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L. Sholl
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MINI 02 - Immunotherapy (ID 92)
- Event: WCLC 2015
- Type: Mini Oral
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:P. Forde, S.J. Antonia
- Coordinates: 9/07/2015, 10:45 - 12:15, Four Seasons Ballroom F3+F4
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MINI02.01 - Pulmonary Large Cell Carcinoma and Solid Adenocarcinoma Are Highly Mutated with Frequent Expression of PDL1 (ID 2257)
10:45 - 12:15 | Author(s): L. Sholl
- Abstract
- Presentation
Background:
Large cell carcinoma (LCC) is an uncommon lung tumor that arises predominantly in smokers and shares many features of solid adenocarcinoma (ADC). 40% of LCC/solid ADC harbor mutations in KRAS; EGFR and ALK alterations are rare in this tumor type. The majority of these tumors, however, lack one of the commonly queried oncogenic driver alterations, thus therapeutic options are limited for patients with this tumor type. Immunomodulatory therapies, including targeting PDL1, have shown promise in a variety of tumor types. Tumor neo-antigens, including those induced by smoking, are associated with mutational burden and may predict susceptibility to cytolytic immune response; in addition, high PDL1 expression in non small cell lung carcinoma has been associated with response to anti-PDL1 drugs. Given the high prevalence of smoking in patients with LCC and solid ADC, we hypothesize that these tumors may be amenable to immunomodulatory therapy and sought to define the frequency of PDL1 expression in tumors lacking an oncogenic driver mutation.
Methods:
This study was restricted to 27 LCC and solid ADC known to be negative for KRAS, EGFR, ALK and ROS1 alterations. Hybrid capture targeted next generation sequencing (NGS) on an Illumina HiSeq 2500 was performed using a cancer genomic assay to detect mutations, copy number variations (CNVs) and structural variants. The assay captures exonic sequences of 275 cancer genes and 91 introns across 30 genes for rearrangement detection. Findings were compared to an institutional cohort of 732 consecutive lung tumors sequenced on the same platform. Immunohistochemistry for PDL1 was performed using a rabbit monoclonal antibody (Cell Signaling Technologies) at 1:100 dilution following pretreatment with citrate buffer/pressure cooker and detected using the Envision + polymer system (DAKO). Immunostaining was considered positive in the tumor component or the inflammatory component if ≥5% of the cells showed membranous staining.
Results:
Of the 27 tumors tested, 26 were resected from smokers. NGS revealed an average of 14.9 mutations per case for LCC/solid ADC cohort versus 8.1 mutations in the overall cohort of lung tumors (p<0.0001). 11 cases (41%) were positive for PD-L1. 7 cases (26%) showed strong, diffuse staining (≥70% of cells) for PD-L1. The inflammatory component was positive for PD-L1 in 25 cases (93%). Two cases with strong expression of PD-L1 by immunohistochemistry (>90% of cells) showed focal amplification of CD274 by NGS.
Conclusion:
LCC and solid ADC are strongly associated with a smoking history and harbor a significantly higher average mutational burden than other lung tumors. 41% of LCC/solid ADC are positive for PDL1 by immunohistochemistry with 26% showing very strong PDL1 expression and nearly all cases showing some degree of positivity in the associated inflammatory infiltrate. In some cases, high PDL1 expression is associated with focal amplification of CD274, the gene encoding PDL1. These findings suggest that LCC/ solid ADC is likely to have smoking-associated neo-antigen expression and that PDL1-directed immunotherapies may be a promising therapeutic approach in this otherwise poorly-characterized lung tumor.
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MINI 13 - Genetic Alterations and Testing (ID 120)
- Event: WCLC 2015
- Type: Mini Oral
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 2
- Moderators:Y. Koh, R.K. Thomas
- Coordinates: 9/08/2015, 10:45 - 12:15, 205+207
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MINI13.03 - Characterization of MET Gene and MET Protein Expression in Lung Cancer (ID 2155)
10:45 - 12:15 | Author(s): L. Sholl
- Abstract
- Presentation
Background:
Activation of the MET signaling pathway can propel the growth of cancer cells in non-small cell lung cancer (NSCLC). Increased MET gene by amplification and/or polysomy can cause MET protein overexpression; less common causes include mutations, translocations, and alternative RNA splicing. Clinical trials using MET as a biomarker for selection of lung cancer patients who might most benefit from targeted therapy have experienced variable outcomes. We aimed to characterize the relationship between MET protein overexpression and MET amplification or mean copy number alterations in patients with NSCLC.
Methods:
The Lung Cancer Mutation Consortium (LCMC) is performing an ongoing study of biomarkers with patients with NSCLC from 16 cancer center sites across the United States. For this analysis, 403 cases had complete data for MET protein expression by immunohistochemistry (IHC, monoclonal antibody SP44, Ventana) and MET gene amplification by fluorescence in-situ hybridization (FISH, MET/CEP7 ratio). Pathologists evaluated MET expression using the H-score, a semi-quantitative assessment of the percentage of tumor cells with no, faint, moderate, and/or strong staining, ranging from 0-300. Spearman's correlation was used to analyze the correlation between MET protein expression (H-scores) and FISH results (MET/CEP7 ratio (N=403) and MET copy number (N=341). Protein overexpression using 5 different cut-offs was compared with amplification defined as MET/CEP7 ≥ 2.2 and high mean copy number defined as ≥ 5 MET gene copies per cell using the Fisher’s exact test. Cox Proportional Hazards models were built to examine the associations of these different definitions of positivity with prognosis, adjusting for stage of disease.
Results:
MET protein expression was significantly correlated with MET copy numbers (r=0.17, p=0.0025), but not MET/CEP7 ratio (r=-0.013, p=0.80). No significant association was observed between protein overexpression using a commonly used definition for MET positivity (“at least moderate staining in ≥ 50% tumor cells”) and MET amplification (p=0.47) or high mean copy number (p=0.09). A definition for MET protein overexpression as “≥ 30% tumor cells with strong staining” was significantly associated with both MET amplification (p=0.03) and high mean copy number (p=0.007), but a definition of “≥ 10% tumor cells with strong staining” was not significantly associated with either. Definitions of protein overexpression based on high H-scores (≥200 or ≥250) were associated with high MET mean copy numbers (p=0.03 and 0.0008, respectively), but not amplification (p=0.46 and 0.12, respectively). All 5 definitions of MET protein overexpression demonstrated a significant association with worse prognosis by survival analyses (p-values ranged from 0.001 to 0.03). High MET copy number (p=0.045) was associated with worse prognosis, but MET amplification was not (p=0.07).
Conclusion:
Evaluation of NSCLC specimens from LCMC sites confirms that MET protein expression is correlated with high MET copy number and protein overexpression is associated with worse prognosis. Definitions of MET protein overexpression as “an H-score ≥250” and “≥30% tumor cells with strong staining” were significantly associated with high mean MET copy number. It may be worth reevaluating the performance of MET as a biomarker by different definitions of positivity to predict response to MET-targeted therapies.
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MINI13.15 - Discussant for MINI13.11, MINI13.12, MINI13.13, MINI13.14 (ID 3340)
10:45 - 12:15 | Author(s): L. Sholl
- Abstract
- Presentation
Abstract not provided
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MTE 24 - Rising to the Challenge of Testing Small Biopsy Specimens (Ticketed Session) (ID 76)
- Event: WCLC 2015
- Type: Meet the Expert (Ticketed Session)
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:
- Coordinates: 9/09/2015, 07:00 - 08:00, 105
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MTE24.01 - Rising to the Challenge of Testing Small Biopsy Specimens (ID 2011)
07:00 - 08:00 | Author(s): L. Sholl
- Abstract
- Presentation
Abstract:
The clinical benefits of biomarker-driven targeted cancer therapy are clear in many tumor types, particularly in lung adenocarcinoma. At this time, use of targeted agents is predicated on access to tumor tissue; in lung cancer patients this tissue is often limited in quantity, a product of minimally invasive procedures using narrow-gauge biopsy needles intended to reduce complications. Current clinical practice has forced pathology labs to revisit approaches to tissue handling, diagnosis, and molecular platform selection. Coordinated efforts are critical to optimizing the handling of small biopsies, from the time of request, to the actual procedure, to receipt in the pathology lab. Ideally, the requesting physician clearly communicates the need for genomic studies to both the operator, who may consider the need/feasibility of multiple biopsies, and pathologist, who may perform rapid on site evaluation. Interventional techniques designed to improve the chances of obtaining material amenable to molecular diagnostics should be implemented whenever possible, including combined core biopsy and fine needle aspiration,[1]and preferential sampling of soft tissue components of bony metastases when possible, in order to avoid the need for specimen decalcification. In the future, in vivo microscopy may be used to guide the biopsy location.[2] Routine histology practices, such as cutting slides from a paraffin block in an iterative fashion only after a pathologist’s review, often requires “refacing” to optimize the plane of section and can waste valuable tumor tissue. The need for genomics studies should be clearly indicated to the receiving pathology laboratory, thereby facilitating histology protocols designed to conserve tissue. Such protocols may include embedding each tissue core in an individual block, delivering “up-front” unstained sections for use in immunohistochemistry and molecular studies, and/or triaging slides to a dedicated molecular diagnostics workflow. As an example, from a single 18-gauge core needle biopsy containing 30% tumor, 15-20 unstained slides can typically generate enough material to perform a diagnostic workup, immunohistochemistry and/or FISH for ALK and ROS1 rearrangements, and adequate DNA for hybrid capture next generation sequencing.[3]With careful histology embedding and sectioning, material will still remain in the block for future studies. International guidelines have recognized the centrality of molecular testing to the clinical management of lung cancer patients and have responded with recommendations for judicious use of immunohistochemical studies in pathology workups and introduced new diagnostic categories intended to eliminate ambiguous classifications, particularly for small biopsy specimens.[4]These guidelines advise against use of the term “non small cell lung carcinoma” whenever possible and recommend use of TTF-1 and p63 or p40 immunohistochemical stains as first line markers for distinguishing between adenocarcinoma and squamous cell carcinoma.[5] Simultaneous evaluation of fine needle aspirates and core biopsies with an optimized IHC protocol may significantly reduce rates of the NSCLC diagnosis and generate additional “testable” material.[6] In institutions that use touch imprints for rapid evaluation of core needle biopsies, careful specimen handling is required to maintain tumor cell adequacy in the needle biopsy specimen.[7] When cytology material is available for testing, cell blocks are preferred because a physical record of the sample can be retained in the form of a stained glass slide.[4] However, in many circumstances the most cellular material is in the form of a smear; these preps can generate abundant and high quality DNA and so should be considered for use in molecular testing, particularly if the slide can be scanned and a digital image archived. Validation of alternative specimens such as cytology smears for FISH can provide additional tissue for in situ assays when core needle or cell block specimens are inadequate.[8] In the molecular diagnostics lab, a plethora of testing platforms are available, many of which are evolving to accept low DNA inputs. Many targeted amplicon-sequencing based assays can accept as little as 10ng of input DNA, whereas hybrid capture approaches typically require around 50ng or more for targeted sequencing panels and whole exome sequencing. However, in today’s practice, it is rare that a single test can provide comprehensive analysis of all the desired targets; indeed, confirmation of individual events detected by sequencing by FISH or immunohistochemistry may be needed. Therefore, an interdisciplinary effort to optimize tumor sampling and conserve tissue is required to ensure successful and comprehensive molecular profiling of lung tumors. References 1. Poulou LS, Tsagouli P, Ziakas PD, Politi D, Trigidou R, Thanos L. Computed tomography-guided needle aspiration and biopsy of pulmonary lesions: A single-center experience in 1000 patients. Acta Radiol. 2013;54(6):640-645. 2. Hariri LP, Mino-Kenudson M, Applegate MB, et al. Toward the guidance of transbronchial biopsy: Identifying pulmonary nodules with optical coherence tomography. Chest. 2013;144(4):1261-1268. 3. Austin MC, Smith C, Pritchard CC, Tait JF. DNA yield from tissue samples in surgical pathology and minimum tissue requirements for molecular testing. Arch Pathol Lab Med. 2015. 4. Lindeman NI, Cagle PT, Beasley MB, et al. Molecular testing guideline for selection of lung cancer patients for EGFR and ALK tyrosine kinase inhibitors: Guideline from the college of american pathologists, international association for the study of lung cancer, and association for molecular pathology. Arch Pathol Lab Med. 2013;137(6):828-860. 5. Travis WD, Brambilla E, Noguchi M, 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):668-684. 6. Sigel CS, Moreira AL, Travis WD, et al. Subtyping of non-small cell lung carcinoma: A comparison of small biopsy and cytology specimens. J Thorac Oncol. 2011;6(11):1849-1856. 7. Rekhtman N, Kazi S, Yao J, et al. Depletion of core needle biopsy cellularity and DNA content as a result of vigorous touch preparations. Arch Pathol Lab Med. 2015;139(7):907-912. 8. Betz BL, Dixon CA, Weigelin HC, Knoepp SM, Roh MH. The use of stained cytologic direct smears for ALK gene rearrangement analysis of lung adenocarcinoma. Cancer Cytopathol. 2013;121(9):489-499.
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ORAL 17 - EGFR Mutant Lung Cancer (ID 116)
- Event: WCLC 2015
- Type: Oral Session
- Track: Treatment of Advanced Diseases - NSCLC
- Presentations: 1
- Moderators:P. Meldgaard, E. Felip
- Coordinates: 9/08/2015, 10:45 - 12:15, Four Seasons Ballroom F3+F4
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ORAL17.07 - Mechanisms of Acquired Resistance to AZD9291 in EGFR T790M Positive Lung Cancer (ID 1365)
10:45 - 12:15 | Author(s): L. Sholl
- Abstract
Background:
AZD9291 is an irreversible, mutant-selective epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) developed to have potency against both EGFR-sensitizing mutations and T790M. In the ongoing Phase I study of AZD9291 (AURA, NCT01802632), the response rate in patients with T790M positive lung cancer with disease progression on previous EGFR-TKI was >60%, with a preliminary median progression-free survival of >10 months. The molecular mechanisms underlying acquired resistance to AZD9291 are currently under investigation.
Methods:
Plasma genotyping was performed on patients from AURA who had progressed on AZD9291 if they had detectable T790M pre-AZD9291, as assessed by tumor or plasma genotyping, and if they had plasma collected at progression available for analysis. Cell-free DNA (cfDNA) was extracted from plasma taken at progression. Droplet digital PCR (ddPCR) was performed for EGFR exon 19 deletions, L858R, T790M, and C797S. For further exploration, next-generation sequencing (NGS) of an amplicon panel was performed on available progression cfDNA. Lastly, targeted NGS was performed on available resistance biopsy specimens.
Results:
Plasma specimens were available following disease progression on AZD9291 from 40 patients with tumors positive for T790M through tumor (33) or plasma genotyping (7). Twenty-six progression cfDNA specimens were positive for an EGFR-sensitizing mutation by ddPCR, and were deemed eligible for initial resistance analysis. Of these, 12 (46%) had no detectable T790M in plasma despite presence of the EGFR-sensitizing mutation, suggesting overgrowth of an alternate resistance mechanism. Seven patients had detectable C797S on ddPCR (27%), all with detectable T790M; of 14 with detectable T790M at resistance, C797S was only detected with EGFR exon 19 deletions (7/9) and not L858R (0/5, p=0.02). Plasma NGS was performed on 12 cases with acquired resistance that were T790M positive pretreatment. Exon 19 deletion/T790M/C797S were detected in four cases, with two of these harboring two different DNA mutations encoding for C797S. One case lost T790M and exhibited HER2 copy number gain (6.3 copies); a tumor biopsy from a separate case underwent aCGH at Institute Gustave Roussy and was also found to have focal HER2 amplification with loss of T790M. Targeted NGS was performed on resistance biopsies from a total of 10 patients from four centers with T790M positive biopsies pre-AZD9291. Six cases maintained T790M, with three harboring exon 19 del/T790M/C797S. In four cases with loss of T790M, one harbored BRAF V600E and one harbored PIK3CA E545K.
Conclusion:
Complementary genomic analysis of plasma and tumor DNA provides insight into the diverse molecular mechanisms of acquired resistance to AZD9291 in EGFR-mutant lung cancer. Our studies show that a majority of cases maintained T790M at resistance, at times acquiring a new C797S mutation in those with EGFR exon 19 deletion. Loss of T790M at progression may be mediated by overgrowth of cells harboring HER2 amplification, BRAF V600E, or PIK3CA mutations. These data highlight the need for investigation of combination therapies to effectively prevent or treat the complexity of drug resistance in EGFR-mutant lung cancer.
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ORAL 37 - Novel Targets (ID 146)
- Event: WCLC 2015
- Type: Oral Session
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:S.S. Ramalingam, E. Thunnissen
- Coordinates: 9/09/2015, 16:45 - 18:15, Mile High Ballroom 4a-4f
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ORAL37.07 - Lung Cancer Mutation Consortium Pathologist Panel Evaluation of MET Protein (ID 2129)
16:45 - 18:15 | Author(s): L. Sholl
- Abstract
- Presentation
Background:
MET is a receptor tyrosine kinase with frequently activated signaling in lung cancers. Multiple studies indicate that MET overexpression correlates with poor clinical prognosis. Tumors with MET amplification and overexpression may respond better to MET inhibitors than tumors with low expression. The prevalence of MET overexpression in lung cancer cohorts has varied from 20%-80%, as has the proportion of patient’s testing positive for prospective clinical trials with entry based on MET overexpression. The Lung Cancer Mutation Consortium (LCMC) Pathologist Panel endeavored to standardize evaluation of MET protein expression with “Round Robin” conferences.
Methods:
508 FFPE non-small cell lung cancer specimens were stained by immunohistochemistry for MET protein expression (SP44 antibody, Ventana). Seven pathologists from LCMC sites with specialized training in MET scoring evaluated 78 Aperio-scanned images of MET-stained slides in two successive rounds of 39 different cases per round. The percentage of tumor cells with membranous and/or cytoplasmic staining at different intensities were evaluated with H-scores ranging from 0 to 300. Overall group and individual pathologist’s scores were compared with intraclass correlation coefficients (ICCs). Between rounds, a “Round Robin” teleconference was conducted to review discordant cases and improve consistency of scoring. Steps to improve scoring included: review of a Roche MET training document, sharing pictures of cases with concordant scores (Figure 1), and provision of H&E images for the second round to facilitate identification of tumor areas. Figure 1
Results:
The overall average MET H-score for the 78 cases was 165.3 (H-score range: 42.5-279.7). The average H-score was <125 for 14 specimens, 125-175 for 35 specimens, and >175 for 29 specimens. The overall group ICC comparing the consistency of H-scores from all 7 pathologists improved from 0.50 (95% confidence interval: 0.37-0.64, “fair” correlation) for the first scoring round to 0.74 (95% confidence interval: 0.64-0.83, “good” correlation) for the second round. A comparison of the individual pathologist’s ICCs demonstrated improved individual scoring consistency for all seven pathologists between rounds with an average of 0.64 (“moderate” correlation, range 0.43-0.76) for the first round and 0.82 (“almost perfect” correlation, range 0.75-0.93) for the second round.
Conclusion:
Development of standardized, reproducible strategies for evaluation of complex biomarkers, such as MET, are critical to clinical trial design. The consistency of scoring for MET protein expression and other biomarkers may be improved by continuous training and communication between pathologists with easy access to H&E images and other visual aids.
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