Sabato 27 ottobre 2012 - Pacini Editore

Pathologica 2012;104:359-420 POSTEr
BIOLOGIA MOLECOLArE
ThinPrep cytology is a valuable and efficient
method in detecting egfr mutations in lung
adenocarcinomas
V. D’Alicandro, E. Melucci, B. Casini, V. Dimartino, C. Ercolani,
A. Di Benedetto, C.A. Amoreo, M. Filippetti, M. Milella, P. Visca,
E. Pescarmona, M. Mottolese
Regina Elena Cancer Institute, Rome, Italy
Introduction. Lung cancer is one of the leading causes of cancerrelated
deaths worldwide and adenocarcinoma is recently becoming
the most frequent histologic type among non-small-cell lung
cancers (NSCLC) in many countries. Epidermal growth factor
receptor (EGFR) is a transmembrane receptor forming dimers
on ligand binding which stimulates signals through receptor
autophosphorylation. The activation of these intracellular pathways
facilitates malignant transformation and tumor progression.
EGFR, expressed in more than 40% of NSCLC, is mutated in
about 12-13% of cases in Caucasian population, mainly in the
adenocarcinoma histotype. Metastasic NSCLC patients have a
relatively high probability of achieving an objective response to
EGFR tyrosine kinase inhibitors (TKIs) such as gefitinib or erlotinib,
only when EGFR gene presents activating mutations in the
tyrosine kinase domain, mainly in exons 19 and 21. In NSCLC the
clinical application of TKIs targeting the EGFR requires the analysis
of gene mutational status 1 . Although cytology is a very common
and useful approach to evaluate patient eligibility to TKIs,
the limited number of tumor cells present in the samples may
constitute a bias for molecular analysis. In the majority of cases,
DNA was extracted from Papanicolau smears or cell blocks 2 and
only in very few studies from ThinPrep processed samples 3 .
Aims. The aims of this study was to evaluate the reliability of
ThinPrep processed cytological specimens, obtained from primary
or metastatic lung adenocarcinoma, in detecting EGFR
gene mutations and to verify whether EGFR mutations rate in
ThinPrep processed cytological samples presented an accuracy
and sensitivity similar to bioptic samples.
Materials and methods. DNA was extracted from 66 Liquid
Based Cytology (LBC) cases including 33 CT-guided lung, 15
ultrasound-guided supraclavear or mediastinic lymph nodes and
5 visceral metastases fine needle aspirates (FNA), 7 bronchial
washings, 5 pleural effusions and 1 sputum and from histological
specimens including 32 bronchial and 28 pleural biopsies
retrospectively selected. Exons 19 and 21 was analyzed by direct
sequencing.
Results. The overall specimen insufficiency rate in cytological
and bioptical samples was similar (12.1% vs 11.7%). In our series
of 57 valuable LBC (55% of malignant cells in Papanicolau
smears) we found 7 (12.3%) EGFR mutations, 3 deletions in the
exon 19 and 4 in exon 21 (L858R). 7 (13.2%) out in 53 valuable
biopsies, 4 in the exon 21 and 3 in the exon 19. (Tab. I).
Tab. I. Comparison between EGfR mutations in biopsies and liquid Based
cytology samples.
SPECIMENS EGFR WT
EGFR
MUTATED
NON
VALUABLE
SPECIMENS
TOT.
BiOpSiES 46 7 (13.2%) 7 (11.7%) 60
cYtologY 51 7 (12.3%) 8 (12.1%) 66
Conclusions. These results, obtained using LBC, are highly
concordant with those obtained on bioptic material with an overlapping
number of non valuable cases. Our findings provided
evidence that ThinPrep processed cytological specimens are suitable
for DNA extraction and subsequent EGFR mutation analysis
by direct sequencing; nevertheless novel and more sensitive
techniques (i.e. qPCR) than direct sequencing may enhance LBC
potential in detecting EGFR mutations in NSCLC.
references
1 Pao W, Miller VA. Epidermal growth factor receptor mutations,
small-molecule kinase inhibitors, and non-small-cell lung cancer: current
knowledge and future directions. J Clin Oncol 2005;23:2556-68.
2 van Eijk R, Licht J, Schrumpf M, et al. Rapid KRAS, EGFR, BRAF
and PIK3CA Mutation Analysis of Fine Needle Aspirates from Non-
Small-Cell Lung Cancer Using Allele-Specific qPCR. PLoS One
2011;6:e17791.
3 Lozano MD. Assessment of Epidermal Growth Factor Receptor and
K-Ras Mutation Status in Cytological Stained Smears of Non-Small
Cell Lung Cancer Patients: Correlation with Clinical Outcomes. The
Oncologist 2011;16:877-85.
B-cell clonality and CD20 expression in classical
Hodgkin Lymphoma
L. Marra, M. Cerrone, G. Liguori, V. Gigantino, G. Aquino,
F. Tisci, V. Relli, L. La Sala, C. Santonastaso, A.R. De Chiara,
R. Franco, G. Botti
Pathology Department, National Cancer Institute “Fondazione G. Pascale”,
Naples, Italy
Introduction. Hodgkin Lymphoma is defined as a special kind of
B-cell lymphoma, which usually contains a small number of scattered
Hodgkin and Reed-Sternberg (HRS) cells in an abundant
background non neoplastic inflammatory and accessory cells.
B-cell lymphoma lack of Ig expression and the clonal B-cell
nature is difficult to be demonstrated, even if in non Hodgkin
Lymphoma, where it requires both immunohistochemical profiling
and monoclonality assessment.
Modern single-cell microdissection technique and molecular genetic
study demonstrate that HRS cell are derived from germinal
center B-cell. Some studies report that HRS cells are positive for
CD20 in 5-58% of CHL cases.
Recents studies revealed that CD20 expression is an independent
poor prognostic factor for the failure free survival (FFS) and
overall survival (OS) in cHL patients, but until now, the role of
CD20 expression is controversial.
Today the presence of clonal B-cell receptor rearrangements has
been considered more supportive of B-cell non-HL. However, the
evolution of a clonal B HRS cell population during the primary
local manifestation of HD and persistence and dissemination of
this clone could indicate that a more advanced B- cell clonality
suggest a more aggressive behavior.
In this study, we have correlated CD20 expression in HRS cell
with B- clonality to evaluate advanced abortive B cell differentiation
in CHL.
Material. We have analyzed 80 paraffin-embedded samples cHL
affects. CD20 expression was determined by immunohistochemistry.
The scoring criteria for the staining intensity of CD20 were
based on those described by Rassidakis and Tzankov. On the
sample with CD20 high expression of HRS cell was performed
clonality analysis using the BIOMED2 control gene primer set.
Results and conclusion. CHL is genotypically considered a Bcell
lymphoma, the classical B-cell marker CD20 is expressed only
in ~20-30% of such cases. In our study we have observed that
44% of samples with high score of CD20 expression, described
by Rassidakis and Tzankov, were monoclonal and all samples
with absence of CD20 expression have polyclonal pattern.
Χ 2 – Test shown association between score 3 sec Tzankov and
B-clonality.
Our results indicated that the presence routine PCR methods are
sensitive enough to detect small numbers of malignant cells in
cHL. We propose the use of clonality in association with immunochemistry
to identify cHL with B cell differentiation with
significant prognostic implication.