we provided an integrative
clinicopathologic and molecular characterization of a series of
pancreatic ITPNs and associated adenocarcinomas. Our ndings
highlight that these lesions represent a distinct entity among
pancreatic neoplasms. In the context of pancreatic intraductal/cystic
lesions, correct identication of ITPNs is crucial given their distinctive
clinicopathologic features, genomic and transcriptomic proles, and
potential for target-enrichment strategies for precision oncology.
All data/information are available in the manuscript and in the Supplementary
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CL: study conception and design; TS, S-MH, LAB, LC, GMar, GMal, AP, RS, NH, CJ, AS,
CL: provided original material for the study; TS, S-MH, LAB, LC, GMar, GMal, AP, RS,
NH, PM, CJ, MM, AS, CL: clinical analysis; TS, S-MH, LAB, LC, NH, VA, AS, CL: histological
analysis; AM, MS, DA, CS, CC, RTL, AS, CL: molecular analysis; all authors: data
elaboration, discussion and interpretation; AM, MS, CL: paper writing; all authors: nal
editing and approval of the present version.
This study is supported by Associazione Italiana Ricerca sul Cancro (AIRC IG n. 26343);
Fondazione Cariverona: Oncology Biobank Project Antonio Schiavi(prot. 203885/
2017); Fondazione Italiana Malattie Pancreas (FIMP-Ministero Salute
J38D19000690001); Italian Ministry of Health (RF CO-2019-12369662: CUP:
The authors declare no competing interests.
This study has been approved by the Verona Ethics Committee, date of approval:
0408-2020, project 2610-CESC, code: MN-2019.
Supplementary information The online version contains supplementary material
available at
Correspondence and requests for materials should be addressed to Aldo Scarpa or
Claudio Luchini.
Reprints and permission information is available at
Publishers note Springer Nature remains neutral with regard to jurisdictional claims
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Modern Pathology
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Modern Pathology
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Aims: Intraductal tubulopapillary neoplasm (ITPN) of the pancreas is a recently recognized pancreatic tumor entity. Here, we aimed to determine the most important features with a systematic review coupled with an integrated statistical approach. Methods and results: PubMed, SCOPUS and Embase were searched for studies reporting data on pancreatic ITPN. The clinicopathological, immunohistochemical, and molecular data were summarized. Then, a comprehensive survival analysis and a comparative analysis of the molecular alterations of ITPN with those of pancreatic ductal adenocarcinoma (PDAC) and intraductal papillary mucinous neoplasm (IPMN) from reference cohorts (including the International Cancer Genome Consortium- ICGC dataset and The Cancer Genome Atlas- TCGA program) were conducted. The core findings of 128 patients were as follows - 1) Clinicopathological parameters: pancreatic head is the most common site; presence of an associated adenocarcinoma was reported in 60% of cases, but with rare nodal metastasis. 2) Immunohistochemistry: MUC1 (>90%) and MUC6 (70%) were the most frequently expressed mucins. ITPN lacked the intestinal marker MUC2; unlike IPMN, it did not express MUC5AC. 3) Molecular landscape: Compared with PDAC/IPMN, the classic pancreatic drivers KRAS, TP53, CDKN2A, SMAD4, GNAS, and RNF43 were less altered in ITPN (p<0.001), whereas MCL amplifications, FGFR2 fusions, and PI3KCA mutations were commonly altered (p<0.001). 4) Survival analysis: ITPN with a "pure" branch duct involvement showed the lowest risk of recurrence. Conclusions: ITPN is a distinct pancreatic neoplasm with specific clinicopathological and molecular characteristics. Its recognition is fundamental for its clinical/prognostic implications and for the enrichment of potential targets for precision oncology.
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Prognostically relevant RNA expression states exist in pancreatic ductal adenocarcinoma (PDAC), but our understanding of their drivers, stability, and relationship to therapeutic response is limited. To examine these attributes systematically, we profiled metastatic biopsies and matched organoid models at single-cell resolution. In vivo, we identify a new intermediate PDAC transcriptional cell state and uncover distinct site- and state-specific tumor microenvironments (TMEs). Benchmarking models against this reference map, we reveal strong culture-specific biases in cancer cell transcriptional state representation driven by altered TME signals. We restore expression state heterogeneity by adding back in vivo-relevant factors and show plasticity in culture models. Further, we prove that non-genetic modulation of cell state can strongly influence drug responses, uncovering state-specific vulnerabilities. This work provides a broadly applicable framework for aligning cell states across in vivo and ex vivo settings, identifying drivers of transcriptional plasticity and manipulating cell state to target associated vulnerabilities.
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Infidelity to cell fate occurs when differentiated cells lose their original identity and either revert to a more multipotent state or transdifferentiate into a different cell type, either within the same embryonic lineage or in an entirely different one. Whilst in certain circumstances, such as in wound repair, this process is beneficial, it can be hijacked by cancer cells to drive disease initiation and progression. Cell phenotype switching has been shown to also serve as a mechanism of drug resistance in some epithelial cancers. In pancreatic ductal adenocarcinoma (PDAC), the role of lineage infidelity and phenotype switching is still unclear. Two consensus molecular subtypes of PDAC have been proposed that mainly reflect the existence of cell lineages with different degrees of fidelity to pancreatic endodermal precursors. Indeed, the classical subtype of PDAC is characterised by the expression of endodermal lineage specifying transcription factors, while the more aggressive basal-like/squamous subtype is defined by epigenetic downregulation of endodermal genes and alterations in chromatin modifiers. Here, we summarise the current knowledge of mechanisms (genetic and epigenetic) of cell fate switching in PDAC and discuss how pancreatic organoids might help increase our understanding of both cell-intrinsic and cell-extrinsic factors governing lineage infidelity during the distinct phases of PDAC evolution.
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The molecular carcinogenesis of intraductal tubulopapillary neoplasms (ITPN), recently described as rare neoplasms in the pancreato-biliary tract with a favorable prognosis despite a high incidence of associated pancreato-biliary adenocarcinoma, is still poorly understood. To identify driver genes, chromosomal gains and losses, mutational signatures, key signaling pathways, and potential therapeutic targets, the molecular profile of 11 biliary and 6 pancreatic ITPNs, associated with invasive adenocarcinoma in 14/17 cases, are studied by whole exome sequencing (WES). The WES of 17 ITPNs reveals common copy number variants (CNVs) broadly distributed across the genome, with recurrent chromosomal deletions primarily in 1p36 and 9p21 affecting the tumor suppressors CHD5 and CDKN2A, respectively, and gains in 1q affecting the prominent oncogene AKT3. The identified somatic nucleotide variants (SNVs) involve few core signaling pathways despite high genetic heterogeneity with diverse mutational spectra: Chromatin remodeling, the cell cycle, and DNA damage/repair. An OncoKB search identifies putative actionable genomic targets in 35% of the cases (6/17), including recurrent missense mutations of the FGFR2 gene in biliary ITPNs (2/11, 18%). Our results show that somatic SNV in classical cancer genes, typically associated with pancreato-biliary carcinogenesis, were absent (KRAS, IDH1/2, GNAS, and others) to rare (TP53 and SMAD4, 6%, respectively) in ITPNs. Mutational signature pattern analysis reveals a predominance of an age-related pattern. Our findings highlight that biliary ITPN and classical cholangiocarcinoma display commonalities, in particular mutations in genes of the chromatin remodeling pathway, and appear, therefore, more closely related than pancreatic ITPN and classical pancreatic ductal adenocarcinoma.
Kground and Aims Pancreatic ductal adenocarcinoma (PDAC) arises from several types of premalignant lesions, including intraductal tubulopapillary neoplasm (ITPN); however, the molecular pathogenesis of ITPN remains unknown. Methods We performed studies with Hnf1b-CreERT2; Ptenf/f; Arid1af/f mice to investigate the consequence of genetic deletion of Arid1a in adult pancreatic ductal cells in the context of oncogenic PI3K/Akt pathway activation. Results Simultaneous deletion of Arid1a and Pten in pancreatic ductal cells resulted in the development of ITPN, which progressed to PDAC, in mice. Simultaneous loss of Arid1a and Pten induced dedifferentiation of pancreatic ductal cells and YAP/TAZ pathway activation. Consistent with the mouse data, TAZ expression was found elevated in human ITPNs and ITPN-derived PDACs but not in human intraductal papillary mucinous neoplasms, indicating that activation of the TAZ pathway is a distinctive feature of ITPN. Furthermore, pharmacological inhibition of the YAP/TAZ pathway suppressed the dedifferentiation of pancreatic ductal cells and development of ITPN in Arid1a and Pten double knockout mice. Conclusion Concurrent loss of Arid1a and Pten in adult pancreatic ductal cells induced ITPN and ITPN-derived PDAC in mice through aberrant activation of YAP/TAZ pathway, and inhibition of YAP/TAZ pathway prevented the development of ITPN. These findings provide novel insights into the pathogenesis of ITPN-derived PDAC and highlight the YAP/TAZ pathway as a potential therapeutic target.
Background Extrahepatic cholangiocarcinoma (ECC) is classified into two subtypes based on anatomic origin: distal extrahepatic (DECC) and perihilar (PHCC) cholangiocarcinoma. This study aimed to shed light on its genomic and transcriptomic profiles. Research design and methods The genomic alterations of 99 ECC (47 PHCC and 52 DECC) were investigated by next-generation sequencing of 96 genes. A subgroup of cases, representative of each subtype, was further investigated using transcriptomic analysis. Bioinformatics tools were applied for clustering and pathway analysis and defining the immune composition of the tumor microenvironment. Results PHCC had more frequent KRAS mutations (p=0.0047), whereas TP53 mutations were more common in DECC (p=0.006). Potentially actionable alterations included high-tumor mutational burden and/or microsatellite instability (7.1%), PI3KCA mutations (8.1%), and MYC (10.1%) and ERBB2 amplification (5.1%). The transcriptomic profiles showed the presence of three distinct clusters, which followed the anatomic origin and differed in the type of intra-tumor immune cells. DECC appeared to contain two distinct tumor subgroups, one enriched for druggable alterations and one lacking actionable opportunities. Conclusions This study provides new insights into the molecular landscape and the actionable alterations of ECC. Our findings represent a step toward improved ECC molecular taxonomy and future therapeutic strategies for precision oncology.
PURPOSE To examine the association of gain-of-function (GOF) and non–gain-of-function (non-GOF) TP53 mutations with prognosis of metastatic right-sided (RCC) versus left-sided colorectal cancer (LCC). METHODS This cohort study included patients with metastatic colorectal cancer (CRC) who had next-generation sequencing performed from November 2017 to January 2021. We defined R175H, R248W, R248Q, R249S, R273H, R273L, and R282W as GOF and all other mutp53 as non-GOF. We used Cox regression modeling to examine the association between GOF and non-GOF mutp53 and overall survival (OS), adjusting for age, sex, ethnicity, performance status, Charlson comorbidity index and receipt of chemotherapy. RESULTS Of total 1,043 patients, 735 had tumors with mutp53 and 308 had wild-type p53 (wtp53). GOF was associated with worse OS than non-GOF mutp53 only in LCC (hazard ratio [HR] = 1.66 [95% CI, 1.20 to 2.29]), but not in RCC (HR = 0.79 [95% CI, 0.49 to 1.26]). Importantly, RCC was associated with worse OS than LCC only in the subset of patients whose CRC carried non-GOF (HR = 1.76 [95% CI, 1.30 to 2.39]), but not GOF mutp53 (HR = 0.92 [95% CI, 0.55 to 1.53]) or wtp53 (HR = 0.88 [95% CI, 0.60 to 1.28]). These associations were largely unchanged after also adjusting for RAS, BRAF, and PIK3CA mutations, and microsatellite instability-high. CONCLUSION Poorer survival of patients with metastatic RCC versus LCC appeared to be restricted to the subset with non-GOF mutp53, whereas GOF versus non-GOF mutp53 was associated with poorer survival only among patients with LCC. This approach of collectively classifying mutp53 into GOF and non-GOF provides new insight for prognostic stratification and for understanding the mechanism of sidedness-dependent prognosis. If confirmed, future CRC clinical trials may benefit from incorporating this approach.
Hepatoid tumors (HT) are rare neoplasms, morphologically resembling hepatocellular carcinoma, which arise in several organs other than the liver. A comprehensive molecular profile of this group of neoplasms is still lacking. Genomic characterization of 19 HT from different organs (3 colon, 4 esophagogastric, 4 biliary, 6 genitourinary, 2 lung) was performed using a multigene next-generation sequencing panel. NGS unraveled a composite molecular profile of HT. Their genetic alterations were clearly clustered by tumor site: i) colorectal HT displayed microsatellite instability, high tumor mutational burden, mutations in ARID1A/B genes and NCOA4-RET gene fusion (2/3 cases); ii) gastric HT had TP53 mutations (2/4); iii) biliary HT displayed loss of CDKN2A (3/4) and loss of chromosome 18 (2/4); iv) genital HT showed gain of chromosome 12 (3/6); v) lung HT had STK11 somatic mutations (2/2). The only commonly mutated gene occurring in HT of different sites was TP53 (8/19 cases: 2 colon, 2 esophagogastric, 2 biliary, 1 genital, 1 lung). This study shows that most genetic alterations of HT were clustered by site, indicating that context matters. The novel potential targets for HT precision oncology are also clustered based on the anatomic origin. This study shed light into the biology of these rare cancers, and may have important consequences for treatment decision and clinical trial selection for HT patients.
HER2 is one of the most important proteins of the epidermal growth factor receptor (EGFR) family, whose alterations include amplification, overexpression and gene mutation. Growing attention has been given to HER2 as a biomarker for prognosis, an indicator for treatment response and a target for new drugs. Tumors with HER2 alterations have been well studied in multiple locations as distinct entities for treatment, especially breast cancer, gastric cancer, lung cancer and colorectal cancer. These four cancers are the leading causes of cancer incidence and cancer-related death worldwide. The present study details the landscape of HER2 amplification/overexpression and mutations and gives an up-to-date analysis of current clinical trials in the four cancers mentioned above. Different HER2-altered cancers not only respond differently to HER2-targeting therapies but also display diverse survival outcomes. Even in the same type of cancer, HER2 amplification/overexpression differs from HER2 mutation in terms of clinicopathologic features and treatment strategies. As an emerging strategy in cancer treatment, immune checkpoint inhibitors demonstrate distinct outcomes in HER2-altered breast cancer, gastric cancer and lung cancer.
The FDA granted accelerated approval for pralsetinib on September 4 for NSCLC and December 1, 2020 for thyroid cancer, for: 1) adult patients with metastatic RET fusion-positive non-small cell lung cancer (NSCLC), 2) adult and pediatric patients {greater than or equal to}12 years of age with advanced or metastatic RET-mutant medullary thyroid cancer (MTC) who require systemic therapy, and 3) adult and pediatric patients {greater than or equal to}12 years of age with advanced or metastatic RET fusion-positive thyroid cancer who require systemic therapy and who are radioactive iodine-refractory (if radioactive iodine is appropriate). Approval was based on the results of a multicenter, open-label, multi-cohort clinical trial (ARROW, NCT03037385), demonstrating substantial overall response rates (ORR) and durable responses in patients with RET-altered tumors. ORRs within the approved patient populations ranged from 57% (95% CI: 46, 68) in patients with RET fusion-positive NSCLC previously treated with platinum chemotherapy to 89% (95% CI: 52, 100) in patients with RET fusion-positive thyroid cancer, with response duration of at least 6 months in most responders. The product label includes warnings and precautions for pneumonitis, hypertension, hepatotoxicity, hemorrhagic events, tumor lysis syndrome, risk of impaired wound healing, and embryo-fetal toxicity. This article summarizes the major considerations during the FDA review leading to the approval of pralsetinib.