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S. Dacic

Moderator of

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    MS 05 - Tumor Heterogeneity (ID 23)

    • Event: WCLC 2015
    • Type: Mini Symposium
    • Track: Biology, Pathology, and Molecular Testing
    • Presentations: 4
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      MS05.01 - Overview of Tumor Heterogeneity (ID 1864)

      14:20 - 14:40  |  Author(s): C. Swanton

      • Abstract
      • Presentation
      • Slides

      Abstract:
      Despite advances in genomic technologies, most advanced solid tumors remain incurable and drug resistance is almost inevitable with limited biomarkers available to personalize therapy. Two important lessons have emerged from the comprehensive genomic analyses of cancers, which may provide an explanation for difficulties that have been encountered in biomarker development. First, each tumor contains an individual assortment of multiple genomic aberrations, few of which are shared between patients with the same histopathological tumor subtype. Second, emerging evidence suggests that these anomalies appear to vary both spatially and temporally within the tumor, indicating substantial intratumor heterogeneity. Increasingly, molecular evidence suggests that intratumor heterogeneity may contribute to tumor growth through a branched (polytypic) rather than a linear pattern of tumor evolution. Branched evolutionary growth and intratumor heterogeneity results in coexisting cancer cell subclones with variegated genotypes and associated functional phenotypes that may be regionally separated within the same tumor or distinct within one biopsy and alter in dominance over time. Variegated phenotypes, resulting from intratumoral genetic heterogeneity and the emergence of new subclones at relapse, are likely to have important implications for developing novel targeted therapies and for preventing the emergence of drug resistance. Intratumor heterogeneity and tumour sampling bias, resulting from single biopsy-driven biomarker discovery and validation approaches, may also contribute to the recently reported failures in implementation of robust biomarkers in the clinical setting. Clinical trial efforts taking into account tumour heterogeneity and its relevance to lung cancer will be addressed.

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      MS05.02 - How Does Tumor Heterogeneity Affect Molecular Testing on Biopsy Samples - Diagnostic vs. Rebiopsy (Resistance) (ID 1865)

      14:40 - 15:00  |  Author(s): K. Kerr

      • Abstract
      • Presentation
      • Slides

      Abstract:
      There are many different manifestations of heterogeneity within lung tumours. The three main considerations are: Morphologically, all of the neoplastic cells in a tumour are not identical. These structural differences may be due to differential protein expression and cellular differentiation, in turn the result of either differential expression of wild type genes, post-transcriptional modification or expression of altered genes (mutation, fusion genes etc). Nuclear morphology varies at least in part due to alteration in chromosome structure and number. Genetically, tumour cell populations are heterogeneous, as mentioned above but also it can be demonstrated that there may be heterogeneity related to driver mutations and other functionally important changes which may not necessarily be deterministic of cell morphology. Compositely speaking, tumours are made up of more than just neoplastic cells; stromal cells, immune cells and vasculature for example may account for much of the tumour bulk. Potentially, all three of these may impact upon molecular testing practice in the initial diagnostic phase and at re-biopsy. Molecular testing may be executed in many different ways and may seek many different molecular changes, such that the potential for heterogeneity making an impact on testing is considerable. Some molecular tests involve the morphological examination of a histological or cytological slide for the presence or absence of a particular factor. Proteins are normally assessed at a morphological level using immunohistochemistry. In situ hybridisation can be used to visualise and assess the presence of specific mRNAs. The same technique is used to assess DNA; specific gene copy numbers (gene amplification, polysomy), the creation of new ‘fusion genes’ during rearrangement using break-apart probes etc. These techniques require the molecular signal to be visualised in the cells of interest (usually the tumour cells); morphological and compositional tumour heterogeneity greatly impact the ease with which these techniques are executed. In lung cancer molecular testing, most current interest is in mutation testing. Compositional heterogeneity is a significant practical issue and drives recommendations that samples are pathologically assessed before extraction and mandates steps be taken to maximize the proportion of the sample for extraction that is tumour (macro- or microdissection techniques are often used). The dilution of mutant alleles by wild type alleles from non-neoplastic tissue may lower the mutation allele frequency below the threshold for detection. Are therapeutically important mutations such as those in exons 18-21 of EGFR heterogeneously expressed in tumour cells? This remains a matter of some controversy. Some have argued that since these are addictive driver mutations, they are present from the start of tumourigenesis and therefore present in every tumour cell, as determined by clonal expansion of the neoplastic cell population. Studies which demonstrate mutations in some areas of extracted tumour but not in others, are criticized by failing to use sufficiently sensitive techniques to detect mutations which are over-diluted by non-neoplastic DNA. It is known that selective amplification of mutant alleles (MASI) is heterogeneous in tumours and this may lead to apparent heterogeneity of mutation (detectable in some areas and not in others) when the number of mutant alleles per tumour cell varies in different parts of the tumour, and those areas with fewer mutant alleles are not detected due to poor test sensitivity. This explanation for apparent mutational heterogeneity has been challenged by some studies, however, which have appeared to demonstrate heterogeneity, even when highly sensitive techniques are used. Heterogeneity appears to be associated with lower response rates to EGFR TKIs in EGFR mutant tumours. Discrepancy has been reported in mutational findings between synchronous primary tumour and metastatic deposits. These findings are not universal for EGFR mutations, but when present, tend to involve a mutated primary with wild type metastases more often than the reverse. Data are few but could influence biopsy strategies. More than one mutation may be present in a lung cancer. In the context of molecular aberrations commonly tested for (EGFR, KRAS mutation; ALK rearrangement), double mutations are described but are rare. It is rather more common, for example for double or even triple EGFR mutations to be found in the same tumour sample. For example, in the author’s laboratory, double EGFR mutations are found in 13.8% of EGFR-mutated cases; triple mutations in 0.6%. KRAS double mutations are exceptionally rare (0.8%). The presence of more than one mutation, often at different allelic frequencies (such as can be estimated in many studies), implies different clones of cells bearing different mutations, and from this comes the concept of minor clones of therapeutically resistant cells which are responsible for some, though probably not all disease recurrences on TKI therapy. The best known scenario fitting this ‘minor clone’ hypothesis is the emergence of tumour bearing the EGFR T790M resistance mutation, as well as the original sensitizing mutation, for which an EGFR TKI was given. Resistant minor clones of MET amplified cells may be an alternative source of recurrent, EGFR TKI resistant disease. Similarly with ALK mutated or KRAS mutated recurrences during ALK TKI therapy for ALK rearranged tumours. This increasingly recognised outcome in patients treated with EGFR or ALK TKIs is now driving re-biopsy of recurrent disease into standard of care. Testing approaches and strategy for recurrent disease are still evolving and are driven by this concept of minor clone heterogeneity. Another finding in the re-biopsy setting is histological subtype transformation. Whilst the initial EGFR or ALK altered tumour is almost always adenocarcinoma, recurrent disease may be small cell, sarcomatoid or even squamous cell carcinoma. Little is known about the mechanism of this transformation; emergent clones of different histology or differential stem cell differentiation? There are also emergent data demonstrating that where recurrence occurs at multiple sites, detectable resistance mechanisms may vary. In a broader sense, heterogeneity of sensitivity to particular therapies, amongst tumour cells, is a major driver of treatment resistance and/or relapse, and effectively why there are so very few instances of true cure of lung cancer as a consequence of systemic therapy. The development of effective treatment strategies to overcome recurrence will require a better understanding of how tumour heterogeneity influences this process.

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      MS05.03 - Genomic Evolution and Tumor Heterogeneity (ID 1866)

      15:00 - 15:20  |  Author(s): D. Sidransky, E. Izumchenko, M. Brait, W. Westra

      • Abstract
      • Presentation
      • Slides

      Abstract:
      Adenocarcinomas represent the most frequent subtype of lung cancer[1], and they are usually discovered late in the course of the disease even in the setting of vigilant radiographic and cytologic screening[2]. Despite improvements in molecular diagnosis and targeted therapies, the average 5 year-survival rate for lung adenocarcinoma remains only 15%[3]. Novel strategies based on the detection of genetic markers offer new hope for improved risk assessment, early cancer detection, therapeutic intervention and tumor surveillance, but the impact of these strategies has been limited by an incomplete understanding of the biology of lung cancer, particularly in its early developmental stages. Disappointingly, relatively few genetic alterations critical to the development of lung adenocarcinomas are currently recognized, and the timing and manner by which these alterations initiate and drive glandular neoplasia remains to be delineated. Recent refinements in the histologic classification of lung adenocarcinomas provide greater resolution of the sequential steps of glandular lung neoplasia[4]. Atypical adenomatous hyperplasia (AAH) is a microscopic discrete focus of cytologically atypical type II pneumocytes and/or Clara cells[5-6]. Once dismissed as a reactive change, AAH is now regarded as the first histologic step in a morphologic continuum culminating in the fully malignant adenocarcinoma. The link between AAH and invasive adenocarcinoma is strong and compelling: 5-20% of lungs resected for primary adenocarcinomas also harbor AAH, and AAH harbors some of the same genetic and epigenetic alterations found in adenocarcinomas including KRAS mutations, EGFR mutations, loss of heterozygosity at 9q and 16p, TP53 mutations, and epigenetic alterations in the WNT pathway. Like AAH, adenocarcinoma in situ (AIS) (formerly known as bronchioloalveolar carcinoma, BAC) is recognized as a non-invasive form of glandular neoplasia, but one that exhibits increased size, cellularity and morphologic atypia. In effect, it represents a next step in the continuum towards malignant adenocarcinoma. Minimally-invasive adenocarcinoma (MIA) is defined as a small adenocarcinoma (≤ 3cm) with a predominantly lepidic pattern and invasion of 5 mm or less in any one focus[4]. Invasive growth is present, albeit so limited that these carcinomas have been associated with 100% disease free survival[7,8]. This enhanced delineation of early glandular neoplasia provides a rational histologic framework for studying the timing of genetic alterations driving the early stages of lung tumorigenesis. “Branched evolutionary tumor growth” is the concept that cancers evolve by a repetitive process of clonal expansion, genetic diversification and clonal selection within the adaptive landscapes of tissue ecosystems[9]. In this study, to determine whether this phenomenon is operational during early stages of tumor progression, we evaluated lung glandular neoplasms spanning the full spectrum of early histologic progression using next generation sequencing (NGS) of coding regions from 125 well-characterized cancer-driving genes. We specifically targeted multifocal AAHs and advancing zones of histologic progression within individual AISs and MIAs. This multi-region sequencing revealed that clonal expansion is an early event that can be confirmed even in the earliest recognized step in glandular neoplasia. Moreover, the identification of significant genetic alterations such as KRAS mutations, loss of P53 activity and EGFR activation points to the presence of functionally relevant “drivers” that empower territorial expansion of subclones en route to malignancy. Importantly, these driver alterations are potentially measurable in clinical samples. Using ultra-sensitive droplet digital PCR (ddPCR), mutant DNA associated with early lesions was detected in a patient’s plasma and sputum providing proof of principle that even the earliest stages of glandular neoplasia can be detected via analysis of circulating DNA (circDNA). Our study provides the unique insight into the genetic alterations that initiate and drive the progression of lung glandular neoplasia and underlines the need for precise definition of these events to improve proper diagnosis and early detection of tumors. Identification of mutational features which characterize relevant lesions that actually progress to cancers will allow to better predict the fate of these early lesions and tailor the right therapy to prevent the progression. 1. Colby T. V., Koss M. N. & W., T. in Tumors of the Lower Respiratory Tract (eds Colby T. V., Koss M. N., & Travis W. D.) 91-106 (Armed Forces Institute of Pathology Washington DC, 1994). 2. Frost, J. K. et al. Early lung cancer detection: results of the initial (prevalence) radiologic and cytologic screening in the Johns Hopkins study. The American review of respiratory disease 130, 549-554 (1984). 3. Imielinski, M. et al. Mapping the hallmarks of lung adenocarcinoma with massively parallel sequencing. Cell 150, 1107-1120, doi:10.1016/j.cell.2012.08.029 (2012). 4. Travis, W. D. et al. Diagnosis of lung adenocarcinoma in resected specimens: implications of the 2011 International Association for the Study of Lung Cancer/American Thoracic Society/European Respiratory Society classification. Archives of pathology & laboratory medicine 137, 685-705, doi:10.5858/arpa.2012-0264-RA (2013). 5. Weng, S. Y., Tsuchiya, E., Kasuga, T. & Sugano, H. Incidence of atypical bronchioloalveolar cell hyperplasia of the lung: relation to histological subtypes of lung cancer. Virchows Archiv. A, Pathological anatomy and histopathology 420, 463-471 (1992). 6. Chapman, A. D. & Kerr, K. M. The association between atypical adenomatous hyperplasia and primary lung cancer. British journal of cancer 83, 632-636, doi:10.1054/bjoc.2000.1317 (2000). 7. Borczuk, A. C. et al. Invasive size is an independent predictor of survival in pulmonary adenocarcinoma. The American journal of surgical pathology 33, 462-469, doi:10.1097/PAS.0b013e318190157c (2009). 8. Yim, J. et al. Histologic features are important prognostic indicators in early stages lung adenocarcinomas. Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc 20, 233-241, doi:10.1038/modpathol.3800734 (2007). 9. Greaves, M. & Maley, C. C. Clonal evolution in cancer. Nature 481, 306-313, doi:10.1038/nature10762 (2012).

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      MS05.04 - Hetergeneity and Drug Resistance (ID 1867)

      15:20 - 15:40  |  Author(s): S. Peters

      • Abstract
      • Presentation

      Abstract not provided

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Author of

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    ED 09 - Tissue Is the Issue: Improving Diagnostic Yield in the Age of Minimally Invasive Procedures (ID 9)

    • Event: WCLC 2015
    • Type: Education Session
    • Track: Community Practice
    • Presentations: 1
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      ED09.01 - Optimal Biopsy, Challenges (ID 1806)

      14:20 - 14:40  |  Author(s): S. Dacic

      • Abstract
      • Presentation
      • Slides

      Abstract:
      The majority of patients with lung cancer present in advanced stages, and small biopsy and cytology specimens most frequently provide the only tumor material for diagnosis. Furthermore, the same sample is also needed for molecular studies that guide treatment and management. Lung carcinomas often are diagnosed by minimally invasive techniques and specimens are obtained by bronchoscopic or transthoracic approaches. The choice of the procedure depends on the location, accessibility of the mass and other clinical parameters. Since most adenocarcinomas are peripherally located, the transthoracic approach is often chosen to provide diagnostic samples. Squamous cell carcinoma is most frequently centrally located and the bronchoscopic approach is more common. If cytology specimens, including pleural fluids, are obtained, cell blocks should be prepared. Despite the best efforts, the lesional tissue or the most representative area of the tumor may not be obtained in some cases due to sampling issues. Even when the tumor is sampled, poor tumor differentiation or insufficient characteristic morphological features in the tumor sample will cause the difficulty in rendering a specific diagnosis and would prompt pathologists to use immunohistochemistry. Use of immunohistochemistry greatly reduced the number of lung carcinoma cases classified as non-small cell carcinoma, NOS to only 3%. .In addition to immunohistochemistry, cytology samples should be interpreted in conjunction with histology of small biopsies whenever possible. Immunohistochemistry should be limited only to cases when classification is uncertain and every effort should be made to preserve as much material as possible for molecular studies. There are few strategies besides limited immunohistochemistry panels how this could be accomplished. One approach is to cut unstained slides from a paraffin block after initial hematoxylin-eosin stain sections were obtained. It is essential that histology technicians limit facing of the block and place only one tissue section per slide. Another approach is to have each core biopsy tissue fragment placed into separate blocks during specimen processing so only one block can be used for immunohistochemistry and all of the blocks can be used for molecular studies. Same laboratories in order to avoid tumor microdissection from the unstained slides prefer to core paraffin blocks by 1-mm needles after diagnostic work up. Formalin-fixed, paraffin-embedded tissue samples and cytology aspirates can be used for various testing platforms including next generation sequencing. Each molecular laboratory should establish criteria for specimen adequacy for molecular studies taking into account the specific testing platforms, while surgical pathologists should assess the specimen adequacy. Although PCR-based methods can detect mutations from a single cell, a low copy number DNA template can generate sequence artifacts leading to false results. Therefore, the assessment of adequacy is essential to avoid assays failures and false positive/negative results. Estimates of tumor content from H&E stained sections vary between pathologists and there is no true standard. Acceptable specimens should have a sufficient amount of tumor cells, but also a small proportion of admixed non-neoplastic cells, and no necrosis.[116] If the specimen is inadequate, a new specimen needs to be procured although in this situation decision regarding specimen type is often difficult and depends on many factors including the patient’s health. Alternate non-invasive highly sensitive methods so called “liquid biopsies” have been developed to detect the presence of cancer specific mutations in circulating DNA in blood samples. This approach may result in significant changes in the management of lung cancer patients and may replace invasive procedures. Until then, it is essential that each institution develops its own strategy that addresses the collection and processing of lung cancer samples. At the same time, pathology departments must implement the procedures that would precisely define how to spare the tumor tissue for molecular testing, and how to provide clinically acceptable turnaround time for molecular testing results. It is also essential to define how to integrate diagnostic interpretation and molecular results in a single pathology report. References: Travis WD et al. Diagnosis of lung cancer in small biopsies and cytology: implications of the 2011 International Association for the Study of Lung Cancer/American Thoracic Society/European Respiratory Society classification. Arch Pathol Lab Med 2013; 137(5):668-84. Lindeman NI. et al. Molecular testing guideline for selection of lung cancer patients for EGFR and ALK tyrosine kinase inhibitors: guideline from CAP/IASLC/AMP. J Thorac Oncol 2013:8(7):823-59. Francis G, Stein S.Circulating cell-free tumor DNA in the management of cancer. Int J Mol Sci. 2015;1 (6):14122-42

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