Aurélie Cazes

Pulmonary Resection for Metastases of Colorectal Adenocarcinoma

BACKGROUND Surgery is a safe and effective treatment for patients with lung metastases from colorectal carcinoma. Combining chemotherapy and surgery seems to prolong survival time after metastasectomy. Our purpose was to review the effectiveness of surgery with time and evolving managements. METHODS The records of 127 patients were retrospectively analyzed. The characteristics of primary cancer, lung metastases, resections, and associated therapy were studied according to their incidence on survival. RESULTS There were 74 male and 53 female patients (mean age, 65 years); 223 operations were performed and 314 metastases were resected. Completeness of surgery (n = 117) was the main factor for prolonged survival (5- and 10-year survival, 41% and 27%, versus 0%). There was no factor of significantly better prognosis, but a tendency to higher survival rates was observed in cases of single metastasis, in patients undergoing several lung operations, and in patients in whom liver metastases were previously removed. Three of 7 patients with mediastinal lymph node involvement survived more than 5 years; 58 patients were operated on before January 2000, and 59 between January 2000 and December 2007. Five-year survival rates were 35.1% versus 63.5%, respectively (p = 0.0096), probably related to better selection with modern workup, more frequent use of chemotherapy, and repeated pulmonary resections. CONCLUSIONS Different treatment protocols were reported in the literature and in our series with time, resulting in better survival rates and a more aggressive surgical tendency. The beneficial role of such combined therapy justifies further research, including prospective trials.

Orchestration and Prognostic Significance of Immune Checkpoints in the Microenvironment of Primary and Metastatic Renal Cell Cancer

Purpose: Clear cell renal cell carcinoma (ccRCC) has shown durable responses to checkpoint blockade therapies. However, important gaps persist in the understanding of its immune microenvironment. This study aims to investigate the expression and prognostic significance of immune checkpoints in primary and metastatic ccRCC, in relation with mature dendritic cells (DC) and T-cell densities. Experimental Design: We investigated the infiltration and the localization of CD8+ T cells and mature DC, and the expression of immune checkpoints (PD-1, LAG-3, PD-L1, and PD-L2) in relation with prognosis, in 135 primary ccRCC tumors and 51 ccRCC lung metastases. RNA expression data for 496 primary ccRCC samples were used as confirmatory cohort. Results: We identify two groups of tumors with extensive CD8+ T-cell infiltrates. One group, characterized by high expression of immune checkpoints in the absence of fully functional mature DC, is associated with increased risk of disease progression. The second group, characterized by low expression of immune checkpoints and localization of mature DC in peritumoral immune aggregates (tertiary lymphoid structures), is associated with good prognosis. Conclusions: The expression of the immune checkpoints and the localization of DC in the tumor microenvironment modulate the clinical impact of CD8+ T cells in ccRCC. Clin Cancer Res; 21(13); 3031–40. ©2015 AACR.

Competitive allele specific TaqMan PCR for KRAS, BRAF and EGFR mutation detection in clinical formalin fixed paraffin embedded samples

BACKGROUND The development of targeted therapies has created a need for robust molecular characterization of cancer and it has become a challenge to validate methods to ensure accuracy in tumor mutation testing. METHODS The current study was designed to evaluate KRAS, BRAF and EGFR genotyping by Competitive Allele Specific hydrolysis probes (TaqMan) PCR technology (CAST), on suboptimal formalin fixed paraffin embedded (FFPE) tumor samples. Assays were calibrated on FFPE samples and a minimal quantification cycle (Cq) cut-off was determined to standardize analyses and avoid over-interpretation of degraded material. Sensibility, specificity and blinded clinical sample screenings (n=63) were evaluated. RESULTS CAST PCR allowed efficient amplification of FFPE samples, probes were highly specific and all assays had a sensibility inferior to 1% except for the EGFR p.T790M assay. 60/63 samples were correctly typed. The three missed mutations were EGFR exon 19 deletions that were not recognized by the DEL19 assays that were used. CONCLUSIONS This technology is less laborious and prevent crossover of PCR products as compared to multistep methods. TaqMan® Mutation Detection assay is an important technology to consider in the field of mutation detection for KRAS, BRAF and EGFR point mutation screening. Assay calibration on FFPE samples may prevent erroneous interpretations that will ultimately harm clinical oncology practice.

Very Late Heart Transplant Rejection Is Associated with Microvascular Injury, Complement Deposition and Progression to Cardiac Allograft Vasculopathy

In heart transplants, the significance of very late rejection (after 7 years post‐transplant, VLR) detected by routine endomyocardial biopsies (EMB) remains uncertain. Here, we assessed the prevalence, histopathological and immunological phenotype, and outcome of VLR in clinically stable patients. Between 1985 and 2009, 10 662 protocol EMB were performed at our institution in 398 consecutive heart transplants recipients. Among the 196 patients with >7‐year follow‐up, 20 (10.2%) presented subclinical ≥3A/2R‐ISHLT rejection. The VLR group was compared to a matched control group of patients without rejection. All biopsies were stained for C4d/C3d/CD68 with sera screened for the presence of donor‐specific antibodies (DSAs). In addition to cellular infiltrates with myocyte damage, 60% of VLR patients had evidence of intravascular macrophages. C4d and/or C3d‐capillary deposition was found in 55% VLR EMB. All cases of VLR associated with microcirculation injury had DSAs (mean DSAmax−MFI = 1751 ± 583). This entity was absent from the control group (p < 0.0001). Finally, after a similar follow‐up postreference EMB of 6.4 ± 1 years, the mean of CAV grade was 0.76 ± 0.18 in the control group compared to 2.06 ± 0.26 in the VLR group respectively, p = 0.001). There was no difference in patient survival between study and control groups. In conclusion, VLR is frequently associated with complement‐cascade activation, microvascular injury and DSA, suggesting an antibody‐mediated process. VLR is associated with a dramatic progression to severe CAV in long‐term follow‐up.

Early T Cell Signalling Is Reversibly Altered in PD-1+ T Lymphocytes Infiltrating Human Tumors

To improve cancer immunotherapy, a better understanding of the weak efficiency of tumor-infiltrating T lymphocytes (TIL) is necessary. We have analyzed the functional state of human TIL immediately after resection of three types of tumors (NSCLC, melanoma and RCC). Several signalling pathways (calcium, phosphorylation of ERK and Akt) and cytokine secretion are affected to different extents in TIL, and show a partial spontaneous recovery within a few hours in culture. The global result is an anergy that is quite distinct from clonal anergy induced in vitro, and closer to adaptive tolerance in mice. PD-1 (programmed death -1) is systematically expressed by TIL and may contribute to their anergy by its mere expression, and not only when it interacts with its ligands PD-L1 or PD-L2, which are not expressed by every tumor. Indeed, the TCR-induced calcium and ERK responses were reduced in peripheral blood T cells transfected with PD-1. Inhibition by sodium stibogluconate of the SHP-1 and SHP-2 phosphatases that associate with several inhibitory receptors including PD-1, relieves part of the anergy apparent in TIL or in PD-1-transfected T cells. This work highlights some of the molecular modifications contributing to functional defects of human TIL.

Multiple Lung Cancers Prognosis : What About Histology?

BACKGROUND Among multiple lung cancers (MLC), some may have similar histologic classification. Demonstrating that the second tumor is a metastasis would change the stage and consequently the management. Our purpose was to reconsider this consequence. METHODS We reviewed 234 patients (194 male and 40 female, from 37 to 83 years of age) with synchronous and metachronous non-small cell MLC. Surgery consisted of a potentially curative complete resection with lymphadenectomy. Patients with similar histologic MLC (considered as metastasis) were compared with those with different histologic classification in terms of MLC chronology, type of resection, pT and pN, stage, and overall survival. RESULTS There were 116 metachronous (ipsilateral, n = 48; contralateral, n = 68) and 118 synchronous MLCs (bilateral, n = 10; same lobe, n = 57; other lobe, n = 51). Pneumonectomy was performed in 77 patients, lobectomy in 103, and lesser resection in 54. Histologic classification was similar in 57.9% of patients and different in 42.1%. The 5-year survival rates tended to be lower in patients with synchronous MLCs (23.4% versus 31.6%; p = 0.07). They were higher when synchronous MLCs were located in the same lobe than if they were located in another lobe (29.9% versus 15.6%; p = 0.022). Whatever the type of MLC, the 5-year survival rates were not correlated with similar or different histologic classification. CONCLUSIONS Our analysis supports that surgery is safe and warranted in MLC patients even if synchronous MLCs present ominously. Changing the staging by establishing the diagnosis of metastasis is probably an important issue warranting further biologic research, but according to our results this diagnosis must not in any case preclude surgery.

Histologic subtypes, immunohistochemistry, FISH or molecular screening for the accurate diagnosis of ALK-rearrangement in lung cancer: A comprehensive study of Caucasian non-smokers

EML4-ALK adenocarcinomas constitute a new molecular subgroup of lung tumours that respond very well to crizotinib, an ALK inhibitor. However, the diagnosis of ALK rearrangement in lung cancer is challenging. The aim of this study was to compare the diagnostic accuracy of five different methods in a series of 20 EGFR(wt/wt) lung adenocarcinomas from non- or light- smokers. Multiplex RT-PCR was considered as gold standard and identified four ALK-rearranged tumours among the 20 tested tumours. qRT-PCR got an interpretability rate of 100% and accurately typed all 20 tumours. qRT-PCR from corresponding formalin-fixed paraffin-embedded (FFPE) specimens got an interpretability rate of 65%. Out of the four previously identified ALK-rearranged cases, three were interpretable and two were retrieved using FFPE qRT-PCR. ALK break-apart FISH got an interpretability rate of 60% and accurately typed all of the twelve remaining cases. Anti-ALK immunohistochemistry (IHC) accurately typed all twenty tumours using a cut-off value of strong staining of 100% tumour cells. The 16 non ALK-rearranged tumours got no/light staining in 13 cases, and a moderate staining of 80-100% tumour cells in 3 cases. We then analysed four solid signet-ring lung adenocarcinomas. FFPE qRT-PCR, FISH and immunohistochemistry were concordant in three cases, with positive and negative results in respectively one and two cases. The fourth case, which was positive by FISH and immunohistochemistry but negative by RT-PCR, was shown to have a non-EML4-ALK ALK-rearrangement. As various factors such as RNA quality, fixation quality and type of ALK rearrangement may impede ALK screening, we propose a combined FISH/molecular biology diagnostic algorithm in which anti-ALK immunohistochemistry is used as a pre-screening step.

Recurrent inactivating mutations of ARID2 in non-small cell lung carcinoma.

In eukaryotic cells, DNA is packaged into chromatin and this compact storage in the nucleus promotes transcriptional repression of genes. Chromatin remodeling complexes such as the SWI/SNF complex are involved in making DNA accessible to transcription factors and thereby are implicated in the regulation of gene expression. Mutations and altered expression of chromatin remodeling complex genes have been described in cancer cells. Indeed, non‐small cell lung cancer cell lines have been shown to harbor mutations in SMARCA4 (BRG1), a member of the SWI/SNF complex, but evidence has been less clear in primary tumors. Recently, inactivating mutations in AT‐rich interaction domain 2 (ARID2) were found in liver cancer related to HCV infection and in melanoma. Here, we explored, using a genome‐wide strategy and subsequent sequencing of targeted genes, whether chromatin remodeling is implicated in primary lung adenocarcinoma. Two genes were individualized from the genome screening as homozygously deleted in a set of samples: JARID2 and ARID2. Subsequent analysis of the entire coding sequences showed that ARID2 loss‐of‐function mutations were found in 5% of nonsmall cell lung cancers, thereby constituting one of the most frequently mutated genes in this cancer type after TP53, KRAS, EGFR, CDKN2A and STK11.

Analysis of Spontaneous Tumor-Specific CD4 T-cell Immunity in Lung Cancer Using Promiscuous HLA-DR Telomerase-Derived Epitopes: Potential Synergistic Effect with Chemotherapy Response

Purpose: To investigate the presence and impact of spontaneous telomerase-specific CD4 T-cell responses in cancer patients. Experimental Design: A multistep approach was used to design novel pan-HLA-DR–restricted peptides from telomerase. T-cell clones isolated from cancer patients were used to characterize the polarization of telomerase-specific CD4 response. The presence of spontaneous CD4 T-cell response against telomerase was monitored in 84 metastatic non–small cell lung cancer (NSCLC) patients before first-line chemotherapy (CT) using IFN-γ ELISPOT assay. Then we analyzed the impact of the pretherapeutic telomerase-specific CD4 T immunity on clinical outcome in patients according to their respective response to CT. Results: We described four novel telomerase-derived CD4 epitopes referred as universal cancer peptides (UCP) that effectively bind to most commonly found human MHC class II alleles. UCP-specific CD4 T-cell repertoire is present in human and UCP-specific CD4 T-cell clones generated from cancer patients exhibited high avidity and are Th1 polarized. Significant frequency (38%) of naturally occurring UCP-specific T-cell responses were detected before CT in advanced NSCLC but not in healthy volunteers. This response was shown to significantly increase overall survival (OS) of patients responding to CT (Median OS: 53 vs. 40 weeks, P = 0.034). Conclusions: These results show for the first time a potential synergistic effect of telomerase-specific CD4 T-cell response with CT response in NSCLC and underline the potential role of tumor-specific CD4 T-cell response on the efficiency of conventional anticancer therapy. Clin Cancer Res; 18(10); 2943–53. ©2012 AACR.

Pathologic classification of antibody-mediated rejection correlates with donor-specific antibodies and endothelial cell activation

BACKGROUND Humoral immune responses during heart transplantation may result in antibody-mediated rejection (AMR), which is now taken into account on endomyocardial biopsy (EMB) specimens and ranked according to the pathologic AMR (pAMR) grades of the International Society for Heart and Lung Transplantation classification. This classification might benefit from new immunohistological markers and validation by others biomarkers, namely donor-specific antibodies (DSA). METHODS From the 293 protocol EMBs performed in 113 patients in our institution during a 1-year period for this prospective study, 280 EMB specimens were available with both histology and immunohistochemistry. C4d and labeling of intravascular cells by cluster of differentiation (CD) 68 were performed on paraffin sections. Available sera (n = 150) concomitant of EMB specimens were tested for the presence of DSA. All of the pAMR+ EMB specimens, along with a set of randomized pAMR0 EMB specimens, were immunolabeled for mammalian target of rapamycin (mTOR) effectors, phosphorylated 70 S6-kinase (p70S6K) and phosphorylated S6 ribosomal protein (pS6RP). RESULTS AMR was diagnosed in 37 EMB specimens (13.2%): 1 pAMR1(I+), 27 pAMR1(H+), and 9 pAMR2. The proportion of DSA-positive EMB varied according to the pAMR grade, with pAMR0, pAMR1(H+), and pAMR2 EMB presenting 17.6%, 77.3%, and 100% of DSA-positivity, respectively. Among the 30 pAMR+ specimens with available DSA testing and the 30 pAMR0 randomized specimens, mTOR pathway immunohistochemistry showed endothelial cell positivity for p70S6K in 17 pAMR+ EMB specimens (56.7%) and in 1 pAMR0 EMB specimen (3.3%). pS6RP was detected in 8 pAMR+ EMB specimens (26.7%) and in 1 pAMR0 EMB specimen (3.3%). CONCLUSIONS p70S6K and pS6RP immunohistochemistry afford new markers of AMR on EMB specimens because their expression is correlated with microcirculation inflammation and DSA. The correlation of DSA with pAMR grade suggests that this grading system is valid.