Sabato 27 ottobre 2012 - Pacini Editore

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and CD56 are the best immunostains for neuroendocrine
tumors. Of note, when singly considered all these antibodies
may show aberrant expression among various differentiations
(i.e., p63 may stain about 20-30% of adenocarcinomas). Said
that, combination of these markers may display tumor differentiation
in more than 90% of poorly differentiated NSCLC.
Overall, there is agreement in considering the coordinated
expression for TTF-1 and p63 as the most reasonable panel
in terms of sensitivity and specificity. Despite a limited but
consistent body of evidence, p40 (an antibody reacting only
with truncated dominant-negative isoforms - DeltaN-p63 of
p63) seems to have a higher specificity than conventional
transactivated p63 in distinguishing squamous cell carcinoma,
and p63-positive adenocarcinomas turned-out negative
when exploiting p40 (Pelosi et al. J Thorac Oncol 2012;
Bishop et al. Mod Pathol 2012). Of interest, on molecular
ground miRNA-205 expression parallels results obtained
by conventional morphology and/or immunohistochemistry.
Considering the high cost and laboratory equipment required
for miRNA-205 determinations, however, it is our opinion
that miRNA expression should not be considered a practical
substitute for more conventional characterization of NSCLC
by either immunohistochemistry or morphology. Whenever
there is any doubt about the histologic type, it is correct to ask
for immunostains. At the moment TTF-1 plus p63 is probably
the best panel, but it is important to follow some basic rules
to limit the need for further immunostains or misleading diagnoses,
then preserving tissue for molecular analyses. In any
case, histologic type plays a major role and it is also helpful
in guiding molecular analyses. Napsin A for adenocarcinoma
and p40 and desmocollin-3 for squamous cell carcinoma are
the most appropriate choices if further stains are necessary.
CK7 and 34betaE12 are less specific because they are shared
by squamous cell carcinoma and adenocarcinoma in up to 50-
60% of cases. While no specific immunostains are available
in determining the origin of a squamous cell carcinoma, in
case of a clear-cut adenocarcinoma on morphology immunostains
should be advised only if a differential diagnosis with
metastatic extrapulmonary adenocarcinoma is concerned. In
general, the more stains you demand, the more complicated
the case becomes, considering that some clear-cut pulmonary
adenocarcinomas express a complex phenotype with multiple
markers simultaneously present.
references
Travis WD, et al. J Thorac Oncol 2011;6:244-85.
Bishop JA, et al. Clin Cancer Res 2010;16:610-9.
Matoso A, et al. Appl Immunohistochem Mol Morphol 2010;18:142-9.
Mukhopadhyay S, Katzenstein ALA. Am J Surg Pathol 2011;35:15-25.
Lebanony D, et al. J Clin Oncol 2009;27:2030-7.
Del Vescovo V, et al. Am J Surg Pathol 2011;35:268-75.
Pelosi G, et al. J Thorac Oncol 2012;7:281-90.
Bishop JA, et al. Mod Pathol 2012;25:405-15.
Righi L, et al. Cancer 2011;117:3416-23.
Nizzoli R, et al. J Thorac Oncol 2011;6:489-93.
Reckthman N, et al. J Thorac Oncol 2011;6:451-8.
Reckthman N, et al. Clin Cancer Res 2012;18:1167-76.
CONGRESSO aNNualE di aNatOmia patOlOGiCa SiapEC – iap • fiRENzE, 25-27 OttOBRE 2012
Molecular diagnosis in cytological samples of
nsclc: appropriateness and reliability
G. Fontanini
Department of Surgery, University of Pisa
Molecular test are mandatory in non-small cell lung cancer
(NSCLC) to identify the patients most likely to benefit from
therapies with EGFR inhibitors. In the approximately 70% of
patients with NSCLC the only pathologic material guiding
systemic therapy may be small biopsy or cytology specimens;
the performance characteristics of cytology in NSCLC predictive
marker testing are not well established. In this study we
showed the accuracy of cytologic specimens submitted for
EGFR molecular testing.
Within most advanced laboratory it is now common to see
a fusion of cytological and molecular analysis such as DNA
sequencing with a significant impact on how diseases are diagnosed
and treated in clinical practice.
DNA sequencing technique offers a higher genetic resolution
showing small variations in their nucleotide content. The
clinical use of these variations is fully recognized in pharmacogenetics
where variations in genes predict whether a patient
will have a good response to a drug, a bad response to a drug,
or no response at all.
Molecular cytology is influenced by an increase in the technical
sophistication of next generation techniques impacting on the
overall sensitivity of genetic testing maximizing accuracy and
reproducibility and providing a wide range of testing options.
The key warning of molecular cytology regards the quality of
specimens that has an important impact on diagnostic results.
In fact, cytological samples are exposed to some criticisms as
low cellularity and cellular heterogeneity that may have an
effect on the downstream molecular results. Optimization and
standardization of cytological sample preparation methods is
necessary to preserve bio-molecular integrity and to ensure a
correct molecular result.
As a consequence, the major contribution to obtain adequate
material for molecular diagnostic is the correct and appropriate
sampling: stained smears from the aspirate are evaluated by a
cytopathologist for cellularity and diagnostic yield. Molecular
testing is feasible if: the number of neoplastic cells is higher than
100 (for instance the proportion of neoplastic cells versus non
neoplastic cells will determine which sequencing techniques will
be used and the type of cell dissection) and 2) the sample yielded
sufficient quantity and quality of DNA for mutational analysis.
Moreover each laboratory should take into account a further
internal validation, analyzing in parallel a large number of
cytological samples together with the matching histological
samples. A sequencing technique is suitable for molecular
cytology if the mutations detected in cytological specimens
are the same to that find in surgical specimens in each case.
In our experience the genotyping techniques with a higher
level of accuracy in molecular cytology are pyrosequencing
and real-time PCR based genotyping. In fact, both these techniques
have a sensitivity ranging from 1 to 5% and are able
to identify almost all gene mutational variants. Using these
techniques the overall percentage on neoplastic cells is not
a limit if a strictly selected cell microdissection (cell enrichment)
is performed. Definitely the major limitation for an
adequate molecular test on a cytological sample is the number
of available cells and as a consequence the low concentration
and overall quality of DNA; the use an extremely sensitive
techniques (for instance real-time PCR based genotyping)
could minimize but not completely avoid this issue.