Genes and Pathology of Non-Small Cell Lung Carcinoma Shingo Sakashita,a Mai Sakashita,a and Ming Sound Tsaoa,b While histopathology has traditionally been the cornerstone of treatment decisions in the management of lung cancer patients, the complexity and heterogeneity of histological classification has had a limited impact in the routine practice of oncology. This has changed dramatically in the last few years, owing to discoveries of genomic aberrations and results of clinical trials of novel and targeted therapies. These discoveries have resulted in a new way of classifying non-small cell lung cancer (NSCLC), based on the occurrence of putative or proven driver and targetable genomic changes. The rapidity by which the landscape of mutation and genomic changes is being identified also has led to a new paradigm and approaches to pathological diagnosis of NSCLC. In this context, international consortia have proposed new classifications of lung adenocarcinoma and guidelines for molecular testing in lung cancer and have provided concrete recommendations on new ways to practice lung cancer pathology. Semin Oncol 41:28-39 & 2014 Elsevier Inc. All rights reserved.

D

uring the last decade, we have witness a paradigm shift in the treatment of non-small cell lung cancer (NSCLC). Unlike previous thinking that NSCLC respond similarly to all types of therapies regardless of histological types, the use and effectiveness of new generations of targeted therapies and chemotherapies are more defined by the histology and/or molecular features of the patient tumors (Table 1). This has led to the need for pathologists to make more precise histological subtyping of NSCLC. It is no longer sufficient or acceptable to use the terms “NSCLC not otherwise specified” (NOS) or “large cell carcinoma” on biopsies prior to performing immunohistochemical staining to define specific histological types of NSCLC. In this context, the multidisciplinary experts of the International Association for the Study of the Lung cancer (IASLC), the American Thoracic Society (ATS), a

Princess Margaret Cancer Centre, University Health Network, Ontario, Canada. b Department of Laboratory Medicine and Pathobiology, University of Toronto, Ontario, Canada. Conflicts of interest: none. Dr Sakashita is partially supported by the Terry Fox Foundation STIHR in Molecular Pathology of Cancer at CIHR grant TGT-53912 and Ontario Institute of Cancer Research. Dr Tsao is the M. Qasim Choksi Chair in Lung Cancer Translational Research at the Princess Margaret Cancer Centre and University of Toronto. This work is also supported in part by the Ontario Ministry of Health and Long Term Care. Address correspondence to Ming Sound Tsao, MD, FRCPC, 610 University Ave, Toronto, Ontario M5G 2M9, Canada. E-mail: ming. [email protected] 0093-7754/ - see front matter & 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1053/j.seminoncol.2013.12.008

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and the European Respiratory Society (ERS) published a report that proposed not only a new lung adenocarcinoma classification system but also a series of recommendations on good practices for making diagnoses based on small biopsies and/or cytology specimens, including approaches to ensure sufficient materials are available for testing of molecular predictive markers for targeted therapies.1 More recently, the College of American Pathologists (CAP), IASLC, and the Association of Molecular Pathologists (AMP) published a guideline on molecular testing in lung cancer. 2–4 Here, we highlight the issues in NSCLC classification, the changes and recommendations introduced in the new classification for adenocarcinoma, the relevance of molecular classification, and the challenges that remain to be addressed in future studies.

HISTOLOGIC CLASSIFICATION OF NSCLC In the 2004 World Health Organization (WHO) classification of lung cancer,5 malignant epithelial lung cancers are divided into eight major subtypes: squamous cell carcinoma, small cell carcinoma, adenocarcinoma, large cell carcinoma, adenosquamous carcinoma, sarcomatoid carcinoma, carcinoid tumor, and salivary gland tumors. However, for treatment purposes, most tumors are divided into two major subtypes: small cell lung carcinoma (SCLC) and NSCLC, as prognosis and treatment approaches to a majority of NSCLCs (except neuroendocrine tumors) are similar, and histologic subtypes within NSCLC do not significantly affect treatment decisions. However, clinical trials involving new therapies in NSCLC have introduced histology as an important factor that may influence treatment efficacy or adverse effects. Seminars in Oncology, Vol 41, No 1, February 2014, pp 28-39

Genes and pathology of non-small cell lung carcinoma

Table 1. Current Therapies for Advanced Non-Small Cell Lung Cancer Patients Agent

Molecular Selection

Histology Selection

Bevacizumab None Pemetrexed

Non-squamous

Gefitinib Erlotinib Afatinib

EGFR kinase domain mutation (first-line)

Testing in nonsquamous

Crizotinib

ALK gene Testing in nonrearrangesquamous ment

Bevacizumab is contraindicated in patients with lung squamous cell carcinoma due to an increased risk of fatal hemoptysis.6 Pemetrexed is approved for the treatment of non-squamous NSCLC only, as studies have shown the lack of efficacy in squamous cell carcinoma.7 In addition, known molecular markers of targeted therapies occur preferentially in an adenocarcinoma or mixed carcinoma with adenocarcinoma component. Thus, histology may be used to streamline molecular testing. Based on these considerations, more precise tumor subtyping assumes

29

greater importance in the more selective treatment of NSCLC patients. Histological diagnosis is based on morphological appearances of tumor cells on hematoxylin and eosin (H&E)-stained sections (Figure 1), at times supplemented by certain immunohistochemical markers. The morphological features of adenocarcinoma differentiation are gland structure formation and/or mucin production. In contrast, the morphologic features of squamous differentiation are intercellular bridges, single cell keratinization, and keratin or pearl formation. However, in poorly differentiated tumors and especially small biopsies or cytology specimens, these features may not be readily apparent. This, and the realization that NSCLC subtyping has not been previously considered as crucial for treatment decisionmaking, could have contributed to the increased use in clinical practice of the terms NSCLC-NOS or large cell carcinoma, as revealed in several cancer registry data.8,9 An important consideration is that the WHO classifications traditionally were developed to classify resected tumors, and their application to biopsy and cytology samples has always been implied but recognized as highly accurate.10 However, multi-observer studies have demonstrated good concordance among expert pathologists but poorer agreement among community pathologists.11,12 These data suggest the need for more specialized practice of pulmonary pathology or additional reliable markers for more accurate subclassification of NSCLC.

Figure 1. Histology of adenocarcinoma and squamous cell carcinoma. Adenocarcinoma is characterized by formation of glandular structures (A) by tumor cells with or without mucin secretion (B). Squamous cell carcinoma is characterized by presence of keratin pearl formation (C) and/or intercellular bridge (D). [A: 100x, B: 400x, C: 200x, D: 400x original magnification, respectively].

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S. Sakashita, M. Sakashita, and M. Sound Tsao

PROPOSED IASLC/ATS/ERS NEW CLASSIFICATION FOR ADENOCARCINOMA The rationale for proposing the new IASLC/ATS/ ERS classification system of lung adenocarcinoma involved multiple issues in the 2004 WHO classification.5 Although it is recognized that lung adenocarcinomas commonly contain a heterogeneous mixture of histological growth patterns, the fact that 480% of adenocarcinomas are classified as “mixed type” ignored potentially valuable information that could be associated with the different histological patterns. As a case in point, it has been reported that some early adenocarcinomas with mainly bronchioloalveolar growth pattern behave like non-invasive carcinomas with 100% survival when completely resected.13,14 Because the cells in these tumors grow lepidicly along pre-existing alveolar septae without clear evidence of stromal invasion, it was implied that they represent a preinvasive (in situ) stage of some adenocarcinomas. Furthermore, tumors with a focal invasive area that is limited to r5 mm in greatest diameter also have been associated with nearly 100% disease-free survival.15,16 These are rationale for creating new categories of adenocarcinoma-in-situ (AIS) and minimally invasive adenocarcinoma (MIA). At the same

time, the new classification also recommended the discontinuation of the use of the term “bronchioloalveolar carcinoma” (BAC), as it was commonly and inappropriately applied to advanced-stage metastatic adenocarcinoma, despite having very different clinical, radiologic, and molecular characteristics.17–23 It was also recognized that mucinous BAC often demonstrates more aggressive clinical behavior than nonmucinous noninvasive BAC, and careful histological examination commonly identified areas of invasive patterns. Therefore, in the new classification, while the previous diagnosis of nonmucinous (noninvasive) BAC was changed to AIS, mucinous BAC was changed to invasive mucinous adenocarcinoma. The categories of mucinous AIS and MIA were retained, but these are recognized as very rare. To derive maximum potential information from the histologic patterns demonstrated in invasive adenocarcinoma, the new classification recommended that they be classified based on the presence of the “predominant” pattern (Table 2).1,24,25 Subsequent to its publication, several independent groups have reported the clinical relevance of the new classification, as subtypes have been correlated with different prognoses and molecular features.14,26,27

Table 2. Comparing the Newly Proposed and the 2004 WHO Classifications of Lung Adenocarcinoma 2012 IASLC/ATS/ERS Preinvasive lesions Atypical adenomatous hyperplasia Adenocarcinoma in situ (r3 cm) Nonmucinous Mucinous Mixed mucinous/nonmucinous Minimally invasive adenocarcinoma (r3 cm lepidic tumor with r5 mm invasion) Nonmucinous Mucinous Mixed mucinous/nonmucinous Invasive adenocarcinoma Lepidic predominant (with 45 mm invasion) Acinar predominant Papillary predominant Micropapillary predominant Solid predominant with mucin production Variants of invasive adenocarcinoma Invasive mucinous adenocarcinoma Colloid Fetal (low and high grade) Enteric

2004 WHO Preinvasive lesion Atypical adenomatous hyperplasia Bronchioloalveolar carcinoma (BAC)

None Invasive adenocarcinoma Mixed Mixed/acinar Mixed/papillary Mixed Mixed/solid with mucin/clear cell Mucinous BAC/signet ring Mucinous carcinoma (colloid) Fetal Mucinous carcinoma (colloid)

Genes and pathology of non-small cell lung carcinoma

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Table 3. Sensitivity and Specificity of Immunohistochemical Markers for Subtype Differentiation Histology Type

Marker

Sensitivity

Specificity

Adenocarcinoma

TTF-1 p63 p40 CK5/6

54%–84% 92%–99% 100% 84%

92%–100% 74%–96% 98% 79%

Squamous cell carcinoma

Data from Ocque et al,28 Loo et al,29 and Terry et al.31

APPLICATION OF NEW CLASSIFICATION IN SMALL BIOPSY SAMPLES The IASLC/ATS/ERS report for the first time also made recommendations on how to report NSCLC diagnoses in small biopsy and cytology specimens.1 It is recommended that “the term NSCLC-NOS should be used as little as possible and be applied only when a more specific diagnosis is not possible by morphology and/or special stains,” and that “the term large cell carcinoma should not be used for diagnosis in small biopsy or cytology specimens and should be restricted to resection specimens where the tumor is thoroughly sampled to exclude a differentiated component.” For more precise histologic typing, a limited panel of markers is recommended for routine application in biopsy/cytology samples (Table 3). These include TTF-1 and mucin for adenocarcinoma, and p63 and CK5/6 for squamous cell carcinoma.28–31 More recently, p40 also has been recommended as a new marker of squamous cell carcinoma, as it appears to show better sensitivity and specificity than p63.32,33 When small biopsy specimens showing poorly differentiated NSCLC are associated with these markers, it was recommended that the term “favor adenocarcinoma/ squamous cell carcinoma” be used. It was also recommended that cell blocks are routinely made from cytology including effusion specimens, and only a limited number of immunohistochemical studies should be performed in order to preserve samples for molecular testing.

GENOMIC PATHOLOGY OF LUNG ADENOCARCINOMA Rapid advances in sequencing technologies have provided unprecedented opportunities to dissect the genetic and genomic changes that underlie the heterogeneity and complexity of clinical behavior of lung cancers. The identification of mutations in the tyrosine kinase (TK) domain of epidermal growth factor receptor (EGFR) and the demonstration that their presence identifies lung adenocarcinoma patients who are sensitive to small-molecule

EGFR kinase inhibitors have highlighted the concept of “oncogene addiction” or “driver mutations,”34,35 which have revolutionalized approaches to the diagnosis and treatment of lung cancer.36,37 To date, EGFR TK domain mutations, rearrangements involving the anaplastic lymphoma kinase (ALK)38 and ROS1 genes,39–41 are recognized as bona fide therapeutic targets in lung adenocarcinoma. Kinase inhibitors against these aberrantly activated receptors have become standard therapies for lung cancer patients whose tumors carry these mutations.36,42 To demonstrate the complexity of genomic aberrations in lung cancer, Ding et al43 conducted an all exon/splice site sequencing of 623 known cancer genes in 188 lung adenocarcinomas. This revealed 1,013 nonsynonymous mutations in 163 of 188 tumors, including 915 point mutations, 12 dinucleotide mutations, 29 insertions, and 57 deletions, with the insertion/deletions (indels) ranging from 1–23 nucleotides. A set of 26 genes designated as highfrequency mutation genes were identified. These included known tumor-suppressor genes (P53, STK11, NF1, ATM, APC, CDKN2A, RB1, INHBA), known oncogenes (KRAS, NRAS), potentially oncogenic tyrosine kinase receptors (EGFR, ERBB4, FGFR4, EPHA3, EPHA5, NTRK1, KDR, NTRK3, PDGFRA, LTK, PAK3), and others with undetermined roles (LRP1B, PTPRD, GNAS, ZMYND10/ BLU, SLC38A3). Weir et al44 analyzed copy number alteration (CNV) using significantly recurrent events single-nucleotide polymorphism (SNP) array for 371 lung adenocarcinomas. They found 57 significantly recurrent gene CNVs including large-scale copy number gains or losses involving autosomal chromosomal arms. Thirty-one focal amplifications and deletions were also identified; only six were associated with mutations known at the time of the study. The most commonly amplified chromosomal region was 14q13. 3, found in approximately 12% of samples, and within this was NK2 homeobox1 (NKX2-1) more commonly known as the thyroid transcription factor 1 (TTF1) gene, which is a lineage-specific transcription factor responsible for lung development.

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More recently, intense efforts using whole exome/genome next-generation sequencing technologies have identified additional genes with potential putative “driver” mutations, including ERBB2, AKT1, MET, LMTK2, CTNNB1, NOTCH2, SMARCA4, KEAP1, ARID1A, U2AF1, and RBM10.45–48 Some of these genes are involved in chromatin modification and DNA repair pathways.48 Massively parallel RNA sequencing studies also have identified new fusion genes that could be driver oncogenes. Aside from ALK and ROS1, these include RET, FGFR2, AXL, MAP4/3K3, and PDGFR1, as well as novel metabolic enzymes.41,48–50 An integrative analysis of lung adenocarcinoma at the genome, transcriptome, and epigenome levels from six non-EGFR and nonALK mutated female never-smoker patients also identified 47 somatic mutations and 19 fusion transcripts, many of which were involved in regulating mitotic progression.51 Overall, these results provide preliminary insights into the complexity of genomic aberrations in lung adenocarcinoma without apparent known driver mutations, including the existence of many rare aberrations that still need validation for their biological and clinical significance.

GENOMIC PATHOLOGY OF LUNG SQUAMOUS CELL CARCINOMA Until recently, little was known about the genomic changes in lung squamous cell carcinoma. Studies using array comparative genomic hybridization (a-CGH) and high-density SNP arrays revealed high-frequency gains on chromosomal arms 2p, 3q, 5p, 7, 8p, 8q, 11q, 12q, 13q, 14q, 17q, 19p, 19q, and 20q.52–58 Genes that were of potential biologic and therapeutic interest and located on 3q included PIK3CA, TERC, and SOX2. Bass et al56 and Hussenet et al58 reported that SOX2 amplification is found in up to 20% of squamous cell carcinomas of the lung, and its expression promotes stem cell–related gene expression and proliferation of basal tracheal and squamous carcinoma cells. Focal amplification on 8p has implicated FGFR1 as a driver oncogene in squamous cell carcinoma.59 Kan et al60 used mismatch repair detection (MRD) technology to identify somatic mutations in 441 human primary tumor samples, including 57 lung adenocarcinomas and 63 squamous cell carcinomas. Aside from TP53, they found high frequency mutations on the GRM8, a member of the metabotropic receptor family, and on brain-specific angiogenesis inhibitor 3, a member of the G-protein–coupled receptor family gene. A more comprehensive analysis has been conducted by The Cancer Genome Atlas (TCGA) project, the goal of which is to define somatic genomic changes in more than 20 different

S. Sakashita, M. Sakashita, and M. Sound Tsao

types of cancer. The first TCGA project on lung cancer was on squamous cell carcinoma. The results showed that 75% of squamous cell carcinomas have mutated/deleted/amplified genes, including PI3KCA (16%), PTEN (8%), AKT1-3 (20%), FGFR1-3 (12%), EGFR (9%), ERBB2 (4%), BRAF (4%), NOTCH (13%), and RAS (6%).61 TP53 mutations/deletion were found in approximately 80% and alteration of CDK2NA (p16INK4A)/Rb function in 72% of the tumors studied. Importantly, this study reported mutations of KEAP1 and CUL3 or amplification of NFE2L2, which putatively result in upregulation of oxidative stress response mechanism in 34% of squamous cell carcinomas. The study also found alternation of genes involved in squamous cell differentiation in 44% of samples, including amplification and overexpression of SOX2 (21%) and TP63 (16%), and loss-of-function mutations in NOTCH1/2, ASCL4, and FOXP1. The study also for the first time reported loss-of-function mutations in HLA-A class 1 histocompatibility gene, which may potentially play a role in deregulation of immune function. Wang et al62 have independently validated the occurrence of loss-of-function mutations in NOTCH1/2 in squamous cell carcinoma of skin and lung. These mutations mostly occurred on the extracellular EGF-like repeats, the juxta membranous heterodimerization domain, and the intracellular RAM domain that binds the downstream transcription factor RBPJ.62

COMPARISON TO GENOMIC PATHOLOGY OF SMALL CELL LUNG CANCER Genomic analysis of SCLC is challenging as these tumors are rarely treated by surgery, resulting in a lack of abundant tissue for high-throughput genomic analysis. Voortman et al analyzed SCLC tumor and cell line, bronchial carcinoids, and gastrointestinal carcinoma using array comparative genomic hybridization (aCGH).63 They showed that, compared with carcinoid, the karyotypes of SCLC tumors and cell lines were highly aberrant. Furthermore, high copy number (CN) gains were detected in SCLC tumors and cell lines in cytogenetic bands encoding JAK2, FGFR1, and MYC family members. Since in those samples, the CN of these genes exceeded 100, it was suggested that they could represent driver mutations and potential drug targets, but further validation is required. Peifer et al performed exome sequencing (n ¼ 27), SNP array analysis (n ¼ 63), transcriptomic sequencing (n ¼ 15), and whole genome sequencing (n ¼ 2) in resected SCLCs.64 The very high rate of 7.4 proteinchanging mutations per million basepairs (MB) detected was consistent with tobacco-induced carcinogenesis and was comparable to those found in

Genes and pathology of non-small cell lung carcinoma

lung squamous cell carcinoma (median 8.1/MB)61 and smoker adenocarcinoma (9.8–10.5/MB),47,48 as compared to never-smoker adenocarcinoma (0.6–1.7/MB). They found inactivation of TP53 and RB in all cases of SCLC, supporting the crucial role of these two tumor-suppressor genes in the initiation of SCLC. Mutations were noted in PTEN, SLIT2, and EPH7, as well as amplification of SOX2, Cyclin E1, and FGFR1 (6%). MYC family gene amplification was found in 16% of tumors, a majority involving MycL1 and MycN. Interestingly, the same group has reported that SCLC cell lines with MYC amplification were dependent on Aurora kinase B, offering a potential therapeutic strategy in subset of SCLC patients.65 In addition, they found mutations involving CREBBP, EP300, and MLL genes, which implicates genomic alteration of histone acetylation and epigenetic modifying enzymes. Rudin et al66 also conducted exome, transcriptome, and CN analyses on 36 primary SCLC tumors and 23 cell lines. Aside from TP53 and RB, they found hot spot mutations involving protein kinases and phophatases, Ras family regulators, chromatinmodifying enzymes, and G-protein–coupled receptors. Consistent with Peifer et al, they also found high-frequency (27%) amplification of SOX2, implicating its important role in the biology and pathogenesis of this tumor. In both studies, many novel fusion genes generated from translocations were identified, but their biological significance remains to be established. These preliminary results suggest that SCLC and NSCLC may share some common potential therapeutic targets, as well as distinct aberrations that could be novel specific targets for SCLC. As SCLC is not treated primarily by surgery, SCLC samples are not as readily available. Therefore, global efforts to profile more SCLC tumors are desired, including nonresectable advanced SCLC cases.

EGFR MUTATIONS EGFR mutations, which are associated with high objective responses to single-agent TK inhibitor (TKI) therapy in lung adenocarcinoma, are preferentially observed in a specific subset of patients: females of East Asian ethnicity who have never smoked and who have well-differentiated adenocarcinoma with lepidic, papillary, or acinar histology.36,67–70 In adenocarcinoma, the majority of these mutations have been identified in exons 18–21 of the EGFR gene.71–73 These mutations can be roughly classified into three major categories: inframe deletion in exon 19, insertion mutations in exon 20, and missense mutations in exons 18–21, with the most frequent mutations being the deletion in exon 19 and the exon 21 L858R mutations. Most

33

exon 20 insertions are associated with lack of response to first-generation EGFR TKIs.74,75 T790M mutation in exon 20 has been regarded mainly as an acquired resistance to EGFR TKI, but many exist in minor clones of tumor cells in untreated primary tumors with sensitizing mutations.37,76 These clones would expand and become easily detectable during TKI treatment as a majority of sensitive tumor cells involute. For the selection of patients to receive firstline EGFR TKI, minimum screening should include the common deletions in exon 19 and L858R mutations. However, screening for sensitizing mutations on exon 18 (eg, on E709K/A/G and G719A/S/ C/D) and on exon 21 (L861Q) are also recommended.

ECCHINODERM MICROTUBULE-LIKE 4–ANAPLASTIC LYMPHOMA KINASE FUSION GENE Ecchinoderm microtubule-like 4–anaplastic lymphoma kinase fusion (EML4-ALK) fusion is formed as the result of a small inversion within the short arm of chromosome 2 that joins intron 13 of EML4 to intron 19 of ALK [inv(2)(p21;p23)]. This generates an oncogenic fusion encoding a constitutively activated protein tyrosine kinase.38,77,78 The EML4-ALK fusion is a rare abnormality detected in 3%–13% of patients with adenocarcinomas.38,77,79,80 Several variants that are distinguished by the translocation sites (at different introns) on the EML4 gene have been identified.39,81 Although the EML4-ALK fusion is the most common, other less common variant fusions have been reported, including translocations with other chromosomes (KIF5B-ALK, TFG-ALK).39,81 The NPM-ALK translocation that has been well characterized in anaplastic large cell lymphoma has not been reported in lung cancer. Compared with patients with wild-type ALK and EGFR, patients with the EML4-ALK fusion gene tend to be younger, light or never smokers, and diagnosed at an advanced clinical stage.38,79,82 Because it is a fusion gene, fluorescence in situ hybridization (FISH) analysis is the gold standard for making the diagnosis. Since immunohistochemistry (IHC) is simpler and significantly less expensive than FISH, IHC may be useful for screening purposes.83,84 However, ALK IHC has to be optimized to increase the sensitivity of ALK protein detection, as the protocol used to detect ALK in lymphoma cases often results in false negative staining.83,84 Using IHC optimized to screen for ALK gene rearrangements, several studies have reported very high sensitivity of IHC to detect FISHverified ALK gene–rearranged lung adenocarcinoma.81,85–87 While the false negative rate of optimized ALK IHC in detecting FISH-positive cases is unknown, there are several reports of IHC-positive

34

but FISH-negative patients who responded to ALK inhibitor crizotinib.88

ROS1 ROS1 is a receptor tyrosine kinase (RTK) of the insulin receptor family. ROS1 is normally expressed in the lung and other organs, although its physiological function remains unclear. Chromosomal rearrangements involving the ROS1 gene were originally described in glioblastoma where ROS1 (chromosome 6q22) is fused to the FIG gene located immediately adjacent to ROS1 locus. Recently, ROS1 fusions were identified as potential driver mutations in NSCLC.39–41,89–93 Several ROS1 fusion partners have been discovered, including CD74, solute carrier family 34 (type II sodium/phosphate contransporter), member 2 (SLC34A2), ezrin (EZR), leucine-rich repeats and immunoglobulin-like domains 3 (LRIG3), syndecan 4 (SDC4), tropomyosin 3 (TPM3), and fused in glioblastoma (FIG).39– 40,41,90,92–96 ROS1-rearranged lung carcinoma is rare, with an estimated prevelance of approximately 1%– 2.5%.40,90,96–98 Reports to date suggest that ROS1rearranged lung cancers have similar demographics as ALK-rearranged lung cancer and tend to be younger, female adenocarcinoma patients with less smoking history.40,98 They have overall survival rates similar to those of the ROS1 fusion-negative cancer patients.98 Significantly, in vitro data suggested that ROS1-rearranged cancers respond to the ALK inhibitors and a recent clinical trial revealed marked crizotinib activity in this molecular subclass.40,90,95,97,99 More recently, one acquired resistance mechanism to crizotinib, a G2032R mutation, also has been reported.100

S. Sakashita, M. Sakashita, and M. Sound Tsao

amplification is a poor prognostic marker in squamous cell carcinoma patients, but this finding has not been confirmed in other studies.103 Current trials of FGFR1 inhibitors are specifically targeting NSCLCs harboring FGFR1 amplification.104,105

PI3KCA The PI3K pathway is a signal transduction pathway that is central to cell survival, metabolism motility, and angiogenesis. Several investigators have shown that abnormalities of PI3K/PTEN/AKT/mTOR are more common in squamous cell carcinoma than in adenocarcinoma of the lung,106–110 possibly suggesting an increased reliance on this pathway. In studies for which more detailed histological information is available, the mutation rate for PI3KCA in squamous cell carcinoma ranges from 3.6%– 6.5%. 109,110 Phosphatidylinositol4,5-bisphosphate 3-kinase, catalytic subunit alpha (PI3KCA) amplification also appears to be prevalent in squamous cell carcinoma. Several inhibitors of the PI3K pathway are undergoing evaluation in various solid tumors. These include inhibitors of the various isoforms of PI3K, AKT1, and mTOR, and dual inhibitors of PI3K/ mTOR. Of importance to these trials is the possible coexistence of PIK3CA mutations with aberrations in other oncogenes such as KRAS, BRAF, FGFR, and EML4-ALK in lung adenocarcinoma.111 The extent to which this overlap occurs in squamous cell carcinoma remains to be seen and will determine whether targeting these events is likely to be effective via combinational versus a monotherapy approach.

FGFR1

MOLECULAR TESTING IN NSCLC

The fibroblast growth factor receptor (FGFR) is a transmembrane receptor tyrosine kinase that participates in the regulation of embryonal development, cell proliferation, differentiation, and angiogenesis. Amplification at 8p12 was observed in multiple studies of squamous cell lung cancer56,59 and FGFR1 has been identified as a potential candidate gene in this region. Dysregulation of the FGFR1-4 signaling has been described in multiple cancers, with overexpression seen in breast and prostate cancers, as well as myeloma, and point mutations in sarcoma, bladder, and endometrial cancers among others.101,102 In lung cancer, FGFR1 amplification was enriched in squamous cell and small cell carcinomas in comparison to adenocarcinomas. The initial report suggested that FGFR1 was amplified in approximately 20% of squamous carcinoma cells,59 but more recent data suggested high amplification occurs in 10% or less. Choi et al also reported that FGFR1

As predictive biomarkers are becoming integral in the use of targeted therapies to treat lung cancer patients, there is a need to develop a multidisciplinary and evidence-based guideline for molecular testing. The CAP, IASLC, and AMP have recently collaborated to issue such a guideline.2–4 Following systematic review of the literature and consensus meeting as well as public consultation, 37 guideline items addressing 14 topics and 15 recommendations were included in the publication. The major recommendations include the use of testing for EGFR mutations and ALK gene rearrangement to guide patient selection for therapy with an EGFR or ALK inhibitor, respectively, as clinical factors alone are not sufficiently sensitive or specific to be used as patient selection methods. It is also recommended that testing be done on all advanced NSCLC patients with an adenocarcinoma component, regardless of histologic grade, while reserving testing in squamous

Genes and pathology of non-small cell lung carcinoma

carcinoma only to patients with clinical characteristics that have been associated with higher rates of presence of these genetic aberrations, such as neversmokers or younger patients. The guideline also emphasized the importance of: (1) pathologists’ involvement in the selection and quality assessment of samples to be tested, (2) routine preparation of cell blocks from cytology specimens, (3) conservation of small diagnostic biopsy samples for molecular testing during the initial diagnostic workup to arrive at the diagnosis, (4) avoidance of using samples that have been processed in acid or heavy metal fixatives or decalcifying solutions, (5) encouragement for use of analytic methods or enrichment procedure to increase the sensitivity of detecting mutations in low tumor cellularity samples, and (6) performance of testing in accredited laboratories that can assure reasonable test completion and reporting time and that follow recognized laboratory quality control an assurance policies/procedures. There was also a strong recommendation that the testing for patients with advanced disease be performed at the time of diagnosis or at recurrence after previous resection of early-stage tumors, and that a testing strategy be developed by local laboratories in collaboration with a multidisciplinary clinical team.

FUTURE PERSPECTIVES The discovery of EGFR mutations has revolutionized the concept and strategy for personalizing lung cancer treatment, using the identification of specific driver genetic aberrations as predictive biomarkers to select therapies for individual patients. While currently only EGFR mutation and ALK gene rearrangement are recognized as the markers that need to be tested, based on the availability of proven effective therapies for tumors with these aberrations, rapid discoveries of additional driver oncogenes and their corresponding effective therapies will expand the molecular testing menu. During the last two decades, as pathologists have become highly efficient and accurate in rendering diagnosis on small amounts of materials obtained by less invasive biopsy procedures, the need for more tissues for molecular testing have led to the re-evaluation of approaches to balance the two conflicting needs. There is now recognition that minimal panel of IHC markers should be used to improve the accuracy of histologic diagnosis and tumor classification, while conserving tissues for molecular predictive biomarker testing. At the same time, as more markers need to be tested, it becomes abundantly evident that availability of tissue becomes an issue. No longer will sequential testing be feasible or cost- and timeeffective for treatment decisions. There is little doubt that the future of pathology practice will not only

35

involve pathologists making accurate histopathological diagnoses but also reflex testing of therapeutically relevant molecular markers using multiplex molecular assay platforms such as next-generation sequencing.

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