Pathology Research International N = 165 Correct deﬁnitive Subtyping (n = 148) Diﬃculty in Subtyping (n = 17)(2) Table 1: Factors contributing to diﬃculty of cytologic subtyping of adenocarcinoma and squamous cell carcinoma. Cellularity Diﬀerentiation(1) Histologic type Low 82% 18% High 94% 6% Well-moderately 87% 13% Poorly 69% 31% ADC 93% 7% SQC 70% 30% 3 (1) Grade of diﬀerentiation was based on resected specimens. (2)Diﬃculty in subtyping encompasses cases which were incorrectly classiﬁed, unclassiﬁed, or underclassiﬁed/subtype favored by cytology. diagnostic phase while the lesion can still be readily sam- pled. 4. Accurate Morphological Diagnosis of Subtypes of NSCLC Morphology still remains the cornerstone in lung cancer classiﬁcation. The World Health Organization classiﬁcation of lung tumors through the 1999 edition did not address lung cancer diagnosis based on small biopsies and cytology [12]. In the 2004 World Health Organization classiﬁcation, cytology was addressed for the ﬁrst time, with descriptions of the morphological criteria for each type of pulmonary carcinoma [13]. In the new revised proposal [14], an entire section is dedicated to the classiﬁcation of lung tumors based on small biopsy material including FNAB. This highlights the importance and recognition of the role that FNAB plays in the diagnosis and management of pulmonary carcinomas. Lung cancer histological subtypes that are morpholog- ically recognizable on cytology specimens are ADC, SQC, and SCLC, as well as carcinoid tumors. Other types of lung carcinoma such as large cell carcinoma and other rare variant as fetal type and colloid adenocarcinoma may be suspected on the basis of pure morphology but usually require evaluation of the surgically resected specimen for the ﬁnal diagnosis. Historically, it has been important to accurately identify SCLC as the treatment is diﬀerent from NSCLC. Classical morphological features of SCLC such as nuclear molding, frequent mitoses, and absence of nucleoli are often distorted on a small biopsy specimen showing extensive crush artifact. In this setting, cytology has an edge over histology because of better preservation and fewer artifacts [15]. Cytology is highly accurate and a well-recognized method to distinguish SCLC from NSCLC. In a study of 259 consecutive lung FNAs by Delgado et al., SCLC was distinguished from NSCLC with accuracy of 96% [16]. Unlike the distinction of SCLC and NSCLC, feasibility of NSCLC subtyping in cytology has been controversial. However, based on a recent study from our institution, cytology provides several advantages over surgical specimens for the subtyping of NSCLC [17]. The key morphologic criteria for ADC versus SQC are glandular architecture versus keratinization, respectively. The Papanicolaou (Pap) stain has exquisite sensitivity for even minimal keratinization aiding in the distinction of SQC from ADC. The morphologic patterns which emerge in tumor smears provide a clue to a tumor subtype which may not be apparent in surgical Table 2: Sensitivity and speciﬁcity of cytologic tumor subtyping. n = 183 SQC versus non-SQC ADC versus non-ADC Sensitivity Speciﬁcity Accuracy 87% 98% 98% 79% 97% 93% specimens (Figure 1). In addition, due to immediate ﬁxation, cytology provides greater nuclear and cytoplasmic resolution than histology. While in the majority of cases a line of diﬀerentiation can be clearly identiﬁed by morphology, diﬃculties arise in a subset of cases. In a recent study, which included 165 cases with paired FNAB and resection diagnosis of ADC and SQC, we described some of the limiting factors for the interpreta- tion and accurate classiﬁcation in cytology specimens. The strongest predictors for diﬃculty in subtyping were poor diﬀerentiation of the tumor where distinguishing morpho- logic features are not apparent (Table 1), followed by scant cellularity. Nonkeratinizing poorly diﬀerentiated squamous cell carcinoma in particular is subject to misclassiﬁcation by FNAB [18]. Another diﬃculty is presented by tumors with mixed histology, but true adenosquamous carcinomas are infrequent with reported incidence of 2 to 3% in published surgical series [19]. Despite these limitations, using morphology and occa- sional immunocytochemistry we observed that when faced with the need to subclassify NSCLC we performed with high concordance between cytology and resected specimens of 97% and 93% for identifying squamous and adenocar- cinoma, respectively (Table 2). Despite the lower sensitivity for non-keratinizing SQC, the speciﬁcity of this diagnosis is very high, which means that the false-positive classiﬁcation as SQC is extremely rare. Thus having proved overall high accuracy of cytology in distinguishing SQC versus non-SQC it was concluded that cytological specimens are suitable for guiding therapeutic decisions within these diagnostic categories [18]. 5. Use of Immunohistochemistry Stains in the Characterization of NSCLC Subtypes In tumors that do not show clear-cut signs of diﬀerentiation on light microscopy examination (Figure 2) further investi- gation by immunohistochemistry may highlight tumor cell lineage. In biopsies like FNAB, with limited material, the need to conserve material for possible mutational analysis and other prognostic markers obligates the use of the most