Cristina Teixidó

AXL Mediates Resistance to PI3Kα Inhibition by Activating the EGFR/PKC/mTOR Axis in Head and Neck and Esophageal Squamous Cell Carcinomas

Phosphoinositide-3-kinase (PI3K)-α inhibitors have shown clinical activity in squamous cell carcinomas (SCCs) of head and neck (H&N) bearing PIK3CA mutations or amplification. Studying models of therapeutic resistance, we have observed that SCC cells that become refractory to PI3Kα inhibition maintain PI3K-independent activation of the mammalian target of rapamycin (mTOR). This persistent mTOR activation is mediated by the tyrosine kinase receptor AXL. AXL is overexpressed in resistant tumors from both laboratory models and patients treated with the PI3Kα inhibitor BYL719. AXL dimerizes with and phosphorylates epidermal growth factor receptor (EGFR), resulting in activation of phospholipase Cγ (PLCγ)-protein kinase C (PKC), which, in turn, activates mTOR. Combined treatment with PI3Kα and either EGFR, AXL, or PKC inhibitors reverts this resistance.

Rearranged EML4-ALK fusion transcripts sequester in circulating blood platelets and enable blood-based crizotinib response monitoring in non-small-cell lung cancer

Purpose: Non-small-cell lung cancers harboring EML4-ALK rearrangements are sensitive to crizotinib. However, despite initial response, most patients will eventually relapse, and monitoring EML4-ALK rearrangements over the course of treatment may help identify these patients. However, challenges associated with serial tumor biopsies have highlighted the need for blood-based assays for the monitoring of biomarkers. Platelets can sequester RNA released by tumor cells and are thus an attractive source for the non-invasive assessment of biomarkers. Methods: EML4-ALK rearrangements were analyzed by RT-PCR in platelets and plasma isolated from blood obtained from 77 patients with non-small-cell lung cancer, 38 of whom had EML4-ALK-rearranged tumors. In a subset of 29 patients with EML4-ALK-rearranged tumors who were treated with crizotinib, EML4-ALK rearrangements in platelets were correlated with progression-free and overall survival. Results: RT-PCR demonstrated 65% sensitivity and 100% specificity for the detection of EML4-ALK rearrangements in platelets. In the subset of 29 patients treated with crizotinib, progression-free survival was 3.7 months for patients with EML4-ALK+ platelets and 16 months for those with EML4-ALK− platelets (hazard ratio, 3.5; P = 0.02). Monitoring of EML4-ALK rearrangements in the platelets of one patient over a period of 30 months revealed crizotinib resistance two months prior to radiographic disease progression. Conclusions: Platelets are a valuable source for the non-invasive detection of EML4-ALK rearrangements and may prove useful for predicting and monitoring outcome to crizotinib, thereby improving clinical decisions based on radiographic imaging alone.

Concordance of IHC, FISH and RT-PCR for EML4-ALK rearrangements

The echinoderm microtubule-associated protein-like 4 anaplastic lymphoma kinase (EML4-ALK) has emerged as the second most important driver oncogene in lung cancer and the first targetable fusion oncokinase to be identified in 4-6% of lung adenocarcinomas. Crizotinib, along with a diagnostic test-the Vysis ALK Break Apart fluorescence in situ hybridization (FISH) Probe Kit-is approved for the treatment of ALK positive advanced non-small cell lung cancer (NSCLC). However, the success of a targeted drug is critically dependent on a sensitive and specific screening assay to detect the molecular drug target. In our experience, reverse transcription polymerase chain reaction (RT-PCR)-based detection of EML4-ALK is a more sensitive and reliable approach compared to FISH and immunohistochemistry (IHC). Although ALK FISH is clinically validated, the assay can be technically challenging and other diagnostic modalities, including IHC and RT-PCR should be further explored.

BIM and mTOR expression levels predict outcome to erlotinib in EGFR-mutant non-small-cell lung cancer

BIM is a proapoptotic protein that initiates apoptosis triggered by EGFR tyrosine kinase inhibitors (TKI). mTOR negatively regulates apoptosis and may influence response to EGFR TKI. We examined mRNA expression of BIM and MTOR in 57 patients with EGFR-mutant NSCLC from the EURTAC trial. Risk of mortality and disease progression was lower in patients with high BIM compared with low/intermediate BIM mRNA levels. Analysis of MTOR further divided patients with high BIM expression into two groups, with those having both high BIM and MTOR experiencing shorter overall and progression-free survival to erlotinib. Validation of our results was performed in an independent cohort of 19 patients with EGFR-mutant NSCLC treated with EGFR TKIs. In EGFR-mutant lung adenocarcinoma cell lines with high BIM expression, concomitant high mTOR expression increased IC50 of gefitinib for cell proliferation. We next sought to analyse the signalling pattern in cell lines with strong activation of mTOR and its substrate P-S6. We showed that mTOR and phosphodiesterase 4D (PDE4D) strongly correlate in resistant EGFR-mutant cancer cell lines. These data suggest that the combination of EGFR TKI with mTOR or PDE4 inhibitors could be adequate therapy for EGFR-mutant NSCLC patients with high pretreatment levels of BIM and mTOR.

Identification of ALK, ROS1 and RET Fusions by a Multiplexed mRNA-Based Assay in Formalin-Fixed, Paraffin-Embedded Samples from Advanced Non–Small-Cell Lung Cancer Patients

BACKGROUNDnAnaplastic lymphoma receptor tyrosine kinase (ALK), ROS proto-oncogene 1, receptor tyrosine kinase (ROS1), and ret proto-oncogene (RET) fusions are present in 5%-7% of patients with advanced non-small-cell lung cancer (NSCLC); their accurate identification is critical to guide targeted therapies. FISH and immunohistochemistry (IHC) are considered the gold standards to determine gene fusions, but they have limitations. The nCounter platform is a potentially useful genomic tool for multiplexed detection of gene fusions, but has not been validated in the clinical setting.nnnMETHODSnFormalin-fixed, paraffin embedded (FFPE) samples from 108 patients with advanced NSCLC were analyzed with an nCounter-based assay and the results compared with FISH, IHC, and reverse transcription PCR (RT-PCR). Data on response to fusion kinase inhibitors was retrospectively collected in a subset of 29 patients.nnnRESULTSnOf 108 FFPE samples, 98 were successfully analyzed by nCounter (91%), which identified 55 fusion-positive cases (32 ALK, 21 ROS1, and 2 RET). nCounter results were highly concordant with IHC for ALK (98.5%, CI = 91.8-99.7), while 11 discrepancies were found compared with FISH (87.5% concordance, CI = 79.0-92.9). For ROS1, nCounter showed similar agreement with IHC and FISH (87.2% and 85.9%), but a substantial number of samples were positive only by 1 or 2 techniques. Of the 25 patients deriving clinical benefit from fusion kinase inhibitors, 24 were positive by nCounter and 22 by FISH.nnnCONCLUSIONSnnCounter compares favorably with IHC and FISH and can be used for identifying patients with advanced NSCLC positive for ALK/ROS1/RET fusion genes.

Convergent Akt activation drives acquired EGFR inhibitor resistance in lung cancer

Non-small-cell lung cancer patients with activating epidermal growth factor receptor (EGFR) mutations typically benefit from ufeffEGFR tyrosine kinase inhibitor treatment. However, virtually all patients succumb to acquired EGFR tyrosine kinase inhibitor resistance that occurs via diverse mechanisms. The diversity and unpredictability of EGFR tyrosine kinase inhibitor resistance mechanisms presents a challenge for developing new treatments to overcome EGFR tyrosine kinase inhibitor resistance. Here, we show that Akt activation is a convergent feature of acquired EGFR tyrosine kinase inhibitor resistance, across a spectrum of diverse, established upstream resistance mechanisms. Combined treatment with an EGFR tyrosine kinase inhibitor and Akt inhibitor causes apoptosis and synergistic growth inhibition in multiple EGFR tyrosine kinase inhibitor-resistant non-small-cell lung cancer models. Moreover, phospho-Akt levels are increased in most clinical specimens obtained from EGFR-mutant non-small-cell lung cancer patients with acquired EGFR tyrosine kinase inhibitor resistance. Our findings provide a rationale for clinical trials testing Akt and EGFR inhibitor co-treatment in patients with elevated phospho-Akt levels to therapeutically combat the heterogeneity of EGFR tyrosine kinase inhibitor resistance mechanisms.EGFR-mutant non-small cell lung cancer are often resistant to EGFR tyrosine kinase inhibitor treatment. In this study, the authors show that resistant tumors display high Akt activation and that a combined treatment with AKT inhibitors causes synergistic tumour growth inhibition in vitro and in vivo.

Interferon gamma, an important marker of response to immune checkpoint blockade in non-small cell lung cancer and melanoma patients:

Background: Programmed death-ligand 1 (PD-L1) may be induced by oncogenic signals or can be upregulated via interferon gamma (IFN-γ). We have explored whether the expression of IFNG, the gene encoding IFN-γ, is associated with clinical response to the immune checkpoint blockade in non-small cell lung cancer (NSCLC) and melanoma patients. The role of inflammation-associated transcription factors STAT3, IKBKE, STAT1 and other associated genes has also been examined. Methods: Total RNA from 17 NSCLC and 21 melanoma patients was analyzed by quantitative reverse transcription PCR. STAT3 and Rantes, YAP1 and CXCL5, DNMT1, RIG1 and TET1, EOMES, IFNG, PD-L1 and CTLA4, IKBKE and NFATC1 mRNA were examined. PD-L1 protein expression in tumor and immune cells and stromal infiltration of CD8+ T-cells were also evaluated. Progression-free survival and overall survival were estimated. Results: A total of 17 NSCLC patients received nivolumab and 21 melanoma patients received pembrolizumab. Progression-free survival with nivolumab was significantly longer in NSCLC patients with high versus low IFNG expression (5.1 months versus 2 months, p = 0.0124). Progression-free survival with pembrolizumab was significantly longer in melanoma patients with high versus low IFNG expression (5.0 months versus 1.9 months, p = 0.0099). Significantly longer overall survival was observed for melanoma patients with high versus low IFNG expression (not reached versus 10.2 months p = 0.0183). There was a trend for longer overall survival for NSCLC patients with high versus low IFNG expression. Conclusions: IFN-γ is an important marker for prediction of response to immune checkpoint blockade. Further research is warranted in order to validate whether IFNG is more accurate than PD-L1.

RNA Analysis as a Tool to Determine Clinically Relevant Gene Fusions and Splice Variants

CONTEXTn- Technologic advances have contributed to the increasing relevance of RNA analysis in clinical oncology practice. The different genetic aberrations that can be screened with RNA include gene fusions and splice variants. Validated methods of identifying these alterations include fluorescence in situ hybridization, immunohistochemistry, reverse transcription-polymerase chain reaction, and next-generation sequencing, which can provide physicians valuable information on disease and treatment of cancer patients.nnnOBJECTIVEn- To discuss the standard techniques available and new approaches for the identification of gene fusions and splice variants in cancer, focusing on RNA analysis and how analytic methods have evolved in both tissue and liquid biopsies.nnnDATA SOURCESn- This is a narrative review based on PubMed searches and the authors own experiences.nnnCONCLUSIONSn- Reliable RNA-based testing in tissue and liquid biopsies can inform the diagnostic process and guide physicians toward the best treatment options. Next-generation sequencing methodologies permit simultaneous assessment of molecular alterations and increase the number of treatment options available for cancer patients.

Impact of the new EGF receptor and ALK testing guideline on personalized lung cancer medicine

All advanced-stage lung adenocarcinoma patients should be tested for EGF receptor (EGFR) mutations and for EML4–ALK translocations as a guide for selecting them for tyrosine kinase inhibitor (TKI) therapy. The new EGFR and ALK guideline provides useful recommendations for appropriate testing [1]. Lung tumors are the leading cause of cancerrelated mortality worldwide [2]. Almost 80% of cases are non-small-cell lung cancer (NSCLC) and extensive disease is present at the time of diagnosis in the majority of patients. Systemic treatment with chemotherapy has reached a plateau of 10–20% response rates and 4–5 months of progression-free survival (PFS), and has not improved in more than a decade [3–5]. A first milestone in a new era of targeted therapy for this disease was reached with the discovery of a subset of tumors carrying activating mutations of EGFR, which can be selectively blocked with TKIs such as erlotinib and gefitinib. Numerous clinical studies have demonstrated the efficacy of these TKIs in the treatment of EGFR-mutated lung tumors, with 55–83% response rates and 9–13 month PFS [6,7]. A second milestone was the discovery, in 2007, of the EML4–ALK translocation in 6.7% of Japanese NSCLC patients [8]. The translocation was later found in 4–7% of Chinese [9], European [10] and US patients [11]. When treated with a novel ALK inhibitor, crizotinib, these patients showed response rates of 57% with a PFS greater than 6 months [12]. As a result of these new developments, many laboratories worldwide are currently testing lung tumor samples for EGFR mutations and EML4–ALK fusions. In an effort to standardize testing practices, the College of American Pathologists, the International Association for the Study of Lung Cancer and the Association for Molecular Pathology constituted writing and advisory panels, presided by three cochairs, to