Irene Sansano

Analysis of Expression of Programmed Cell Death 1 Ligand 1 (PD-L1) in Malignant Pleural Mesothelioma (MPM)

Background The increasing incidence and poor outcome associated with MPM requires finding effective treatment for this disease. PD1/PD-L1 pathway plays a central role in tumor immune evasion and appears to be predictive and prognostic marker. PD-L1 is expressed in many different human cancers but its role in MPM has yet to be established. The aim of this study is to evaluate the expression of PD-L1 in MPM. Methods 119 MPM patients (p) from two institutions between November 2002 and February 2014 were reviewed. Formalin-fixed, paraffin-embedded tissue was stained with anti-PD-L1 (clone E1L3N). Cases showing more than 1% of tumor cells expression of PD-L1 were considered positive. Results PD-L1 was analyzed in 77 p with tumor tissue available and was positive in 20.7% p (14 samples in membrane, 16 in cytoplasm and 4 in immune infiltrate). PD-L1 intensity was weak in 56.2%, moderate in 25% and strong in 18.7% p. There was a significant relationship between PD-L1 expression and histology (PD-L1 expression 37.5% in no-epithelioid tumor and 13.2% in epithelioid; p=0.033). The median survival in p PD-L1 positive was 4.79 vs 16.3 months in p PD-L1 negative (p=0.012). Conclusions We have shown PD-L1 is expressed in 20% of patients, associated with no epithelioid histology and poor prognostic in MPM. Our results suggest PD-L1 warrants further exploration in selecting p for immunotherapy.

Adaptive resistance to targeted therapies in cancer

It is widely acknowledged that there is a need for molecular profiling in non-small-cell lung cancer. For example, treatment based on EGFR mutation status has attained successful results. However, in spite of excellent initial response to oral EGFR tyrosine kinase inhibitors (TKIs), progression-free survival is still limited. Current research has focused mostly on acquired resistance mechanisms, such as overexpression of AXL and loss of the Mediator MED12. In this review, in contrast, we discuss adaptive, rather than acquired, resistance. Adaptive resistance can occur almost immediately after starting targeted therapy through a rapid rewiring of cancer cell signaling. By losing ERK negative feedback on receptor tyrosine kinase (RTK) expression, cancer cells are exposed to the stimuli of several ligands, and the ensuing activation of several RTKs reprograms all the canonical signaling pathways. The overexpression of several RTKs was observed in breast cancer cell lines treated with a MEK inhibitor and in BRAF(V600E) melanoma cell lines treated with BRAF inhibitors. This rebound effect of overexpression of several RTKs, including ERBB3, also occurs in lung cancers driven by Kras or EGFR mutations when treated with MEK, PI3K or dual PI3K/mTOR inhibitors. Synthetic lethality can be effectively induced by co-targeting these overexpressed RTKs. We speculate that in patients with EGFR mutations, adaptive resistance occurs in a significant proportion of patients. Rebiopsies performed hours after starting treatment with EGFR TKIs can identify which RTKs are overexpressed after treatment. Efficient co-targeting of these RTKs can induce synthetic lethality and help overcome the limited effect of EGFR TKI monotherapy.

Analysis of expression of PTEN/PI3K pathway and programmed cell death ligand 1 (PD-L1) in malignant pleural mesothelioma (MPM).

BACKGROUND Malignant pleural mesothelioma (MPM) frequently express elevated AKT/mTOR activity. Previous reports in gliomas, colon, breast and prostate cancer suggest that PTEN/PI3K pathway may be important for the induction of PD-L1 expression. This study explored the expression of PTEN/PI3K pathway and PD-L1 in MPM and its relationship with the patient́s prognosis MATERIAL AND METHODS Twenty seven consecutive MPM patients were reviewed. Formalin-fixed, paraffin-embedded tissue biopsies were used for immunohistochemical analysis of PTEN/PI3K pathway and PD-L1 RESULTS: Expression of PTEN, mTOR, pAKT, p4EBP1, peif4E, pS6 and FOXO3a was found in 88.5%, 92.3%, 78.3%, 38.5%, 100%, 52.2% and 100% of tumors and PD-L1 in 23%. We found a significant correlation between pAKT, FOXO3a and PD-L1 expression and longer overall survival (p <0.05). We did not identify significant association between the level of PD-L1 expression and alterations in PI3K pathway CONCLUSIONS This study shows PTEN/PI3K pathway and PD-L1 in MPM are frequently activated. Our results suggests that there is not association between PD-L1 and the involvement of the PI3K pathway in MPM.

Activity of HSP90 Inhibiton in a Metastatic Lung Cancer Patient With a Germline BRCA1 Mutation

Abstract Heat shock proteins (HSPs) are molecular chaperones that maintain proteins in their correct conformation to ensure stability and protect carcinoma cells from apoptosis. HSP90 inhibitors (HSP90i) block multiple targets simultaneously, and despite responses in a selected population, no HSP90i have yet been approved. We present a patient with a lung tumor with an exceptional response to cisplatin/gemcitabine in combination with HSP90i, which nowadays continues with HSP90i maintenance after three years. Whole-exome sequencing of the lung tumor unveiled a BRCA1/2 deficiency mutational signature, and mutation analysis confirmed a germline BRCA1 mutation. The striking efficacy of HSP90i plus chemotherapy vs chemotherapy alone was reproduced in a patient-derived xenograft (PDX) model from a breast cancer patient with a BRCA1 mutation (mean tumor volume [SD], No. of tumors: vehicle 8.38 [7.07] mm3, n = 3; HSP90i 4.18 [1.93] mm3, n = 5; cisplatin plus gemcitabine 3.31 [1.95] mm3, n = 5; cisplatin plus gemcitabine plus HSP90i 0.065 [0.076] mm3, n = 6). This case and the PDX demonstrate the efficacy for therapeutic inhibition of HSP90 in a BRCA-mutated patient, opening a new potential avenue for better identifying patients who might benefit most from HSP90i.

Computer-Based Intensity Measurement Assists Pathologists in Scoring Phosphatase and Tensin Homolog Immunohistochemistry — Clinical Associations in NSCLC Patients of the European Thoracic Oncology Platform Lungscape Cohort

Introduction: Phosphatase and tensin homolog (PTEN) loss is frequently observed in NSCLC and associated with both phosphoinositide 3‐kinase activation and tumoral immunosuppression. PTEN immunohistochemistry is a valuable readout, but lacks standardized staining protocol and cutoff value. Methods: After an external quality assessment using SP218, 138G6 and 6H2.1 anti‐PTEN antibodies, scored on webbook and tissue microarray, the European Thoracic Oncology Platform cohort samples (n = 2245 NSCLC patients, 8980 tissue microarray cores) were stained with SP218. All cores were H‐scored by pathologists and by computerized pixel‐based intensity measurements calibrated by pathologists. Results: All three antibodies differentiated six PTEN+ versus six PTEN‐ cases on external quality assessment. For 138G6 and SP218, high sensitivity and specificity was found for all H‐score threshold values including prospectively defined 0, calculated 8 (pathologists), and calculated 5 (computer). High concordance among pathologists in setting computer‐based intensities and between pathologists and computer in H‐scoring was observed. Because of over‐integration of the human eye, pixel‐based computer H‐scores were overall 54% lower. For all cutoff values, PTEN‐ was associated with smoking history, squamous cell histology, and higher tumor stage (p < 0.001). In adenocarcinomas, PTEN‐ was associated with poor survival. Conclusion: Calibration of immunoreactivity intensities by pathologists following computerized H‐score measurements has the potential to improve reproducibility and homogeneity of biomarker detection regarding epitope validation in multicenter studies.

Abstract 53: FGFR1 and SOX2 amplification in squamous cell carcinoma (SCC) of the lung.

SRY-related HMG-box (SOX2) gene is a key transcription factor that coordinates development, differentiation and self-renewal of normal non-alveolar epithelium of the airway. SOX2 amplification has been reported in lung squamous cell carcinoma (SCC). Fibroblast growth factor receptor 1 (FGFR1) is a receptor tyrosine kinase that, upon activation, promotes cell proliferation, survival and apoptotic resistance through PLCγ/PKC, RAS/MAPK and PI3K-AKT pathways, respectively. FGFR1 is also amplified in 15-25% of lung SCC and pre-clinical data with targeted inhibitors showed a growth dependency on FGFR1 amplification either in vitro and in vivo. A clinical trial with BIBF1120 (which also inhibits FGFR1), will be developed in the Netherlands and Spain in second line SCC of the lung with FGFR1 amplified by FISH. Genetic heterogeneity in solid tumors is a major research area and therefore its assessment in SCC of the lung is of great relevance. We have examined FGFR1 and SOX2 gene copy number (GCN) in 76 lung SCC by multiplex ligation-dependent probe amplification (MLPA). Analysis of mutations in DDR2, PIK3CA, BRAF, EGFR, KRAS, and TP53 is ongoing. Genomic DNA (gDNA) was isolated from enriched tumor cells by laser captured microdisection from formalin-fixed paraffin embedded (FFPE) tumor tissue sections. 50-100 ng of gDNA was analyzed in each of the three independent replicates per tumor sample. Two independent probe sets were used for each gene analyzed. For inter-patient GCN value comparisons, the tumor results from each patient was normalized against the GCN values derived from FFPE peripheral blood leukocytes control. We also used MLPA technique to study intra-tumor heterogeneity (TH) by analyzing FGFR1 and SOX2 in different tumor areas. In particular, in 4 cases FGFR1 and SOX2 were co-amplified. Also, TH was examined in serial tumor biopsies and/or resections obtained at different points of disease progression in two patients. Of the 76 patients evaluable for FGFR1 and SOX2 GCN, FGFR1 high amplification was detected in 13/76 (17.10%) and low amplification in 7/76 (9.21%). SOX2 presented high amplification in 38/63 (60.32%) and low amplification in 9/63 (14.28%). Interestingly, 46.15% of the FGFR1-amplified tumors were also co-amplified for SOX2. An important degree of TH was found in 2 of the 4 tumors analyzed. GCN changes in FGFR1, SOX2, PIK3CA, PDGFRa, KDR, EGFR and MET over 10 years follow-up will be presented for one surgically resected SCC lung cancer patient. Frequencies of FGFR1 and SOX2 gene amplifications detected are in agreement with previously published data. Survival according to FGFR1 and SOX2 status will also be presented. FGFR1 and SOX2 co-amplification, together with the TH, warrants further research and could represent a novel therapeutic target. Citation Format: Pedro Mendez, Jose L. Ramirez, Amaya Gasco, Teresa Moran, Enric Carcereny, Miquel Taron, Jose Luis Mate, Irene Sansano, Maria Perez, Monica Botia, Montserrat Tierno, Harry J.M Groen, Rafael Rosell. FGFR1 and SOX2 amplification in squamous cell carcinoma (SCC) of the lung. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 53. doi:10.1158/1538-7445.AM2013-53

Abstract P1-01-29: Intraoperative molecular analysis of sentinel lymph node as a new predictor of axillary status in early breast cancer patients.

The one-step nucleic acid amplification (OSNA) assay (Sysmex Corporation, Kobe, Japan) is a new diagnostic device that uses molecular biological techniques to analyze sentinel lymph node (SLN). Intraoperative SLN assessed by OSNA has been validated as an accurate method for detection of SLN metastasis compared to conventional histological examination. Although recent reports have shown that breast cancer patients with The aim of the present study is to assess the intraoperative positive SLN total tumor load (TTL, defined as the amount of CK19 mRNA copies [copies/μL] in all positive SLNs) obtained by OSNA and to determine whether it is predictive of non-SLNs metastasis independently of the number of affected SLN and the type of surgery. Data were collected during the month of June 2012 from medical records and include age, tumor size and grade, estrogen and progesterone receptor status, HER2 status, Ki67, presence of lymphovascular invasion (LVI), total number of SLN and non-SLN, number of positive and negative non-SLN, size of SLN and non-SLN metastasis, and TTL in each SLN. A total number of 701 patients were recruited, of which 697 (99,4%) met the study selection criteria. Univariate logistic regression showed that, in addition to TTL (p Moreover, the ROC curve analysis showed that, as compared to the number of affected SLN, TTL has a better ROC curve, as measured by the AUC: LogTTL 0.709 (95% CI, 0.667–0.760); number of affected SLN 0.610 (95% CI, 0.570–0.652), p 3 positive SLN the TTL was In conclusion, TTL by OSNA is a newly standardized, automated, and reproducible tool that predicts axillary node status better and independently of the number of affected SLNs and the type of surgery. This value can then help clinicians to personalize surgical treatment. Prospective studies will be carried out to determine the clinical impact of this variable in the management of patients. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-01-29.