Multiplex ligation-dependent probe amplification (MLPA) screening in meningioma.
- [Show abstract] [Hide abstract]
ABSTRACT: BACKGROUND: This study sought to identify genes in nontypical meningiomas with gains in copy number (CN) that correlate with earlier age of onset, an indicator of aggressiveness. METHODS: Among 94 adult patients, 91 had 105 meningiomas that were histologically confirmed. World Health Organization grades I (typical), II (atypical), and III (anaplastic) were assigned to tumors in 76, 14, and 1 patient, respectively. Brain invasion indicated that two World Health Organization grade I meningiomas were biologically atypical. DNA from 15 invasive/atypical/anaplastic meningiomas and commercial normal DNA were analyzed with multiplex ligation dependent probe amplification. The CN ratios (fold differences from normal) for 78 genes were determined. The CN ratio was defined as [tumor CN]/[normal CN] for each gene to normalize results. RESULTS: Characteristic gene losses (CN ratio < 0.75) occurred in >50% of the invasive/atypical/anaplastic meningiomas at 22q11, 1p34.2, and 1p22.1 loci. Gains (CN ratio ≥ 2.0) occurred in each tumor for 2 or more of 19 genes. Each of the 19 genes' CN ratio was ≥2.0 in multiple tumors, and their collective sums (up to 49.1) correlated inversely with age (r = -0.72), minus an outlier. In patients ≤55 versus >55 years, 5 genes (BIRC2, BRAF, MET, NRAS, and PIK3CA) individually exhibited significantly higher CN ratios (P < 0.05) or a trend for them (P < 0.09), with corrections for multiple comparisons, and their sums correlated inversely with age (r = -0.74). CONCLUSIONS: Low levels of amplification for selected oncogenes in invasive/atypical/anaplastic meningiomas were higher in younger adults, with the CN gains potentially underlying biological aggressiveness associated with early tumor development.World Neurosurgery 11/2011; · 1.77 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Recent gene expression and copy number profilings of glioblastoma multiforme (GBM) by The Cancer Genome Atlas (TCGA) Research Network suggest the existence of distinct subtypes of this tumor. However, these approaches might not be easily applicable in routine clinical practice. In the current study, we aimed to establish a proteomics-based subclassification of GBM by integrating their genomic and epigenomic profiles. We subclassified 79 newly diagnosed GBM based on expression patterns determined by comprehensive immunohistochemical observation in combination with their DNA copy number and DNA methylation patterns. The clinical relevance of our classification was independently validated in TCGA datasets. Consensus clustering identified the four distinct GBM subtypes: Oligodendrocyte Precursor (OPC) type, Differentiated Oligodendrocyte (DOC) type, Astrocytic Mesenchymal (AsMes) type and Mixed type. The OPC type was characterized by highly positive scores of Olig2, PDGFRA, p16, p53 and synaptophysin. In contrast, the AsMes type was strongly associated with strong expressions of nestin, CD44 and podoplanin, with a high glial fibrillary acidic protein score. The median overall survival of OPC-type patients was significantly longer than that of the AsMes-type patients (19.9 vs 12.8 months). This finding was in agreement with the Oncomine analysis of TCGA datasets, which revealed that PDGFRA and Olig2 were favorable prognostic factors and podoplanin and CD44 were associated with a poor clinical outcome. This is the first study to establish a subclassification of GBM on the basis of immunohistochemical analysis. Our study will shed light on personalized therapies that might be feasible in daily neuropathological practice. (Cancer Sci, doi: 10.1111/j.1349-7006.2012.02377.x, 2012).Cancer Science 07/2012; 103(10):1871-9. · 3.48 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: NF2 gene alterations may have a clinical impact in non-NF2 vestibular schwannomas (VSs). It has been suggested that NF2 mutations might correlate with clinical expression of VS in NF2 patients. The aim of this study was to analyze the impact of genetic alterations in the NF2 gene on epidemiologic, clinical, and radiologic features of patients with sporadic VS. The association between cigarette consumption and the molecular genetic findings was also studied. The study group consisted of 51 patients who underwent surgery for removal of vestibular schwannoma in our institution between January 2006 and December 2010. Five highly polymorphic microsatellite DNA markers were used to observe the frequency of loss of heterozygosity (LOH) in chromosome 22. The NF2 gene mutations were detected using polymerase chain reaction amplification and denaturing high-performance liquid chromatography analysis (PCR/dHPLC), and direct sequencing of NF2. Multiplex ligation-dependent probe amplification (MLPA) of the NF2 gene was also performed. An NF2 mutation was identified in 49%, 22q LOH in 57%, and MLPA alterations in 13.7% of the cases. One mutational hit was present in 27%, and 2 hits were present in 45% of the tumors. No association was found between the type of NF2 mutation and relevant clinical parameters.The presence of NF2 mutations detected by PCR/dHPLC was associated with no complaint of hearing loss at the time of diagnosis (p = 0.023), with subjective aural fullness (p = 0.022) and with an absence of tumor involvement of the internal auditory canal (p = 0.029). Patients with NF2 mutations had lower mean corrected PTA thresholds compared with those with no NF2 mutation (p = 0.037). Inactivation of the NF2 gene by mutation, MLPA, or LOH was more frequent in smokers when compared with never smokers (p = 0.048). NF2 mutations may play a role in the pathophysiology of hearing loss as well as in the pattern of growth of VS. Cigarette smoking in patients with VS seems to play a role in both the risk of developing the tumor and also in its genetic profile. More studies are needed to corroborate these results and, more broadly, to establish links between molecular and clinical data.Otology & neurotology: official publication of the American Otological Society, American Neurotology Society [and] European Academy of Otology and Neurotology 08/2013; · 1.44 Impact Factor
Letter to the editor
Multiplex ligation-dependent probe amplification (MLPA)
screening in meningioma
Meningiomas, which develop from arachnoid meningeal
cells, represent up to 20e25% of all intracranial tumors,
with an incidence rate of approximately 6 per 100,000 in-
dividuals . Cytogenetically, the main alteration is partial
or total loss of a chromosome 22, found in 70% of the cases
. In addition, the loss of 1p and 14q are cytogenetic
changes associated with the progression of this type of
Molecular studies have shown that approximately half of
these tumors show allelic losses that affect the q12 band in
chromosome 22. The NF2 gene, located in 22q12.2 has
been implicated as a candidate gene in the genesis of me-
ningiomas, acting as a tumor suppressor gene. Alterations
in the NF2 gene appear in familial form, causing neurofi-
bromatosis type 2, which is characterized by schwannomas,
primarily of the eighth cranial nerve, and meningiomas.
Mutations of this gene (most of which are small insertions,
deletions, or missense mutations resulting in a truncated
and nonfunctional protein) have been detected in 60% of
The close association in meningiomas with NF2 gene
mutations and the allelic losses in chromosome 22 suggest
that NF2 is a tumor suppressor gene located in that chromo-
some and involved in this meningioma development .
This gene may also be inactivated by CpG island aberrant
promoter methylation . Loss of NF2 gene function oc-
curs in only one third of meningiomas with loss of hetero-
zygosity of chromosome 22, which suggests the existence
of a second tumor suppressor gene in this region. Some
of the potential candidates are SMARCB1 (alias INI1),
AP1B1 (alias BAM22), LARGE, and MN1 genes [5,6].
Among genes located outside chromosome 22 that may
be implicated in meningioma tumorigenesis are EPB41L3
(alias DAL-1/4.1B) on chromosome 18, IGSF4 (previously
TSLC1) on chromosome 11, TP53 on chromosome 17,
CDKN2A (alias p14ARF) on chromosome 9, TERT (alias
hTERT) on chromosome 5, TGFB1 (alias TGF-b) on chro-
mosome 19, and others [5e7].
Conventional methods based on exon scanning do not
detect large deletions or mutations in noncoding regions,
due to wild-type allele coamplification, as has been de-
scribed for the NF2 gene [8e10]. The multiplex ligation-
dependent probe amplification (MLPA) technique [11,12]
has proven to be a high-resolution gene-dosage assay for
the screening of large deletions and duplications. Previous
studies have confirmed the efficiency of MLPA as a rapid,
reliable, economical, and high-throughput method [13,14].
In this type of assay, the material that is amplified is not the
DNA of the sample, but the probes, after a hybridizatione
ligation step in a multiplex polymerase chain reaction
(PCR) reaction in which specific sequences are simulta-
neously quantified; in consequence, the amplification
depends on the presence of the target sequences in the
MLPA has been used to study abnormalities of 22q 
and NF2 patients in whom intragenic NF2 mutations had
not previously been found by exon scanning . We ap-
plied the MLPA technique to detect deletions and duplica-
tions in a large series of familial and sporadic newly
diagnosed meningiomas. DNA was isolated from the pe-
ripheral blood of five healthy donors as controls and from
54 frozen meningioma tumor samples, using the Wizard ge-
nomic DNA purification kit (Promega, Madison, WI). Nine
of the tumor samples were from patients with a known fam-
ily history of NF2.
Pathological diagnosis was established according to the
WHO classification . The study included 47 WHO grade
I and 7 WHO grade II meningiomas, corresponding to 39
transitional, 9 meningotheliomatous, and 6 other histologic
subtypes of meningioma.
For NF2 analysis, we used a commercial MLPA kit
(SALSA P044 NF2; MRC-Holland, Amsterdam, Nether-
lands). The kit includes single probes for the 17 coding
exons and two probes for the promoter region of the NF2
gene. As a control, it includes 12 probes for different chro-
mosomal locations. Information regarding the probe
sequences and ligation sites can be found at http://
www.mlpa.com. The MLPA protocol was performed as
previously described , using 50 ng of DNA from con-
trol and tumor samples. DNA denaturation and hybridiza-
tion of the SALSA probes was followed by a ligation
reaction and PCR. One microliter of the amplified sample
product was analyzed with the ABI 3100 Avant sequencer
(Applied Biosystems, Foster City, CA), using as an internal
size standard the ROX-500 GeneScan (ABI 401734). Suc-
cessful ligation reaction and identification of samples with
insufficient amounts of DNA were verified using MLPA
internal ligation-independent probes.
Data analysis was performed with MRC-Coffalyser ver-
sion 2 software (MRC-Holland, Amsterdam, Netherlands).
0165-4608/07/$ e see front matter ? 2007 Elsevier Inc. All rights reserved.
Cancer Genetics and Cytogenetics 173 (2007) 170e172
Intranormalization for sample data was first performed on
control probes, and then each tumor sample was normalized
on control probes using data from five control samples. Sin-
gle regression for control and tumor data slope correction
was performed. Normal ratio limits were set at 0.75 and
1.3. Statistical analysis was accomplished using the same
Deletions of the whole NF2 gene were detected in 26 out
of the 54 samples (48.1%) and partial losses in 14 of 54
(25.9%), for a combined 74% showing loss for this gene.
As expected, total or partial losses were higher in sporadic
meningiomas than in the NF2 cases. No specific association
between these results and WHO grading was found. The
partial deletions were diverse, with loss from the promoter
region to exon 7 being the most frequent, found in 5
(11.1%) of the 54 cases, followed by loss from the pro-
moter region to exon 1, in 3 cases (6.7%), one of which
had in addition a deletion in exon 17.
It is in partial losses that the MLPA shows its potential,
given that conventional methods of screening (e.g., single-
strand conformation polymorphism, automated sequencing,
or loss of heterozygosity) cannot detect the defects, because
they are masked by the wild-type allele. This is also appli-
cable to duplications. Of special interest is one sample that
showed deletion for all the probes on the NF2 gene and its
promoter region. The calculated ratio for each of the probes
was between 0.48 and 0.72, except for the probes of exons
9, 10, and 11, for which the calculated ratio was between
0.15 and 0.23. These findings demonstrate the capacity of
the MLPA technique to detect various deletions in each
one of the alleles of a sample; in this particular case, the to-
tal deletion of the NF2 gene in one of the alleles and the
loss of exons 9 to 11 in the other (Fig. 1).
We also found a case with probe ratios compatible with
NF2 gene duplication from the promoter region to exon 12.
A summary of the partial losses and gains for the NF2 gene
is given in Table 1. The combined P-value for all the probes
was 0.05, with 0.08 being the higher individual P-value for
the probes on exons 14 and 17, and also for the control
probe 15q24.3. The 14q13 probe showed deletion in 39
(52.7%) of the 55 tumor samples (P Z 0.07). These results,
together with the previously described frequent loss of this
region in meningioma , made us exclude the probe as
a control; we then analyzed it in the same way as the probes
corresponding to NF2 exons.
We also found 12 duplications for the control probes in
11 tumor samples: 6 samples with duplication of the 4q35
probe, 1 sample with duplication on 5q31.1, 3 samples
(6.7%) with duplication on 9q21.3, and 1 sample with
two duplications for the 11p12 probe. These data do not
seem to indicate a specific association between these dupli-
cations and tumorigenesis or tumor progression in meningi-
oma. Nevertheless, we cannot discard the possibility that
further studies may find genes with oncogenic capacity in
some of these regions.
Fig. 1. MLPA electrophoresis peak-area patterns. (A) Tumor sample showing deletion of the whole NF2 gene and the 14q13 probe. (B) The sizes of the
peaks in exons 9, 10, and 11 show a partial deletion in one allele of a tumor sample that also shows a total deletion of NF2 gene in the other allele. (C)
Normal peak-area pattern in a control sample. Arrows indicate probes with proportional loss. X axis: NF2 gene and control probes ranging from 139 to
400 base pairs with a progressive increase of 9 base pairs. Y axis: Automatic sequencer fluorescent intensity units.
NF2 alterations detected with MLPA in 54 familial and sporadic
del Pr/ex 11
del ex 5/ex 6
del Pr/ex 7
del Pr/ex 1
del Pr/ex 1 and del ex 17
dup ex 1/ex 12
del ex 9/ex 11
del Pr/ex 8
del Pr/ex 13
Abbreviations: del, deletion; dup, duplication; ex, exon; MLPA, multi-
plex ligation-dependent probe amplification; Pr, promoter region.
Letter to the editor / Cancer Genetics and Cytogenetics 173 (2007) 170e172
In summary, our study found a high frequency (74%) of
large alterations (deletions and duplications) in the NF2
gene in sporadic and familial meningiomas using MLPA,
a technique that has proven to be an accurate and simple
method for detecting alterations that escape other screening
methods. In the specific case of tumor samples, in which
the cytogenetic and molecular alterations are usually abun-
dant, we have confirmed that the MLPA technique can dis-
criminate between various possible losses that can be
present simultaneously in both alleles of a gene.
Acknowledgments and dedication
Bello, PhD (June 18, 1957eMarch 4, 2006). The study was
supported bythe followinggrant sponsors:Fondo deInvesti-
gaciones Sanitarias, Ministerio de Sanidad (03/0235 and 05/
0829), and Fundacio ´n MAPFRE Medicina.
Vı ´ctor Martı ´nez-Glez
Carmen Franco-Herna ´ndez
Jesu ´s Lomas
Carolina Pen ˜a-Granero
Jose ´ M. de Campos
Juan A. Rey
Laboratorio de Oncogene ´tica Molecular
Unidad de Investigacio ´n
Hospital Universitario La Paz
Paseo Castellana 261
28046 Madrid, Spain
E-mail address: email@example.com (J.A. Rey)
 Louis DN, Scheithauer BW, Budka H, von Deimling A, Kepes JJ.
Meningiomas. In: Kleihues P, Cavenee WK, editors. Pathology and
genetics of tumors of the nervous system. 2nd ed. World Health Or-
ganization classification of tumours. Lyon: IARC Press, 2000. pp.
 Kleihues P, Louis DN, Scheithauer BW, Rorke LB, Reifenberger G,
Burger PC, Cavenee WK. The WHO classification of tumors of the
nervous system. J Neuropathol Exp Neurol 2002;61:215e25.
 Rey JA, Bello MJ, de Campos JM, Kusak E, Moreno S. Chromo-
somal involvement secondary to ?22 in human meningiomas. Cancer
Genet Cytogenet 1988;33:275e90.
 Lomas J, Bello MJ, Arjona D, Alonso ME, Martinez-Glez V, Lopez-
Marin I, Aminoso C, de Campos JM, Isla A, Vaquero J, Rey JA.
Genetic and epigenetic alteration of the NF2 gene in sporadic menin-
giomas. Genes Chromosomes Cancer 2005;42:314e9.
 Lamszus K. Meningioma pathology, genetics, and biology. J Neuro-
pathol Exp Neurol 2004;63:275e86.
 Lusis E, Gutmann DH. Meningioma: an update. Curr Opin Neurol
 Martinez-Glez V, Bello MJ, Franco-Hernandez C, de Campos JM,
Isla A, Vaquero J, Rey JA. Mutational analysis of the DAL-1/4.1B
tumour-suppressor gene locus in meningiomas. Int J Mol Med
 Bruder CE, Hirvela C, Tapia-Paez I, Fransson I, Segraves R,
Hamilton G, Zhang XX, Evans DG, Wallace AJ, Baser ME,
Rouleau GA, Poptodorov G, Jordanova A, Rask-Andersen H,
Kluwe L, Mautner V, Sainio M, Hung G, Mathiesen T, Moller C,
Pulst SM, Harder H, Heiberg A, Honda M, Niimura M, Sahlen S,
Blennow E, Albertson DG, Pinkel D, Dumanski JP. High resolution
deletion analysis of constitutional DNA from neurofibromatosis type
2 (NF2) patients using microarray-CGH. Hum Mol Genet 2001;10:
 Kluwe L, Nygren AO, Errami A, Heinrich B, Matthies C,
Tatagiba M, Mautner V. Screening for large mutations of the NF2
gene. Genes Chromosomes Cancer 2005;42:384e91.
 Zucman-Rossi J, Legoix P, Der Sarkissian H, Cheret G, Sor F,
Bernardi A, Cazes L, Giraud S, Ollagnon E, Lenoir G, Thomas G.
NF2 gene in neurofibromatosis type 2 patients. Hum Mol Genet
 Schouten JP, McElgunn CJ, Waaijer R, Zwijnenburg D, Diepvens F,
Pals G. Relative quantification of 40 nucleic acid sequences by mul-
tiplex ligation-dependent probe amplification. Nucleic Acids Res
 Sellner LN, Taylor GR. MLPA and MAPH: new techniques for detec-
tion of gene deletions. Hum Mutat 2004;23:413e9.
 Fernandez L, Lapunzina P, Arjona D, Lopez Pajares I, Garcia-
Guereta L, Elorza D, Burgueros M, De Torres ML, Mori MA,
Palomares M, Garcia-Alix A, Delicado A. Comparative study of
three diagnostic approaches (FISH, STRs and MLPA) in 30 patients
with 22q11.2 deletion syndrome. Clin Genet 2005;68:373e8.
 Vorstman JA, Jalali GR, Rappaport EF, Hacker AM, Scott C,
Emanuel BS. MLPA: a rapid, reliable, and sensitive method for de-
tection and analysis of abnormalities of 22q. Hum Mutat 2006;27:
 Leone PE, Bello MJ, de Campos JM, Vaquero J, Sarasa JL,
Pestana A, Rey JA. NF2 gene mutations and allelic status of 1p,
14q and 22q in sporadic meningiomas. Oncogene 1999;18:2231e9.
M, BoltshauserE,Papi L,
Letter to the editor / Cancer Genetics and Cytogenetics 173 (2007) 170e172