4 Figure 2: Poorly diﬀerentiated carcinoma lacking clear signs of diﬀerentiation on light microscopic examination. Further work up using limited panel of antibodies will diﬀerentiate tumor cell lineage in the majority of cases. eﬃcient and limited panel of immunohistochemical stains. Several recent publications have addressed the question of what is the best panel of markers to be used in the distinction between adenocarcinomas and squamous cell carcinoma. Results published by Wu et al. showed the advantages of immunocytochemistry in distinguish- ing poorly diﬀerentiated SQC from SCLC of the lung, and primary adenocarcinoma from metastatic tumors in cytology specimens. In their study, all poorly diﬀerentiated squamous cell carcinomas showed expression of p63 while negative for thyroid transcription factor-1 (TTF-1), whereas adenocarcinomas had the opposite staining pattern. Of note, SCLC had identical p63/TTF-1 expression proﬁle as adeno- carcinoma, but application of standard morphologic criteria and addition of neuroendocrine markers was suﬃcient for accurate classiﬁcation [20]. Nicholson et al., in their recently published work that included 13 FNA cytology specimens, showed that a limited panel of TTF-1, CK5/6, and p63 together with a mucin stain, reﬁned diagnosis of NSCLC to either ADC or SQC in 65% of cases [21]. In summary, most of the work has been concentrated on the expression of 3 markers TTF-1, p63, and a high- molecular-weight cytokeratin (HMWCK). The following algorithm, based on published data and our experience can guide the interpretation of the stains. A TTF-1 negative/p63- positive/HMWCK-positive proﬁle is supportive of SQC, whereas any expression of TTF-1 is supportive of ADC. It is worth mentioning that coexpression of TTF-1 and p63 can be seen in adenocarcinomas. In most cases p63 expression in adenocarcinoma is patchy and weak. Negative staining for both p63 and TTF-1 usually rules out squamous cell carcinoma [22]. Napsin-A is an aspartic proteinase, involved in the maturation of the surfactant protein B and is expressed in the cytoplasm of cells of lung and kidney [23]. The staining is cytoplasmic and is strongly positive in up to 80% of primary lung adenocarcinomas. In the study of Stoll et al., 75 cytology cases were analyzed. It showed that the sensitivity and speciﬁcity of TTF-1 were each 81%. Napsin-A exceeded the speciﬁcity of TTF-1 at 96% with a lower sensitivity of 65%. In the study, the only carcinoma Pathology Research International of nonlung origin in which Napsin-A was detected was renal cell carcinoma, suggesting that Napsin-A can be used as a surrogate marker in work up of poorly diﬀerentiated lung adenocarcinoma or an unknown primary tumor [24]. Desmocolin-3, constitutive protein of desmosomes, is found to be overexpressed in SQC of the lung [25, 26]. In the study by Monica et al., which included 31 cytological specimens originally classiﬁed as NSCLC, staining for desmocolin-3 and TTF-1 was mutually exclusive in tumors [26]. Several authors reported on the high sensitivity and speciﬁcity of miRNA expression in SQC of the lung, and its usefulness in diﬀerentiating ADC from SQC in small specimens [27, 28]. However, a recently published study does not support this observation [29]. The utility of these new approaches compared to standard markers needs to be evaluated further and validated, since a limited panel of immunohistochemical markers can reproduce the results obtained by the new molecular techniques [22]. 6. Adequacy of Material Obtained by FNAB for Molecular Testing In the recent years we have witnessed a revolution in our understanding of the molecular basis of NSCLC. These advances have led to development of multitude of com- mercially available prognostic and predictive biomarkers and targeted therapeutic agents. Despite these advances in treatment, the overall prognosis remains poor in patients with advanced disease. Personalizing therapy based on an individual tumor molecular proﬁle can optimize eﬃcacy with the available agents. Molecular determinants that guide treatment decision-making may have a prognostic or predictive function, and are commonly referred to as prognostic or predictive markers, respectively. Prognostic marker refers to a tumor characteristic that is useful for estimating a patient’s outcome independent of therapeutic decisions. In contrast, predictive markers are useful in making therapeutic decisions. Mutations of EGFR, KRAS, and EML4/ALK translocations are mutually exclusive in lung ADC and identify tumor subsets with unique dependencies and drug sensitivities. KRAS mutation testing is utilized by some institutions to exclude EGFR mutations or an EML4/ALK translocation. In particular EGFR mutation and EML4/ALK fusion gene testing have reached clinical validation and are incorporated into the current treatment paradigm [30]. EML4/ALK testing also appears to have clinical utility in identifying patients who could beneﬁt from referral for a study targeting ALK inhibition, such as ongoing phase 3 studies of the small molecule tyrosine kinase inhibitor crizotinib. There is increasing awareness that the quality of speci- mens, such as cytology, has a profound inﬂuence on molec- ular diagnostic test results. FNAB samples are exposed to a greater variety of cytopreparation methods than resected tis- sue and sample size and heterogeneity may have an eﬀect on the downstream molecular test results [31]. Most molecular techniques including in situ hybridization, polymerase chain reaction (PCR), and transcriptional proﬁling can be done on FNAB specimens [32, 33]. Optimizing and standardizing