Simone Terra

Identification of Independent Primary Tumors and Intrapulmonary Metastases Using DNA Rearrangements in Non–Small-Cell Lung Cancer

PURPOSE Distinguishing independent primary tumors from intrapulmonary metastases in non-small-cell carcinoma remains a clinical dilemma with significant clinical implications. Using next-generation DNA sequencing, we developed a chromosomal rearrangement-based approach to differentiate multiple primary tumors from metastasis. METHODS Tumor specimens from patients with known independent primary tumors and metastatic lesions were used for lineage test development, which was then applied to multifocal tumors. Laser capture microdissection was performed separately for each tumor. Genomic DNA was isolated using direct in situ whole-genome amplification methodology, and next-generation sequencing was performed using an Illumina mate-pair library protocol. Sequence reads were mapped to the human genome, and primers spanning the fusion junctions were used for validation polymerase chain reaction. RESULTS A total of 41 tumor samples were sequenced (33 adenocarcinomas [ADs] and eight squamous cell carcinomas [SQCCs]), with a range of three to 276 breakpoints per tumor identified. Lung tumors predicted to be independent primary tumors based on different histologic subtype did not share any genomic rearrangements. In patients with lung primary tumors and paired distant metastases, shared rearrangements were identified in all tumor pairs, emphasizing the patient specificity of identified breakpoints. Multifocal AD and SQCC samples were reviewed independently by two pulmonary pathologists. Concordance between histology and genomic data occurred in the majority of samples. Discrepant tumor samples were resolved by genome sequencing. CONCLUSION A diagnostic lineage test based on genomic rearrangements from mate-pair sequencing demonstrates promise for distinguishing independent primary from metastatic disease in lung cancer.

Integrated analysis of the genomic instability of PTEN in clinically insignificant and significant prostate cancer.

Patients with clinically insignificant prostate cancer remain a major over-treated population. PTEN loss is one of the most recurrent alterations in prostate cancer associated with an aggressive phenotype, however, the occurrence of PTEN loss in insignificant prostate cancer has not been reported and its role in the separation of insignificant from significant prostate cancer is unclear. An integrated analysis of PTEN loss was, therefore, performed for structural variations, point mutations and protein expression in clinically insignificant (48 cases) and significant (76 cases) prostate cancers treated by radical prostatectomy. Whole-genome mate pair sequencing was performed on tumor cells isolated by laser capture microdissection to characterize PTEN structural alterations. Fluorescence in situ hybridization probes were constructed from the sequencing data to detect the spectrum of these PTEN alterations. PTEN loss by mate pair sequencing and fluorescence in situ hybridization occurred in 2% of insignificant, 13% of large volume Gleason score 6, and 46% of Gleason score 7 and higher cancers. In Gleason score 7 cancers with PTEN loss, PTEN alterations were detected in both Gleason pattern 3 and 4 in 57% of cases by mate pair sequencing, 75% by in situ hybridization and 86% by immunohistochemistry. PTEN loss by sequencing was strongly associated with TMPRSS2-ERG fusion, biochemical recurrence, PTEN loss by in situ hybridization and protein loss by immunohistochemistry. The complex nature of PTEN rearrangements was unveiled by sequencing, detailing the heterogeneous events leading to homozygous loss of PTEN. PTEN point mutation was present in 5% of clinically significant tumors and not in insignificant cancer or high-grade prostatic intraepithelial neoplasia. PTEN loss is infrequent in clinically insignificant prostate cancer, and is associated with higher grade tumors. Detection of PTEN loss in Gleason score 6 cancer in a needle biopsy specimen indicates a higher likelihood of clinically significant prostate cancer.

Immunohistochemical study of 36 cases of pulmonary sarcomatoid carcinoma—sensitivity of TTF-1 is superior to napsin

Immunohistochemistry is often used to distinguish pulmonary sarcomatoid carcinoma from morphologic mimics. Napsin-A is a pulmonary adenocarcinoma marker, but literature on expression in sarcomatoid carcinoma is limited. Thirty-six cases of sarcomatoid carcinoma were stained for napsin, TTF-1, Oscar, CAM5.2, AE1/AE3, desmin, SMA, S-100, CK5/6, calretinin, D2-40, and WT1. Patients were 24 men and 12 women (mean, 70 years; range, 46-93). There were 27 pleomorphic carcinomas, 5 spindle cell carcinomas, 3 carcinosarcomas, and 1 giant cell carcinoma. Cases were positive for at least 1 keratin: AE1/3 was positive in all 36 cases; Oscar, in 34 cases (94%); and CAM5.2, in 32 cases (89%, weaker/more focal). Napsin was positive in 14 cases (39%): 8 diffuse, 3 focal, and 3 rare cells. TTF-1 was positive in 22 cases (61%): 15 diffuse, 3 focal, and 4 rare cells. No cases were napsin positive and negative for TTF-1. Variable staining for mesothelial markers was observed, including positivity for calretinin (12 cases, 33%), WT1 (6 cases, 17%), D2-40 (5 cases, 14%), and CK5/6 (9 cases, 25%). Mesenchymal markers were also sometimes positive (usually focal), including S-100 (4 cases, 11%), desmin (4 cases, 11%), and SMA (7 cases, 19%, 1 diffuse). In conclusion, TTF-1 is more sensitive than napsin for detection of sarcomatoid carcinoma, and no cases were positive for napsin but negative for TTF-1. CAM5.2 is less sensitive than AE1/AE3 and Oscar. Use of a thoughtful immunohistochemical panel is important in the evaluation of sarcomatoid carcinoma because mesothelial and mesenchymal markers can be expressed.

Genomic Rearrangements Define Lineage Relationships between Adjacent Lepidic and Invasive Components in Lung Adenocarcinoma

The development of adenocarcinoma of the lung is believed to proceed from in situ disease (adenocarcinoma in situ, AIS) to minimally invasive disease with prominent lepidic growth (minimally invasive adenocarcinoma, MIA), then to fully invasive adenocarcinoma (AD), but direct evidence for this model has been lacking. Because some lung adenocarcinomas show prominent lepidic growth (AD-L), we designed a study to address the lineage relationship between the lepidic (noninvasive) component (L) and the adjacent nonlepidic growth component representing invasive disease within individual tumors. Lineage relationships were evaluated by next-generation DNA sequencing to define large genomic rearrangements in microdissected tissue specimens collected by laser capture. We found a strong lineage relationship between the majority of adjacent lepidic and invasive components, supporting a putative AIS-AD transition. Notably, many rearrangements were detected in the less aggressive lepidic component, although the invasive component exhibited an overall higher rate of genomic rearrangement. Furthermore, a significant number of genomic rearrangements were present in histologically normal lung adjacent to tumor, but not in host germline DNA, suggesting field defects restricted to zonal regions near a tumor. Our results offer a perspective on the genetic pathogenesis underlying adenocarcinoma development and its clinical management.

Temporal and spatial heterogeneity of programmed cell death 1-Ligand 1 expression in malignant mesothelioma

ABSTRACT Background: Programmed Cell Death 1-Ligand 1 (PD-L1) and Programmed Death Protein 1 (PD-1) blocking antibodies are promising immunotherapies for malignancies. We have previously shown PD-L1 expression in 40% of malignant mesothelioma (MM); however, the temporal and spatial heterogeneity of its expression has not been thoroughly studied. We compared PD-L1 expression between paired primary and metastatic MM. Design: Pathology files (1995–2016) were searched for MM with tissue from multiple sites and/or time points. PD-L1 (clone SP263) expression was reviewed by 2 authors. Mesothelioma cell lines (H2461, One 58, EM-MESO) were cultured with or without vinorelbine or pemetrexed. Following incubation, PD-L1 expression (clone MIH1) was analyzed by flow cytometry. Results: 64 patients (53 men, median age, 64 years) with epithelioid (N = 50), biphasic (N = 11) or sarcomatoid (N = 2) MM or well differentiated papillary mesothelioma (N = 1) (pleural, n = 56; peritoneal, n = 8) were included. Patients had a subsequent specimen from the primary site (n = 48), from a metastasis (n = 6), or both (n = 10). Reviewers agreed on PD-L1 expression in 133 of 151 (88%) specimens. There was agreement of PD-L1 expression between paired primary lesions obtained at separate time points in 47 of 58 (81%) and between paired primary and metastatic lesions in 11 of 16 (69%) cases. A significant increase in PD-L1 expression was observed in all 3 MM cell lines (p < 0.003 each) following exposure to vinorelbine but not to pemetrexed. Conclusion: Overall there is good agreement in PD-L1 expression between paired MM lesions; however, the 19–31% of cases with discordant PD-L1 expression, and the dynamics of PD-L1 expression may limit its use as a predictive biomarker for therapy.

EGFR mediates activation of RET in lung adenocarcinoma with neuroendocrine differentiation characterized by ASCL1 expression

Achaete-scute homolog 1 (ASCL1) is a neuroendocrine transcription factor specifically expressed in 10-20% of lung adenocarcinomas (AD) with neuroendocrine (NE) differentiation (NED). ASCL1 functions as an upstream regulator of the RET oncogene in AD with high ASCL1 expression (A+AD). RET is a receptor tyrosine kinase with two main human isoforms; RET9 (short) and RET51 (long). We found that elevated expression of RET51 associated mRNA was highly predictive of poor survival in stage-1 A+AD (p=0.0057). Functional studies highlighted the role of RET in promoting invasive properties of A+AD cells. Further, A+AD cells demonstrated close to 10 fold more sensitivity to epidermal growth factor receptor (EGFR) inhibitors, including gefitinib, than AD cells with low ASCL1 expression. Treatment with EGF robustly induced phosphorylation of RET at Tyr-905 in A+AD cells with wild type EGFR. This phosphorylation was blocked by gefitinib and by siRNA-EGFR. Immunoprecipitation experiments found EGFR in a complex with RET in the presence of EGF and suggested that RET51 was the predominant RET isoform in the complex. In the microarray datasets of stage-1 and all stages of A+AD, high levels of EGFR and RET RNA were significantly associated with poor overall survival (p < 0.01 in both analyses). These results implicate EGFR as a key regulator of RET activation in A+AD and suggest that EGFR inhibitors may be therapeutic in patients with A+AD tumors even in the absence of an EGFR or RET mutation.

Classic Hodgkin lymphoma involving the endometrium: an extremely rare finding associated with refractory/widespread disease

Classic Hodgkin lymphoma (HL) involving the endometrium is extremely rare as lymphoma involving the female genital tract is uncommon in general and those that do are most commonly non-Hodgkin lymphomas. Here, we present a patient with refractory classic HL where PET scan imaging results led to an endometrial biopsy that confirmed uterine involvement of her disease.

Heterogeneity of Programmed Cell Death-Ligand 1 Expression in Thymic Epithelial Tumors Between Initial Specimen and Synchronous or Metachronous Metastases or Recurrences

Thymic epithelial tumors (TET) are rare malignant neoplasms that have the potential to metastasize, recur, or even have a fatal outcome. While many thymomas have a favorable outcome with a 5-year overall survival of 75.8%, reported 5-year survival rates of thymic carcinomas are only 27.5 to 50.5% [1, 2] Complete resection is the curative treatment of choice, however, high stage TET are often not amenable to complete resection. Therefore, alternate treatments such as anti-PD-1/PD-L1 inhibitors, have been studied in this patient population and remain an area of active investigation. This article is protected by copyright. All rights reserved.

Genomic rearrangements in sporadic lymphangioleiomyomatosis: an evolving genetic story

Sporadic lymphangioleiomyomatosis is a progressive pulmonary cystic disease resulting from the infiltration of smooth muscle-like lymphangioleiomyomatosis cells into the lung. The migratory/metastasizing properties of the lymphangioleiomyomatosis cell together with the presence of somatic mutations, primarily in the tuberous sclerosis complex gene (TSC2), lead many to consider this a low-grade malignancy. As malignant tumors characteristically accumulate somatic structural variations, which have not been well studied in sporadic lymphangioleiomyomatosis, we utilized mate pair sequencing to define structural variations within laser capture microdissected enriched lymphangioleiomyomatosis cell populations from five sporadic lymphangioleiomyomatosis patients. Lymphangioleiomyomatosis cells were confirmed in each tissue by hematoxylin eosin stain review and by HMB-45 immunohistochemistry in four cases. A mutation panel demonstrated characteristic TSC2 driver mutations in three cases. Genomic profiles demonstrated normal diploid coverage across all chromosomes, with no aneuploidy or detectable gains/losses of whole chromosomal arms typical of neoplastic diseases. However, somatic rearrangements and smaller deletions were validated in the two cases which lacked TSC2 driver mutations. Most significantly, one of these sporadic lymphangioleiomyomatosis cases contained two different size deletions encompassing the entire TSC1 locus. The detection of a homozygous deletion of TSC1 driving a predicted case of sporadic lymphangioleiomyomatosis, consistent with the common two-hit TSC2 mutation model, has never been reported for sporadic lymphangioleiomyomatosis. However, while no evidence of the hereditary tuberous sclerosis complex disease was reported for this patient, the potential for mosaicism and sub-clinical phenotype cannot be ruled out. Nevertheless, this study demonstrates that somatic structural rearrangements are present in lymphangioleiomyomatosis disease and provides a novel method of genomic characterization of sporadic lymphangioleiomyomatosis cells, aiding in defining cases with no detected mutations by conventional methodologies. These structural rearrangements could represent additional pathogenic mechanisms in sporadic lymphangioleiomyomatosis disease, potentially affecting response to therapy and adding to the complex genetic story of this rare disease.

Abstract 1129: EGFR-mediated activation of RET in ASCL1+ lung adenocarcinoma

Lung cancer accounts for about 27% of the cancer related deaths in the USA annually. Pathologically, it is a very complex disease broadly classified into small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). NSCLCs are further classified into squamous cell carcinoma, large cell carcinoma and adenocarcinoma. Achaete-scute homolog 1 (ASCL1), an important transcription factor essential in the development of neuroendocrine cells (NE) in lungs is shown to be specifically expressed in lung NE cancers and 10-20% of adenocarcinomas (AD) with NE differentiation (NED), thus suggesting a role in the pathogenesis of these tumors. Our previous study showed that ASCL1 is a regulator of the RET oncogene in AD with NED. RET is a receptor tyrosine kinase with two isoforms in humans: RET9 (short) and RET51 (long). We performed survival analysis to study implications of RET isoforms in ASCL1+ tumors and found that elevated expression of the long RET mRNA was associated with poor survival. Subsequent in vitro experiments demonstrated that treatment with EGF robustly induced phosphorylation of RET in HCC1833 and H1755 cell lines which have high endogenous levels of ASCL1 and RET but not in VMRC-LCD cell line which has high level of ASCL1 but low level of RET. EGF induced phosphorylation of RET was diminished by gefitinib and by EGFR siRNA. Immunoprecipitation results indicated direct binding between EGFR and RET in presence of EGF. Furthermore, a high throughput drug screening found 8 EGFR inhibitors that were 10 - 250 fold more cytotoxic in ASCL1+ compared with ASCL1- AD cells. These results implicate EGFR as a key regulator of RET activation in ASCL1+ AD and suggest that EGFR inhibitors may be therapeutic for this population of patients. Citation Format: Kaustubh N. Bhinge, Yang Lin, Hamed Rahi, Marie Christine Aubry, Aaron Mansfield, Irina Kovtun, Stephen Murphy, Ping Yang, Dennis Wigle, Joanne (Eunhee) Yi, Aqsa Nasir, Simone Terra, Julian Molina, George Vasmatzis, Farhad Kosari. EGFR-mediated activation of RET in ASCL1+ lung adenocarcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1129.