Pathology Research International laboratories. In addition, a positive signal (especially with the most widely used split-signal assay) is often subtle and easily missed. Furthermore, a distinction between neoplastic and nonneoplastic cells can be diﬃcult in the absence of direct histological/immunohistochemical correlation on contiguous slides. Finally, the threshold of 15% is rather arbitrary; indeed, it is not followed in all studies [64]. Reverse transcriptase-polymerase chain reaction (RT- PCR) is another way to look for the fusion protein. Primers that generate an amplicon only when a fusion is present are used [56]. An additional step of sequencing can be added for further validation of the result. A limitation to this assay is that RNA is often degraded in FFPE tissue. A third option is to look for the fusion at the protein level, which is a goal of high clinical relevance since ALK inhibitors are targeting proteins rather than genes. IHC oﬀers in situ information about protein expression and allows correlation with tumor morphology. Besides, it is widely available because of its low cost and simplicity. A number of diﬀerent antibody clones for ALK detection have been described so far [68–70]. It appears that ALK1 clone lacks sensitivity due to cases with high background whereas D5F3 clone has a more preferable signal-to-background ratio [69]. Interestingly, Mino-Kenudson et al. propose an objective, quantitative, and automated assay for ALK detection [69]. Subjectivity in IHC interpretation and positivity threshold detection, as well as insuﬃcient antibody validation, are known limitations of IHC [71, 72]. Martelli et al. [68] reported the presence of EML4-ALK fusion transcripts detected with RT-PCR in normal lung tissue from NSCLC whose tumors were negative for such transcripts. In addition, they failed to detect any protein in IHC, probably because protein levels were too low to be detected. Interestingly, ALK1 along with other clones was used for the detection of the fusion proteins in IHC. Camidge et al. [73] have discovered ALK fusion genes by FISH in normal lung adjacent to ALK-positive or ALK-negative lung tumors. Taken together, these studies underscore the importance of further research in the biology of ALK protein in lung tumors; the possible presence of ALK fusion genes in normal lung might drive the spectrum of adverse events from treatment with crizotinib. 4. Assays for Histologic Classiﬁcation of NSCLC Histological classiﬁcation of NSCLC has been reviewed and changed several times in the past decades according to emerging knowledge on the disease. However, the need for speciﬁc diagnosis of adenocarcinoma or squamous cell carcinoma, the two major types of NSCLC has become clinically relevant only recently. Current NSCLC treatment optimization is histology speciﬁc. In this context, pemetrexed (thymidylate synthase inhibitor) and bevacizumab (anti-angiogenesis monoclonal antibody) are approved for the treatment of non-squamous cell lung carcinomas [74–76]. Histology classiﬁcation is traditionally based on morphological criteria. Morphology was able to classify correctly only 54% of preoperative 5 cytological and tissue samples [77]. Ou and Zell showed that the percentage of patients diagnosed with NSCLC and classiﬁed as “Not Otherwise Speciﬁed” (NOS) was much greater when a cytology specimen was used for diagnosis (39% versus 22.1% overall) in the California Cancer Registry [52]. NOS is a diagnosis that should be strictly limited to small biopsies and cytology specimens [78]. Histological misclassiﬁcation can happen as pathologists agreed with each other in 71.5% of the cases in a study [79]. The adenocarcinoma-speciﬁc agreement rate was 82.9% and the squamous-speciﬁc agreement rate was 91.2%. 4.1. Immunohistochemistry as an Aid for Histology Classiﬁca- tion. Several markers have been shown to be diﬀerentially expressed in adenocarcinomas and squamous cell carcino- mas of the lung. Among them, mucin and thyroid transcrip- tion factor 1 (TTF1) are adenocarcinoma-speciﬁc proteins, whereas p63 and cytokeratins 5/6 (CK 5/6) are squamous cell carcinoma speciﬁc. These markers were applied in a panel of biopsy samples which were classiﬁed as NOS by morphological criteria, and the assay was able to classify 73% of the cases into a histological type. Further comparison with matched surgical resections revealed accuracy of 86% [80]. Since none of these proteins is deﬁnitive when tested as a single marker, a combination of them creates patterns that favor speciﬁc histological types. Furthermore, a quantitative approach that is based on a weighted algorithm of the expression of ﬁve proteins is better at classifying NSCLC NOS into squamous cell carcinoma or adenocarcinoma than TTF1/TP63 staining [81]. Taken together, these studies underscore the value of IHC as a diagnostic that aids correct histological classiﬁcation and, therefore, the optimization of treatment in NSCLC. 4.2. Micro-RNA 205. For the subset of cases which remain unclassiﬁed after morphological criteria and IHC diagnostics have been applied, more sophisticated tests are emerging. Micro-RNA 205 is a small RNA molecule that is expressed in squamous cell carcinomas of the lung but not in lung adenocarcinomas. A diagnostic test based on the expression of micro-RNA 205, micro-RNA 21, and snRNA U6 can predict squamous cell carcinoma with a sensitivity of 96% and a speciﬁcity of 90% [82]. These ﬁndings were validated from a separate study where the micro-RNA-205-based diagnostic was able to predict histology perfectly in a set of poorly diﬀerentiated squamous cell carcinomas and adenocarcinomas [83]. In the same study, the diagnostic was able to predict correctly histology in 20 out of 21 prospectively collected biopsies with matched resections. A possible limitation of the assay is that it requires a tumor cellularity of at least 50% in the block. Indeed, micro-RNA 205 expression can be artiﬁcially altered by contamination from stromal or normal cells. It would be interesting to see whether assessment of micro-RNA 205 with an in situ assay like in situ hybridization (ISH) with micro-RNA- speciﬁc probes can overcome this limitation. It is unknown whether diﬃcult cases that need the aid of sophisticated and expensive tests to be classiﬁed correctly respond the same to pemetrexed as clear-cut cases. In this regard, it would