•We report resistance mechanisms on osimertinib in lung cancer patients.•We analyzed tissue and liquid biopsies collected at progression by NGS.•C797S EGFR mutation and MET amplification were the ...most frequent mechanisms.•Loss of T797M occurred in 68% of the cases.•Patients treated with osimertinib in “real-life” experienced prolonged survival.
The understanding of histo-molecular mechanisms associated with resistance to osimertinib is a critical step to define the optimal treatment strategy in advanced EGFR-mutated Non-Small-Cell-Lung-Cancer (NSCLC).
We performed a multicentric retrospective analysis on a cohort of consecutive patients treated with osimertinib for an advanced EGFR-mutated NSCLC and collected histo-molecular data from plasma and tumor samples at the time of progression. Next-generation sequencing (NGS) was performed for all samples. Best Overall Response Rate (ORR), Progression Free Survival (PFS), Overall Survival (OS) and data on treatment post-progression efficacy were also collected.
Two-hundred and twenty-six patients were included from 9 Academic French Hospitals between April 2015–October 2018. Osimertinib was given in second-line or more in 219 patients (97%). Best ORR was 52% and best central nervous system ORR was 56%. Median PFS and OS were 9.5 months (IQR 4.0-17.2) and 24 months (IQR 12.4-NR) respectively. At the time of analysis, 150 patients (66%) had tumor progression. Among them, 73 contributive samples (56 tumor biopsies) were available. The most frequent molecular alterations were C797S mutation (n = 9 (13%)) and MET amplification (n = 8 (11%)). Histologic transformation occurred in 5 patients (9% of tumor biopsies). In T790M + NSCLC, loss of T790 M occurred in 68% of cases. Median PFS and OS with treatment beyond progression were 6.0 months (IQR 2.0-10.4) and 15.1 months (IQR 6.7-NR) respectively and longer in case of osimertinib continuation beyond progression.
We confirmed the efficacy of osimertinib in patients with advanced EGFR mutation positive NSCLC. At progression, the most frequent molecular alterations were MET amplification and C797S mutation.
During the last few years, determination of microstatellite instability (MSI) status has become a routine part of clinical practice, essentially to detect Lynch syndrome. Recently, MSI testing has ...increased with the development of immunotherapy and has expanded to a large panel of solid tumours. The aim of our work was to evaluate a fully automated system developed by Biocartis, the Idylla MSI Test, which performs an MSI analysis within 150 min.
A comparison between pentaplex PCR, immunohistochemistry and Idylla MSI Test was performed in 53 colorectal carcinoma samples, 7 small intestine adenocarcinomas, 15 duodenal and pancreatic adenocarcinomas, 16 gastric tumours, 15 endometrial adenocarcinomas, 5 ovarian carcinomas and 4 cases of urinary tract tumours using extracted DNA. Limit-of-detection (LOD) experiment was also done using a commercial DNA known to harbour MSI phenotype.
The overall sensitivity was 94% and the overall specificity was 100%. Two invalid and three false-negative results were observed. Our experiments showed that the amount of DNA loaded into the cartridge was decisive and should be superior to 25 ng. LOD comprised between 4% and 8%.
Overall, we have demonstrated that the Idylla MSI Test is a rapid and valid option to detect MSI phenotype which can be used in a large panel of solid tumours.
Bile duct adenomas (BDAs) are benign intrahepatic biliary proliferations consisting of bile ducts arranged in small and tortuous tubules and embedded in a dense fibrous stroma 1. Microscopically, ...tubules are lined by a single layer of cuboidal to columnar cells with nuclei showing neither atypia nor mitoses. BDAs were formerly considered reactive by some authors 2,3. Recurrent BRAF V600E mutations were however further identified in these lesions, supporting the hypothesis that they are true neoplasms 4,5. They are most often incidentally identified during intra-abdominal surgery and the main differential diagnosis of BDAs is metastatic adenocarcinoma, as mentioned by Levin et al. in the first report of bile duct adenomatosis of the liver 6.
Abstract Immune checkpoint inhibition is a new therapeutic strategy that has shown promising efficacy in many cancer types. Significant activity associated with mismatch repair (MMR) deficiency has ...been observed in hypermutated, microsatellite unstable (MSI) metastatic colorectal cancer (CRC). Beyond deficient-MMR tumors, somatic or germline DNA polymerase D1 ( POLD1 ) or DNA polymerase E ( POLE ) alterations cause a hypermutated phenotype in CRC. This recently identified and rare subgroup of proficient-MMR tumors may also benefit from immunotherapy. In this review, we provide a comprehensive overview of recent data on CRC tumors harboring POLD1 or POLE mutations, with a focus on their molecular, histological, and clinical features. We also examine the evidence supporting the development of immune checkpoint inhibitors in this specific subgroup of CRC patients.
Combined hepatocellular-cholangiocarcinoma (cHCC-CCA) is a rare malignancy associated with an overall poor prognosis. We aimed to investigate the immune profile of cHCC-CCA and determine its impact ...on disease outcome.
We performed a multicenter study of 96 patients with cHCC-CCA. Gene expression profile was analyzed using nCounter PanCancer IO 360 Panel. Densities of main immune cells subsets were quantified from digital slides of IHC stainings. Genetic alterations were investigated using targeted next-generation sequencing.
Two main immune subtypes of cHCC-CCA were identified by clustering analysis: an "immune-high" (IH) subtype (57% of the cases) and an "immune-low" (IL) subtype (43% of the cases). Tumors classified as IH showed overexpression of genes related to immune cells recruitment, adaptive and innate immunity, antigen presentation, cytotoxicity, immune suppression, and inflammation (
< 0.0001). IH cHCC-CCAs also displayed activation of gene signatures recently shown to be associated with response to immunotherapy in patients with HCC. Quantification of immunostainings confirmed that IH tumors were also characterized by higher densities of immune cells. Immune subtypes were not associated with any genetic alterations. Finally, multivariate analysis showed that the IH subtype was an independent predictor of improved overall survival.
We have identified a subgroup of cHCC-CCA that displays features of an ongoing intratumor immune response, along with an activation of gene signatures predictive of response to immunotherapy in HCC. This tumor subclass is associated with an improved clinical outcome. These findings suggest that a subset of patients with cHCC-CCA may benefit from immunomodulating therapeutic approaches.
Tumor cells infected by Epstein–Barr virus (EBV) express latency proteins, among which LMP1 is able to increase the level of the anti‐apoptotic protein BCL‐2, today considered as an attractive target ...for therapeutic strategies. Our results suggest that agents targeting BCL‐2, used either alone or in combination with rituximab, represent a novel promising approach for post‐transplant EBV‐positive B lymphoproliferative disorder treatment.
Post‐transplant lymphoproliferative disorders (PTLD) and Burkitt's lymphoma (BL) are B‐cell malignancies strongly associated with Epstein–Barr virus (EBV) infection. In these lymphoproliferative disorders, EBV infection induces an increase in the expression of the anti‐apoptotic protein BCL‐2. Given its chemoprotective effect, BCL‐2 constitutes an attractive target for new therapeutic strategies for EBV‐positive B‐cell malignancies. Here, we show that ABT‐737, a small inhibitor of BCL‐2, BCL‐X(L), and BCL‐w, strongly induced apoptosis in vitro in EBV‐positive lymphoblastoid cell lines (which is a model for PTLD), whereas BL was less sensitive. ABT‐737 reduced tumor growth and increased the overall survival of mice in a xenograft model of PTLD but had no effect on BL xenograft mice. ABT‐737 combined with a low dose of cyclophosphamide, a major component of the conventional CHOP chemotherapy regimen for BL patients, reduced tumor growth during treatment but failed to improve the overall survival of BL xenograft mice. By contrast, the combination of ABT‐737 and rituximab, one of the main options for the treatment of PTLD, was highly efficient and induced approximately 70% remission in PTLD xenograft mice. These results suggest that the use of agents targeting BCL‐2, either alone or in combination with other conventional drugs, represents a novel promising approach for post‐transplant EBV‐positive B lymphoproliferative disorders.
Circulating tumor DNA (ctDNA) has emerged as a good candidate for tracking tumor dynamics in different cancer types, potentially avoiding repeated tumor biopsies. Many different genes can be mutated ...within a tumor, complicating procedures for tumor monitoring, even with highly sensitive next-generation sequencing (NGS) strategies. Droplet-based digital PCR (dPCR) is a highly sensitive and quantitative procedure, allowing detection of very low amounts of circulating tumor genetic material, but can be limited in the total number of target loci monitored.
We analyzed hypermethylation of 3 genes, by use of droplet-based dPCR in different stages of colorectal cancer (CRC), to identify universal markers for tumor follow-up.
Hypermethylation of WIF1 (WNT inhibitory factor 1) and NPY (neuropeptide Y) genes was significantly higher in tumor tissue compared to normal tissue, independently of tumor stage. All tumor tissues appeared positive for one of the 2 markers. Methylated ctDNA (MetctDNA) was detected in 80% of metastatic CRC and 45% of localized CRC. For samples with detectable mutations in ctDNA, MetctDNA and mutant ctDNA (MutctDNA) fractions were correlated. During follow-up of different stage CRC patients, MetctDNA changes allowed monitoring of tumor evolution.
These results indicate that MetctDNA could be used as a universal surrogate marker for tumor follow-up in CRC patients, and monitoring MetctDNA by droplet-based dPCR could avoid the need for monitoring mutations.
Formalin-fixed paraffin-embedded (FFPE) tissue blocks are widely used to identify clinically actionable molecular alterations or perform retrospective molecular studies. Our goal was to quantify ...degradation of DNA occurring during mid to long-term storage of samples in usual conditions. We selected 46 FFPE samples of surgically resected carcinomas of lung, colon, and urothelial tract, of which DNA had been previously extracted. We performed a second DNA extraction on the same blocks under identical conditions after a median period of storage of 5.5 years. Quantitation of DNA by fluorimetry showed a 53% decrease in DNA quantity after storage. Quantitative PCR (qPCR) targeting
KRAS
exon 2 showed delayed amplification of DNA extracted after storage in all samples but one. The qPCR/fluorimetry quantification ratio decreased from 56 to 15% after storage (
p
< 0.001). Overall, remaining proportion of DNA analyzable by qPCR represented only 11% of the amount obtained at first extraction. Maximal length of amplifiable DNA fragments assessed with a multiplex PCR was reduced in DNA extracted from stored tissue, indicating that DNA fragmentation had increased in the paraffin blocks during storage. Next-generation sequencing was performed on 12 samples and showed a mean 3.3-fold decrease in library yield and a mean 4.5-fold increase in the number of single-nucleotide variants detected after storage. In conclusion, we observed significant degradation of DNA extracted from the same FFPE block after 4 to 6 years of storage. Better preservation strategies should be considered for storage of FFPE biopsy specimens.
Cholangiocarcinoma is an aggressive biliary neoplasm lacking effective therapeutic agents. Immunotherapies targeting the PD-L1/PD-1 immune checkpoint have shown encouraging results in solid and ...hematologic cancers in clinical trials. Response to these immunomodulators is correlated with PD-L1 expression. Our goal was to characterize PD-L1 expression in intra-hepatic (iCCA) and perihilar (pCCA) cholangiocarcinomas, and to correlate our results with clinicopathological features, density of tumor-infiltrating lymphocytes (TILs) and PD-1 expression.A series of 58 iCCAs and 41 pCCAs was included in the study. PD-L1, PD-1 and CD3 expression was investigated using immunohistochemistry. Density of TILs was evaluated by immunohistochemistry using a quantitative score of CD3-stained intratumoral lymphocytes.PD-L1 expression by neoplastic cells was observed in 9 cases (9%, 5 iCCAs and 4 pCCAs). PD-L1 positive inflammatory cell aggregates were identified in 46% (n = 46) of the cases (31 iCCAs and 15 pCCAs). PD-L1 expression by either neoplastic or inflammatory cells was associated to high density of CD3-positive TILs (p = 0.01 and p = 0.005, respectively). The number of PD-L1 positive inflammatory cell aggregates was higher in tumors with high PD-1 expression (p < 0.0001).Altogether, PD-L1 in iCCA and pCCA is mainly expressed in tumors with high density of TILs. Our results suggest that CCAs with dense intratumoral lymphocytic infiltration might represent good candidates for PD-L1/PD-1 blocking agents.
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