Lung cancer is the leading cause of cancer-related death

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1 Molecular Diagnostics of Lung Carcinomas Sanja Dacic, MD, PhD N Context. The development of targeted therapies in the treatment of lung carcinoma is a rapidly growing area that requires a precise histologic classification of lung carcinomas and the implementation into clinical practice of testing for predictive biomarkers of therapy response. Molecular testing has added another layer of complexity in the routine workup of rather limited diagnostic tumor tissue. Objective. To review the most important lung carcinoma biomarkers predictive of response and to discuss proposed routine molecular testing in clinical practice. Data Sources. PubMed (US National Library of Medicine) available review articles, peer-reviewed original articles, and experience of the author. Conclusions. Histologic profile, clinical characteristics, and mutational profile of lung carcinoma have all been reported as predictive factors of response to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) and other targeted therapies. Recently published results of large clinical trials indicate that mutational profiling, particularly identification of activating epidermal growth factor receptor (EGFR) mutations, is the best predictor for EGFR-TKI response. Despite all these observations, molecular profiling of lung carcinomas has not been standardized or validated in clinical practice. Rapid development of targeted therapies will probably require molecular testing for a panel of mutations to identify molecular subtypes of non small cell lung carcinomas that will benefit from new therapeutic approaches in personalized patient care. (Arch Pathol Lab Med. 2011;135: ) Lung cancer is the leading cause of cancer-related death in the world. 1 Non small cell lung carcinoma (NSCLC) accounts for 80% to 85% of all lung cancers, with lung adenocarcinoma being the most common histologic type in the United States. Most patients present with advancedstage disease and are not candidates for surgical resection but are subjected to platinum-based chemotherapy and radiation therapy alone, or in combination. Unfortunately, despite these therapies, prognosis is poor, with an overall 5-year survival rate of only 15%. Major advances in the understanding of the molecular pathogenesis of lung cancer have led to new strategies for early detection, diagnosis, staging, and therapy. 2 In recent years, the treatment options have changed from cytotoxic chemotherapies alone to single-agent and combination targeted therapies. A major accomplishment in the treatment of advanced lung cancer has been the discovery of tyrosine kinase inhibitors (TKIs) targeting the epidermal growth factor receptor (EGFR). 3 5 Patients responses to these therapies are variable, and testing for predictive biomarkers of therapeutic response is becoming an important part of the diagnostic workup. Certain clinical characteristics such as female sex, East Asian ethnicity, nonsmoking history, and adenocarcinoma histology have been shown to be associated with an increased likelihood of responsiveness to EGFR-TKI. However, molecular predictors of response, Accepted for publication January 14, From the Department of Pathology, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania. The author has no relevant financial interest in the products or companies described in this article. Reprints: Sanja Dacic, MD, PhD, Department of Pathology, Room PUH C608, University of Pittsburgh Medical Center, 200 Lothrop St, Pittsburgh, PA ( dacics@upmc.edu). such as activating mutations in the tyrosine kinase domain of EGFR, seem to be more reliable predictors of response than clinical characteristics. 6 8 Similarly, other gene alterations that play an important role in adenocarcinoma development have been identified and new targeted therapies are under development (Figure 1). The results of clinical trials with EGFR-TKI have revolutionized the first-line therapy for NSCLC, and first-line treatment with EGFR-TKI is now a standard of care for patients with an EGFR mutation. 9 Unfortunately, not much progress has been made in treatment of other subtypes of NSCLC, such as squamous cell carcinomas, which are usually nonresponsive to EGFR-TKI and may be associated with fatal complications if treated with anti vascular epithelial growth factor therapies. 10,11 Because the histologic profile is one of the predictors of response to targeted therapies, oncologists place high demands on pathologists to precisely subclassify NSCLCs. 12 At the same time, there is a pressure to facilitate and participate in the implementation of biomarker testing in clinical practice. However, as yet, clinical testing for predictive biomarkers in lung carcinoma is neither standardized nor validated in the United States. 13 Recently, the International Association for the Study of Lung Cancer (IASLC) has published the recommendations of the European workshop for EGFR mutation testing in Europe. 9 This review will address the current, most common molecular biomarker testing in lung carcinomas and the need for standardization of molecular testing in the management of patients with lung carcinoma. TUMOR TISSUE SAMPLE It is very unlikely that tumor tissue will be collected for molecular testing only. Since most of the patients with 622 Arch Pathol Lab Med Vol 135, May 2011 Molecular Diagnostics of Lung Carcinomas Dacic

2 Figure 1. Therapies targeting the epidermal growth factor receptor (EGFR) signaling pathway. Abbreviations: AKT, also known as protein kinase B; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; ERK, extracellular signal regulated kinase; HDAC, histone deacetylase; mtor, mammalian target of rapamycin; PDK1, 3- phospoinositide dependent protein kinase-1; PI3K, phosphatidylinositol 3-kinase; PTEN, phosphatase and tensin; RAS, rat sarcoma; TKIs, tyrosine kinase inhibitors; TSG, tumor suppressor gene; VEGF, vascular endothelial growth factor; VEGFR, vascular endothelial growth factor receptor. Reprinted with permission from Chirieac and Dacic. 78 lung carcinoma present at the advanced stage of the disease, no resection specimen will be available and only tissue collected at the initial diagnostic workup will be considered for molecular testing. Therefore, dilemmas regarding tumor sample remain, such as type of specimen, reliability of molecular testing on cytology specimens, biopsy technique for providing the most adequate tumor volume, and tissue processing for diagnostic and molecular workup. None of these questions presently have answers. The European EGFR Working Group has proposed some of the answers to these questions, which could be applied to any molecular assay in lung carcinoma. 9 The main initial goal in any presumable molecular testing is to obtain a high-quality, high-volume tumor specimen, which would in part depend on biopsy technique (Table). The results may be influenced also by the biopsy site, such as primary tumor versus metastatic tumor. Published studies have demonstrated discordance in results of mutational and fluorescence in situ hybridization (FISH) assays between the primary tumor and metastatic disease. Most of the time, clinicians are sampling only the most easily accessible tumor tissue. It is very hard to design a study that specifically looks into the differences in therapy responses based on molecular results obtained from different types of tumor specimens from the same patient. Therefore, we may never know the right answer to this question. For mutational assays, the ratio between malignant and normal cells within the sample is essential for the detection of specific mutations. 19 Manual or laser microdissection of tumor cells may be required to improve reliability and accuracy of the molecular results, particularly direct DNA sequencing. 20 The minimum number of tumor cells that is optimal for mutational analysis is uncertain, but empirically, 50% of tumor cells per sample is sufficient for DNA sequencing. If other, more sensitive mutation methods are used, even less than 10% of tumor cells may be adequate. 9,21 27 Although processing and fixation of lung tumors has not been standardized by experimental and clinical experiences, short fixation times (6 12 hours for biopsy specimens; 8 18 hours for larger resection specimens) in 10% neutral buffered formalin should be optimal for DNA- and RNA-based tests, as well as FISH assays. 28 In contrast, Bouin fixative should be avoided. Most lung cancers are diagnosed with cytology samples, which may be used for molecular analysis, particularly if cell blocks are made. 29,30 POSITIVE PREDICTORS OF RESPONSE TO EGFR-TKI Epidermal Growth Factor Receptor Mutational Analysis. Two treatment strategies to target EGFR have been developed: monoclonal antibodies directed against the extracellular ligand-binding domain of EGFR (eg, cetuximab [Erbitux, ImClone Systems Inc, Biopsy Technique and Volume of Diagnostic Material a 21-g Needle Aspiration 19-g Needle Aspiration Transbronchial Biopsy CT-Guided Needle Biopsy No. of cells per biopsy/ aspiration $100 $150 $300 $500 No. of biopsies Abbreviation: CT, computed tomography. a Modified with permission from Pirker et al. 9 Arch Pathol Lab Med Vol 135, May 2011 Molecular Diagnostics of Lung Carcinomas Dacic 623

3 Branchburg, New Jersey]) and small-molecule TKIs (eg, gefitinib [Iressa, AstraZeneca, London, United Kingdom]) and erlotinib (Tarceva, F. Hoffmann-La Roche, Basel, Switzerland). 3,4,31 35 Activating somatic mutations in the exons 18 through 21 of the tyrosine kinase domain of EGFR have been shown to correlate with a high likelihood of response to EGFR-TKI, including gefitinib and erlotinib, but not to monoclonal antibodies. The status of EGFR and the patient s response to EGFR-TKI have been assessed by various methods, such as immunohistochemistry (protein expression) and FISH/chromogenic in situ hybridization (CISH) (gene copy number changes), but mutational analysis has been shown to be the best predictor of tumor response to EGFR-TKI Therefore, only EGFR mutation testing in NSCLC has been recommended in Europe. 9 Activating predictive mutations target 4 exons (18 21), which encode part of the tyrosine kinase domain of EGFR (the entire kinase domain is encoded by exons 18 24) and are clustered around the adenosine triphosphate binding pocket of the enzyme. 41 The most common are in-frame deletions in exon 19 (45%), followed by a point mutation (CTG to CGG) in exon 21 at nucleotide 2573, which results in substitution of leucine by arginine at codon 858 (L858R) (41%). Therefore, if limited DNA material is available, a reasonable approach would be to test for the most common mutations within exons 19 and 21 only. These mutations occur in approximately 10% of Western patients and up to 50% of Asian patients with lung adenocarcinomas. They also have prognostic significance, because patients with EGFR-mutant adenocarcinomas showed prolonged survival compared with those with EGFR wild-type disease, regardless of the treatment received Various mutational assays have been developed, but direct DNA sequencing is still the most commonly used method. There is no consensus on the best method, because each method has certain advantages and disadvantages. The major advantage of DNA sequencing is that it can detect all mutations, including novel variants and it identifies the exact mutation. In contrast, newer mutational methods (eg, single-strand conformation polymorphism, denaturing high-performance liquid chromatography, and amplification refractory mutation system) may miss mutations that are not assayed but are more sensitive (requiring fewer tumor cells) and are less time-consuming than DNA sequencing. The significance of low-abundance mutations detected in heterogeneous samples with these more sensitive assays is uncertain and these mutations need further evaluation, including a determination of their clinical or predictive significance. Other Methods. Until recently, the significance of EGFR copy number changes determined by FISH was an area of controversy. Initial reports 38,45 had suggested a role for EGFR-FISH results in predicting the patient s response to EGFR-TKI and antibody treatments. Similarly, chromogenic in situ hybridization was suggested as a reliable alternative to FISH in determining EGFR copy number status in NSCLC. 46 A complicating observation was the fact that EGFR-mutated tumors frequently have EGFR gene copy number changes, and until recently, it was difficult to determine which method provides better prediction of the patient s response. However, recent clinical trials have provided supporting evidence that EGFR mutation status is the most relevant marker for treatment selection, and therefore EGFR mutational analysis should be the method of choice in clinical practice. 47,48 Epidermal growth factor receptor immunohistochemistry is not recommended for EGFR testing in lung cancer. However, mutation-specific rabbit monoclonal antibodies have been shown to detect the 2 most common EGFR mutations (exon 19 deletions and exon 21 L858 mutation) (Cell Signaling Technology, Inc, Danvers, Massachusetts). 49,50 The EGFR L858R mutant antibody showed clinically acceptable performance (sensitivity 95%, positive predictive value of 99%, with a positivity cutoff of 1+). 50 The EGFR exon 19 mutant-specific antibody showed reduced sensitivity for exon 19 deletions other than 15 base pairs. 50 This simple assay should be further validated, because it may provide a rapid and cost-effective clinical test for identifying patients with lung cancer who may be responsive to EGFR-targeted therapies. NEGATIVE PREDICTORS OF RESPONSE TO EGFR-TKI Primary Resistance Genetic mutations in the genes encoding proteins involved in the EGFR signaling cascade (KRAS, HER2, BRAF, PI3K, LKB1, SHP2) exist as mutually exclusive somatic mutations with the possible exception of those in PI3K. 51 The presence of these mutations is associated with a lack of response to EGFR-TKIs in the treatment of lung cancer. Therefore, additional mutational analysis of genes other than EGFR may be necessary to improve patient selection for EGFR-targeted therapies. v-ki-ras-2 Kirsten Rat Sarcoma Viral Oncogene Homolog (KRAS). KRAS mutations are found in approximately 30% of adenocarcinomas and 5% of squamous cell carcinomas. 52,53 The EGFR-KRAS signaling cascade in lung cancer is considered a unidirectional linear outside-in signaling cascade. Therefore, KRAS mutations result in a constitutive activation of the RAS/ MAPK pathway independently of EGFR activation. KRAS point mutations, which lead to substitutions of amino acids for glycine on codons 12 and 13, are most frequently observed in former or current smokers. 42,54,55 Distinct KRAS point mutations, such as G12D, have been reported in approximately 15% of never smokers. 55 The most common method to detect KRAS codon 12 and 13 mutations is by polymerase chain reaction amplification of exon 2 and by direct sequencing of the amplicon. All of the previously discussed issues of specimen selection and quality control that affect EGFR mutational analysis apply to KRAS mutational analysis as well. The impact of KRAS mutations on the rate of recurrence of disease and patient survival has been the subject of many studies, which are difficult to interpret because of differences in tumor stage, histologic inclusion criteria, and rather small numbers of patients. Initially, KRAS was considered a poor prognostic marker, but the data have been contradictory. 56,57 59 KRAS has been also perceived as a negative predictive marker of chemotherapy benefits, on the basis of retrospective studies. With the data presently available, it is not possible to determine with certainty if KRAS mutational status is an independent prognostic marker and/or a negative predictive marker of adjuvant chemotherapy benefit. Several clinical trials 57,60,61 have reported an association between KRAS mutations and a lack of response to EGFR- TKI. In contrast to colon cancer, patients treated with anti- 624 Arch Pathol Lab Med Vol 135, May 2011 Molecular Diagnostics of Lung Carcinomas Dacic

4 EGFR monoclonal antibodies, such as cetuximab, showed no difference in response rate regardless of KRAS mutational status. 31,45,62,63 Similarly, the progression-free survival times in the cetuximab-containing arm and the chemotherapy-alone arm were similar between patients with KRAS wild-type and KRAS mutant tumors. Although many laboratories in the United States routinely perform KRAS mutational analysis of lung carcinomas, the clinical question remains whether there is clinically significant difference in the efficacy of EGFR - TKIs between KRAS mutant adenocarcinomas and KRAS wild-type/egfr wild-type adenocarcinomas. Therefore, for patients without EGFR mutations, the value of determining KRAS mutational status in selecting EGFR- TKI or anti-egfr monoclonal antibodies is uncertain. Development of new therapies targeting the downstream RAS effector pathways PI3K/AKT/mTOR and RAS/ RAF/MEK will probably require knowledge about KRAS mutational status. Secondary Resistance to EGFR Inhibitors T790M EGFR Mutation. The T790M EGFR mutation was initially considered to confer secondary resistance to TKI. 64 Using more sensitive mutation detection techniques, studies showed that this mutation may coexist with L858R mutations; this indicated that T790M might in fact confer pretreatment (primary) resistance to EGFR- TKIs. Importantly, the presence of T790M at such a low frequency did not preclude significant responses to therapy with TKI among patients with sensitivity-conferring EGFR mutations, but it was associated with a significant difference in median progression-free survival. Mesenchymal Epithelial Transition Factor. Amplification of mesenchymal epithelial transition factor (MET) proto-oncogene has been identified as a second mechanism of EGFR-TKI resistance. 65 MET amplification, which has been associated with poor prognosis in different retrospective series, is a relatively uncommon event in NSCLC, occurring in 1% to 7% of unselected cases. Nevertheless, in highly selected cohorts of patients, such as those harboring somatic mutations of EGFR, with acquired resistance to EGFR-TKI, MET amplification can be observed in up to 20% of cases (Figure 2). 66 Somatic EGFR mutations and MET amplification are not mutually exclusive events and, therefore, additional studies will be needed to clarify the role of MET in de novo and/or acquired resistance. Different methodologic approaches can be used to determine gene amplification, but for clinical laboratories using formalin-fixed, paraffin-embedded tissue, FISH assay is an optimal choice. New drugs targeting against the MET kinase receptor or its ligand are available and have shown promising results. A treatment approach, including a combination of EGFR and MET TKI, may be an effective strategy and may lead to the clinical investigation of multiple MET TKIs in combination with erlotinib. OTHER TARGETED THERAPIES Anaplastic Lymphoma Kinase Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase originally identified as part of the t(2;5)(p23;35) translocation in a subset of anaplastic large cell lymphomas. 67,68 Subsequent studies 69,70 showed that some solid tumors (inflammatory myofibroblastic tumors, squamous cell carcinomas, and NSCLCs) activate ALK signaling by creating unique oncogenic fusions of the ALK gene (at chromosomal locus 2p23) with a variety of partners through chromosomal translocations; this results in the generation of oncogenic ALK fusion genes and their encoded proteins and leads to constitutive activation of the ALK kinase domain. ALK rearrangements were described in a subset of Asian and white patients with lung adenocarcinomas Most ALK rearrangements in lung adenocarcinoma result from an interstitial deletion and inversion in chromosome arm 2p and result in the EML4-ALK fusion gene product. The oncogenic activity of this fusion product has been effectively blocked by small-molecule inhibitors of ALK. 73,74 Early studies in unselected groups of Asian patients showed that EML4-ALK rearrangement was a relatively rare event occurring in 1% to 7% of lung adenocarcinomas. A recent report 72 has shown a similar observation in early-stage adenocarcinomas in the Western population. However, this rearrangement can be detected in up to 20% of stage IV lung adenocarcinomas, mainly in younger patients with a history of never smoking. None of the ALK-rearranged adenocarcinomas showed coexistent EGFR mutations. Several diagnostic screening tools for EML4-ALK positive tumors have been reported, including reverse transcription polymerase chain reaction (RT-PCR) assays, immunohistochemistry, and FISH. 71,75 77 Several variants that connect various exons of EML4 to ALK have been reported in the literature. Therefore, for optimal and accurate screening of ALK rearrangements, multiplex RT- PCR assays should be designed to detect all of the reported variants. It is uncertain if any clinically significant differences exist between various isoforms, and therefore, RT-PCR as a screening method in this case seems to be less optimal for clinical use. Immunohistochemical detection of EML4-ALK proteins in surgical biopsy, resection, or cytology specimens would be a convenient screening approach for pathologists. Unlike anaplastic large cell lymphoma, immunohistochemical detection of ALK translocation fusion products in lung carcinoma does not necessarily correlate with the presence of translocation. 71,77 However, a recently developed monoclonal antibody (Cell Signaling Technology) shows increased sensitivity for ALK detection, with promise for applicability in the clinical laboratory (Figure 3). 77 This novel immunohistochemical assay correctly identifies ALK-rearranged lung adenocarcinoma with high reproducibility. Because this antibody is not yet available for commercial use, many laboratories are using a FISH break-apart probe to the ALK gene (Vysis LSI ALK Dual Color, Break Apart Rearrangement Probe, Abbott Molecular Abbott Park, Illinois) (Figure 4, A and B). PRACTICAL CONSIDERATIONS FOR MOLECULAR TESTING IN LUNG CARCINOMAS It is obvious that molecular testing of lung carcinoma should be the standard of care, but there are many practical, logistic, and technical questions that need to be addressed. The first is which laboratory should be allowed to perform the testing. Only accredited, CLIA (Clinical Laboratory Improvement Amendments of 1988) certified laboratories with appropriate quality control standards should carry out this type of molecular testing. The Society Arch Pathol Lab Med Vol 135, May 2011 Molecular Diagnostics of Lung Carcinomas Dacic 625

5 Figure 2. Fluorescence in situ hybridization assay with c-met (RP11-163C9, Children s Hospital Oakland Research Institute, Oakland, California) and centromeric (CEP7) probes in lung adenocarcinoma demonstrates amplification of mesenchymal epithelial transition factor (MET) (original magnification 3200). Figure 3. Detection of anaplastic lymphoma kinase (ALK) translocation by immunohistochemistry (original magnification 340). Courtesy of Lucian Chirieac, MD, Brigham and Women s Hospital, Boston, Massachusetts. Figure 4. Break-apart fluorescence in situ hybridization probe to the anaplastic lymphoma kinase (ALK) gene (Vysis LSI ALK Dual Color, Break Apart Rearrangement Probe, Abbott Molecular, Abbott Park, Illinois). A, Normal cells. Cells without translocation show green and orange signals in juxtaposition. B, Example of lung adenocarcinoma with ALK translocation: 1 pair of fused signals (green and orange) with 1 separate green signal and 1 separate orange signal. of European Pathologists has recommended that only laboratories with an analytic success rate of 95% and an accuracy rate of more than 97% be allowed to perform KRAS mutational testing in colon cancer. At the present time, it is uncertain how many laboratories can achieve this standard. Therefore, different laboratories should share protocols and exchange positive and negative control samples to improve overall analytic performance, and they should implement a system for validation of techniques and results. Since molecular workup represents an additional step in the diagnostic workup of available tumor tissue, one of the aims should be to provide results in a timely manner. Therefore, the question is whether molecular testing should be integrated into the workflow of on site pathology laboratories or whether the tissue should be sent to commercial laboratories. The IASLC European Thoracic Oncology Platform Multidisciplinary Workshop has proposed a 626 Arch Pathol Lab Med Vol 135, May 2011 possible scenario for diagnostic and molecular workup of lung cancer cases (Figure 5). This approach has been already used by many large academic centers in the United States, but the question remains as to how lung cancer specimens will be handled by community hospitals that do not have adequate molecular laboratory support. A turnaround time of 2 weeks from diagnostic procedure to the final report has been suggested, but organizing a system that would include multiple parties, from pathology, oncology, and thoracic surgery to reference laboratories, seems to be quite challenging. Testing by outside laboratories also brings up another challenge, such as integration of all available data about the tumor (eg, histology, stage, and mutational profile) in one pathology report and in the patient s medical records, which is simple if testing is performed at a single institution. A common question is who should request the mutational profiling: a treating physician or a Molecular Diagnostics of Lung Carcinomas Dacic

6 Figure 5. Proposed workflow for lung carcinoma by the European EGFR Workshop Group. Abbreviations: EGFR, epidermal growth factor receptor; IHC, immunohistochemistry. Modified with permission from Pirker et al. 9 pathologist? This question emphasizes the importance of close communication between pathologists and treating physicians. Our institution s policy is for pathologists to order mutational profiling at the time of the diagnostic workup. This policy was implemented after discussion with thoracic oncologists and surgeons. The rationale behind reflex testing was to reduce the turnaround time for molecular testing and to provide a complete histologic/molecular profile of the tumor at the time of patient discharge or office visit. SUMMARY The diagnostic and treatment approaches to lung carcinoma, primarily adenocarcinomas, are undergoing a revolution. The classification of lung carcinoma is going beyond small cell carcinoma and NSCLC, and precise subclassification of NSCLC has a direct impact on a patient s management and prognosis. Many new targeted therapies are under development, and the molecular testing of lung carcinoma has become the standard of care in a very short period of time. EGFR mutations have emerged as a key predictive biomarker for EGFR-TKI treatment and should be the primary standard for selection of patients for first-line treatment with EGFR-TKIs. Clinical testing for EGFR mutations and other predictors of response should be optimized and standardized in regard to mutational techniques, specimen and tumor types, and result reporting. Many new drugs are on the horizon, and routine molecular diagnostics for lung cancer is going beyond EGFR mutational profiling. There is already a need for more efficient screening for multiple mutations by using high-throughput/high-sensitivity technology. References 1. 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