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L. Righi
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P1.04 - Poster Session/ Biology, Pathology, and Molecular Testing (ID 233)
- Event: WCLC 2015
- Type: Poster
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:
- Coordinates: 9/07/2015, 09:30 - 17:00, Exhibit Hall (Hall B+C)
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P1.04-104 - Lung Cancer Patients Derived Xenografts: Prospective Molecular Profiling and Potential Evaluation of Drug Resistance (ID 1278)
09:30 - 09:30 | Author(s): L. Righi
- Abstract
Background:
The discovery of “driver mutations” such as the Epidermal Growth Factor Receptor (EGFR) and the Anaplastic Lymphoma Kinase (ALK) has led to a remarkable improvement in the outcomes of lung adenocarcinoma, which accounts 50% of the non-small cell lung cancer (NSCLC) diagnoses. Up today, no druggable molecular targets have been identified for squamous carcinoma or small cell lung cancer, which are still treated with the “one-fits-all” therapeutic approach, as it is for a relevant percentage of adenocarcinomas too. The precise definition of molecular profile and, possibly, the description of predictive factors are research priority in the thoracic oncology field. The vast majority of preclinical data are based on in vitro studies, but cell lines models do not entirely reflect tumour characteristics and are hampered by genetic divergence from primary tumours. Patient derived tumour xenografts (PDTX) are a valuable alternative to closely reproduce tumour biology and to prospectively characterize in vivo mechanisms of cancer growth and therapeutic response. Through the generation of a cohort of lung cancer xenopatients, the project aims to confirm the reliability of such models in this disease and to prospectively characterize its biomolecular features.
Methods:
Metastatic and early stages lung cancer cases are considered for the enrolment. Written informed consent is requested from each patient. Fresh tumour tissue from lung biopsies or lung resections is collected and kept in serum free medium (4° C), embedded in 20% matrigel and subcutaneously engrafted into NSG and NOD SCID mice, within 24 hours from sample collection. The exponentially growing tumours are passaged subcutaneously to other mice for a second passage after which they are archived for subsequent analyses (formalin fixed, snap frozen and RNA later). Each sample from surgical resection is also stored to create a DNA lung cancer bank.
Results:
Fourteen samples from TC-guided lung biopsies and sixty-six from radically resected NSCLC were engrafted in NSG and NOD SCID mice lineage in a 1:1 ratio. Due to the low engraftment rate and high morbidity observed in NGS mice in the first 73 samples, subsequent engraftments and expansions were performed in NOD SCID mice only. The overall engraftment rate in biopsy samples was 0 % in NGS and 7.14 % in NOD SCID mice as opposed to 0 % in NGS and 27,27 % in NOD SCID for surgical samples (50% adenocarcinomas, 44,45% squamous carcinomas and 5,55% sarcomatoid carcinomas). Nineteen samples underwent the second passage: of those, 10 samples have been archived after the second successful passage and will be used for further analyses.
Conclusion:
The trial is still ongoing and a longer follow-up is needed. In biopsy-derived samples, engraftment is deeply limited by the paucity of tissue. The results of this study will possibly confirm the reliability of PDTX in lung cancer and provide prospective biomolecular characterization for different histological types.
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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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P3.04-015 - Immunohistochemistry With 3 ALK Antibodies and Thymidylate Synthase Evaluation of FISH-Positive ALK-Rearranged Lung Adenocarcinomas (ID 1430)
09:30 - 09:30 | Author(s): L. Righi
- Abstract
Background:
ALK-rearranged lung tumors represent approximately 2-7% of all Non-Small-Cell Lung Cancers (NSCLCs). Young age, never/light smoking habit, adenocarcinoma (ADK) histology and good response to chemotherapy with pemetrexed characterize ALK-positive patients (pts). Current treatment strategies in this molecular setting are based on ALK-kinase inhibition with small molecules including first (crizotinib/CZT) and second generation TKIs. The FDA-approved companion diagnostic test for CZT treatment is the Vysis break-apart FISH probe, but several works support the immunohistochemistry (IHC) as a sensitive and specific test. The European Medical Agency (EMA) recently approved CZT for second-line treatment of ALK-rearranged NSCLC as detected by "an accurate and validated ALK assay", thus endorsing IHC for eligibility purposes. Here, we retrospectively assessed ALK status in 28 pts with known FISH-positive ALK-rearranged NSCLC performing IHC with 3 different antibodies in order to assess their diagnostic accuracy as compared to the FISH assay. Moreover, we evaluated thymidylate synthase (TS) expression using real-time polymerase chain reaction (RT-PCR) given the conflicting literature data on pemetrexed sensitivity in those tumors. As a secondary end point we will compare molecular and clinical outcomes.
Methods:
FISH was performed with Vysis break-apart FISH probe. IHC was performed with 3 different antibodies: ALK1 (DAKO), 5A4 (Novocastra) and D5F3 (Ventana/Cell Signaling Technology). For ALK1 and 5A4 an IHC scoring value between 0+ and 3+ was used, as previously proposed, while a positive or negative score was used with D5F3 and Ventana KIT. TS gene expression was measured through Real Time PCR, TaqMan method.
Results:
28 specimens of ALK-rearranged ADK diagnosed between 2010 and 2013 from 7 different Italian Oncology Centres were evaluated. Pts median age at diagnosis was 55 (range: 25-78), 9 pts were female. 25/28 (89.3%) specimens were D5F3 positive. 13/28 (46.4%) had 5A4 3+ positivity, 12 (42.8%) showed 2+ positivity while the remaining 3 were negative. 3/28 specimens (10.7%) had ALK1 3+ score, 9 (32.1%) 2+, 13 (46.5%) 1+ and the remaining 3 (10.7%) were negative. Among the 3 FISH-positive and IHC-negative cases, 2 pts underwent CZT treatment, both progressing within 2 weeks and with low percentage of rearranged tumor cells at FISH testing (16-20%) When considering 3+ and 2+ scores as positive, 12 specimens (42.8%) resulted to be positive with all the 3 antibodies, while score 1+ was observed only with ALK1 in 13 (46.4%). Only 3 cases resulted strongly positive with all clones. TS gene expression median value on 25 cases was 6.27 (range 2,8-14-94). 65% of cases had low expression as compared to a population of ALK-negative lung ADK (personal data).
Conclusion:
IHC proved to be a reliable tool to diagnose ALK-rearranged lung tumors, especially with D5F3 and 5A4 antibodies. As the two IHC negative and FISH positive patients who received CZT didn’t respond to treatment, IHC should be used as screening tool or a confirmatory test in case of low-rearranged FISH-positive cases. TS expression appeared to be lower in ALK-positive lung tumors as compared to ALK-negative lung ADK. Further comparisons between clinical and molecular data are ongoing.
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P3.04-060 - Non Small Cell Lung Cancer in Women: Identification of Molecular Biomarkers Towards Sex Specific Tailored Treatments (ID 1272)
09:30 - 09:30 | Author(s): L. Righi
- Abstract
Background:
Lung cancer is the leading cause of cancer mortality in both men and women in more developed countries, with a four-fold increase in lung cancer in women in US over the past 30 years. This was confirmed in Europe where, in the last 5 years, lung cancer mortality fell in men (−6%) and increased in women (+7%). Several studies documented sex differences in lung cancer in terms of clinical presentation, survival, pathological patterns and treatment related toxicities; younger age at diagnosis, higher frequency of adenocarcinoma histology, different metabolism of tobacco-related carcinogens, differential gene expression are commonly seen in women. Furthermore, previous studies showed in female gender the expression of functional aromatase enzyme in lung tumor tissues as well as the interaction between Estrogen Receptors (ERs) and Epidermal Growth factor Receptor (EGFR) pathways in lung cancer cells. The aim of this study is to collect a prospective series of advanced stage non small cell lung cancers (NSCLC), to identify, through the Next Generation Sequencing (NGS) technology, potential gender sex differences of selected tumor-associated genes, assessing their both mutational status and gene expression levels.
Methods:
One hundred patients, including 50 women and 50 men, with newly diagnosed stage IV NSCLC will be prospectively enrolled. Smoking history, clinical and anamnestical data will be collected for all patients. Female patients will also provide obstetrical-gynecological anamnesis, while men will provide urological one, if present. Formalin fixed, paraffin embedded diagnostic sample of each patient will be collected and sectioned to obtain: a DNA genomic library to define the mutational profile of a selected panel including 50 tumor-associated genes, a mRNA library to obtain gene expression levels of the corresponding transcripts and protein expression of estrogen receptor Beta (ERß) and DNA repair enzyme ERCC1. Immunohistochemistry reaction, for both ERCC1 and ERβ, will be scored according to the H-score method. NGS analyses will be performed by means of the Ion Torrent Personal Genome Machine (PGM, Life Technologies, Grand Island, NE). Tumor tissues will be tested with commercial library kits: Ion AmpliSeq Cancer Hotspot Panel v.2 to investigate 50 cancer-associated genes and significant gene variations will be further confirmed using Sanger Sequencing method; Ion AmpliSeq™ RNA Cancer Panel to define also gene expression of the same 50 cancer-associated genes (Life Technologies). Correlations among mutational profile, transcriptional pattern, protein levels and clinico-pathological characteristics will be assessed.
Results:
Not Applicable
Conclusion:
Lung cancer incidence in women is increasing worldwide and genetic predisposition, sex hormones or specific molecular features could all account for the clinical differences observed between females and males. Up to the current date, the clinical approach to lung cancer treatment does not rely on gender. The identification of differential status of specific biomarkers can deepen knowledge on the molecular basis of this disease, guiding clinicians towards sex-based treatments.
