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A. Moreira
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MINI 01 - Pathology (ID 93)
- Event: WCLC 2015
- Type: Mini Oral
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:W.A. Franklin, A.G. Nicholson
- Coordinates: 9/07/2015, 10:45 - 12:15, Mile High Ballroom 2c-3c
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MINI01.07 - Comparison of Grading Systems Based on Histologic Patterns and Mitotic Activity to Predict Recurrence in Stage I Lung Adenocarcinoma (ADC) (ID 3030)
10:45 - 12:15 | Author(s): A. Moreira
- Abstract
- Presentation
Background:
An established grading system for lung adenocarcinoma does not exist but is greatly needed. The histologic classification proposed by the International Association for the Study of Lung Cancer (IASLC), the American Thoracic Society (ATS) and the European Respiratory Society (ERS) has been shown to define prognostically significant subgroups of lung adenocarcinoma (ADC). Since then, various grading systems based on histologic patterns have emerged as promising methods to further discriminate patient risk of clinical outcomes. The aim of this work is to quantitatively assess the discrimination properties of a set of grading systems proposed in recent years to identify the best grading scale(s) independent of other clinical factors to predict recurrence.
Methods:
We considered five grading systems: (1) single predominant pattern as six subtypes; (2) as three grades of low (lepidic), intermediate (acinar, papillary) and high (micropapillary, solid); (3) two most predominant grades; (4) predominant grade with mitotic grade; and (5) predominant grade with cribriform pattern and mitotic activity criteria. We evaluated the performance of each grading system with the concordance predictive estimate (CPE). The CPE represents the probability that for any pair of patients, the patient with the better predicted outcome from the Cox model had the longer survival time. CPE > 0.80 demonstrates strong performance. To compare the performance of the grading systems, we determined the significance of the differences between the CPEs. Five-year recurrence-free probability (RFP) was derived using the Kaplan-Meier method.
Results:
We applied the grading systems to a uniform large cohort of stage I lung ADC (N=909). The scale based on the single predominant pattern as five subtypes yielded a CPE of 0.63 (95% CI, 0.59-0.67), indicating moderate discrimination. Our analysis showed that grading systems (1), (2), and (3) were not significantly different from each other, suggesting that identifying finer subtypes and second predominant pattern may not improve discrimination. Grading system (4) [CPE, 0.67; 95% CI, 0.63-0.71] yielded a significantly higher CPE than (1), (2) and (3) [p<0.01]. Grading system (5) [CPE, 0.67; 95% CI, 0.63-0.71] was significantly better than (1), (2) and (3) but not (4) [p=0.776]. The lack of improvement in discrimination with the inclusion of cribriform between (4) and (5) can be attributed to the significant relationship between cribriform pattern and mitoses. As the proportion of cribriform pattern increased, the amount of mitotic activity also increased (p<0.001). Under (2), the 5-year RFP of the intermediate grade was 0.81. The addition of cribriform and mitotic counts further classified the intermediate (acinar, papillary) grade such that those with <10% cribriform and low mitotic count had 5-year RFP of 0.89, while the 5-year RFP for the other combinations are between 0.73-0.75.
Conclusion:
Grading systems based on histologic patterns and mitotic activity out-perform those with only histologic pattern. This comparison study suggests that proposed grading systems (4) or (5) provide valuable information in discriminating patients with different risks of disease-recurrence in patients with lung ADC.
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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: 1
- 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): A. Moreira
- 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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ORAL 25 - Biology and Other Issues in SCLC (ID 125)
- Event: WCLC 2015
- Type: Oral Session
- Track: Small Cell Lung Cancer
- Presentations: 1
- Moderators:J. Sage, L. Montuenga
- Coordinates: 9/08/2015, 10:45 - 12:15, 605+607
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ORAL25.07 - DNA Methylation in Small Cell Lung Cancer Defines Distinct Disease Subtypes and Correlates with High Expression of EZH2 (ID 3031)
10:45 - 12:15 | Author(s): A. Moreira
- Abstract
- Presentation
Background:
Small cell lung cancer (SCLC) is an aggressive neuroendocrine lung tumor characterized by extreme plasticity, high metastatic potential, and capacity for acquired resistance to chemotherapy. Despite significant advances in our understanding of SCLC genetics and etiology, the epigenetics of this deadly disease remain under studied. This study profiles DNA methylation in primary SCLC, patient-derived xenografts (PDX) and cell lines at single-nucleotide resolution.
Methods:
This study profiled DNA methylation at single-nucleotide resolution in 47 extensively characterized SCLC samples, including 34 fresh frozen primary SCLC tumors as well as 6 distinct primary patient-derived xenografts and 7 cell lines using the Illumina Human Methylation 450k Bead Chip array. Importantly, 24 primary SCLC in this study have previously been analyzed by whole exome sequencing and RNAseq, allowing integrated analysis of these data types with measurements of DNA methylation. We applied unsupervised clustering, discrete and locally clustered differential methylation analysis, correlation with gene expression, spacial correlation with genomic features, and interrogated the role of the EZH2 methyltransferase in SCLC using bioinformatic and pharmacologic approaches.
Results:
Unsupervised clustering of all samples revealed that PDX clustered with primary SCLC, while cell lines were easily discriminated. We explored this phenomenon further and found that while the top differentially methylated CpGs in both PDX and cell lines were >80% concordant with primary SCLC, only PDX maintained high concordance across larger probe lists. Unsupervised clustering of primary SCLC revealed three distinct subgroups at both the DNA methylation and gene expression levels that correlated with expression of the neurogenic transcription factors ASCL1 and NEUROD1. The chromatin modifier EZH2 was expressed >12-fold higher in SCLC than in normal lung. In addition to the high expression observed in SCLC compared to normal lung, we observed a significant correlation between median EZH2 gene expression and promoter methylation using data from The Cancer Genome Atlas (TCGA). Overall, EZH2 expression in SCLC is greater than or comparable to that of any other tumor type represented in TCGA. EZH2 protein expression was detected by Western blot in 15/17 SCLC PDXs (88%). We assessed the efficacy of the potent EZH2 inhibitor EPZ-5687 in the LX92 SCLC PDX in vivo. EPZ-5687 was well-tolerated and demonstrated remarkable efficacy at 100 mg/kg either QD or BID.
Conclusion:
DNA methylation patterns in primary SCLC are more closely mirrored by those found in PDX, compared to cell lines, including PDX lines of very high passage. Distinct epigenetic subtypes could be observed in SCLC, even among histologically indistinguishable samples with similar mutation profiles. SCLC is notable for consistent high level DNA methylation clustered in promoters containing CpG islands. Promoter methylation in SCLC is distinct from other lung cancers and correlates strongly with high-level expression of the histone methyltransferase gene EZH2. Pharmacologic inhibition of EZH2 in a SCLC PDX markedly inhibited tumor growth. These findings point to a critical role of EZH2 in SCLC tumor biology and support further preclinical efficacy studies in models of SCLC.
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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): A. Moreira
- 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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P3.04 - Poster Session/ Biology, Pathology, and Molecular Testing (ID 235)
- Event: WCLC 2015
- Type: Poster
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 2
- Moderators:
- Coordinates: 9/09/2015, 09:30 - 17:00, Exhibit Hall (Hall B+C)
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P3.04-054 - Validation of PTEN and c-MET Status in Small Biopsy Material and Cytology for Pulmonary Adenocarcinoma (ID 2176)
09:30 - 17:00 | Author(s): A. Moreira
- Abstract
Background:
Targeted therapy in lung cancer is an expanding field. Molecular alterations in phosphatase and tensin homolog (PTEN) and c-MET (mesenchymal epithelial transition proto-oncogene) are potential therapeutic targets. Loss of PTEN expression has been associated with activation of PIK3CA/AKT/mTOR pathway and is associated with sensitivity to mTOR inhibitors. Amplification or overexpression of c-MET is associated with resistance to tyrosine kinase inhibitors and/or poor prognosis. Both PTEN and c-MET can be detected by immunohistochemistry. In this study we evaluated the concordance rate of both antibodies in biopsy material and subsequent excision of the same tumor, since small biopsy and cytology material are the only tissue available for diagnosis in patients with advanced stage. In addition since biopsy material is collected in different fixatives, we also compared the antibody expression in alcohol versus formalin fixed tumors.
Methods:
Pathology database was queried for concurrent biopsy and surgical specimens from 12/2010-7/2014. Surgical core biopsies (n=44) and cytology aspiration biopsies (n=10) with surgical specimens were reviewed to evaluate tumor histology and specimen cellularity. In addition, 8 cases of NSCLC were scrapped and collected in formalin and alcohol fixative. Immunohistochemistry with PTEN antibody (clone 138G6) and c-MET antibody (clone sp44) were performed according to manufactures’ instruction following a rigorous validation using positive and negative controls. PTEN staining was evaluated for complete loss of expression or retention (any cytoplasmic or nuclear stain). c-MET staining was evaluated for the intensityand extent of the staining. Positivity is defined as a strong membranous staining (2-3+) in more than 50% of the tumor cells.
Results:
There was a 90% (19/21) concordance for PTEN expression between biopsy and resection (k=0.76). 6 cases showed loss of expression in the biopsy, among these cases 2 were classified as retained PTEN in the excision. In both cases the excision specimen had partial loss of PTEN. Partial loss of PTEN was seen in 3 other cases with retained PTEN in biopsy. There is a 95.2% (20/21) concordance in c-MET staining (k=0.89). In the discrepant case, the biopsy was deemed positive (2+ > 50% of tumor cells), with a negative excision (1+ >70% of tumor cells). For the cases that were fixed in alcohol and formalin there was a 62% (5/8) concordance for PTEN (k=0.37). 3 cases showed loss of expression in alcohol fixed tissue but retention in formalin fixed material. There was a 37% (3/8) concordance for c-MET between the two fixatives (k=0.21). In the 5 discordant cases, c-MET expression was interpreted as negative in alcohol fixed but was considered positive in formalin fixed tissue due to variability in the intensity of staining.
Conclusion:
Despite a good correlation between biopsy and resection for both markers, our results show there is a greater possibility of discrepant results for PTEN than c-MET because of geographic heterogenetic of expression and scoring criteria. The type of fixative (alcohol vs. formalin) is associated with variability in antibody expression. Therefore evaluation of PTEN and c-MET staining in biopsy material and alcohol fixed tissue should be interpreted with caution, especially when designing clinical trials for potential therapy.
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P3.04-092 - HNF4α Is a Marker for Invasive Mucinous Adenocarcinoma (IMA) and a Prognostic Factor in Stage I Lung Adenocarcinoma (LADC) (ID 3066)
09:30 - 17:00 | Author(s): A. Moreira
- Abstract
Background:
According to the 2015 WHO classification, invasive LADC with prominent apical intra-cytoplasmic mucin and small basally oriented nuclei, formerly referred to as mucinous bronchioloalveolar carcinoma, is classified as IMA. Hepatocyte nuclear factor 4 alpha (HNF4α) is a recently recognized marker for IMA although it is also infrequently positive for other subtypes of LADC. However, the prognostic significance of HNF4α is not known. We investigated the frequency of HNF4α expression in IMA as well as non-IMA subtypes, and the prognostic significance of HNF4α in Stage I LADC.
Methods:
Slides from patients with therapy-naive, surgically resected solitary stage I LADC (1995-2009) were subtyped according to the 2015 WHO classification. Tissue microarrays were constructed from each tumor (n=793), and stained for HNF4α. HNF4α expression intensity (0-3) and distribution (1, 1%-50%; 2, 51%-100%) were summed into a total score (0-5) and dichotomized as negative (score <2) or positive (score ≥2). Comparisons were made with TTF-1 expression. Recurrence-free probability (RFP) was estimated using the Kaplan-Meier method, and multivariate analyses were performed using the Cox proportional hazards model.
Results:
32 cases were identified as IMA. Of all LADC, HNF4α was positive in 68 cases (9%) including72% (n = 23) of IMA, 6% (n = 45) of tumors with non-IMA subtypes (P < 0.001). Among non-IMA subtypes, HNF4α was positive in 6% of lepidic, 4% of papillary, 2% of micropapillary, 7% of solid, and 29% of colloid tumors. HNF4α was positive in 12% of KRAS mutant tumors while it was negative in all EGFR mutant tumors (P < 0.001). HNF4α was more frequently positive in TTF-1 negative tumors (40%) than TTF-1 positive tumors (5%; P < 0.001). The RFP for patients with HNF4α-positive tumors was significantly lower than that for patients with HNF4α-negative tumors (P = 0.002) in the entire cohort. This finding was confirmed in subgroup analysis of patients with non-IMA subtypes (P = 0.009). In multivariate analysis, HNF4α was an independent prognostic factor for recurrence (HR=1.61, 95%CI =1.27-2.02, p<0.001).
Conclusion:
HNF4α expression was significantly associated with IMA histology, negative EGFR mutation status, and TTF-1 negativity. Furthermore HNF4α was also expressed infrequently in non-IMA subtypes, however in these patients it was a significant prognostic factor.
