Françoise Drusch

Portrait of Ependymoma Recurrence in Children: Biomarkers of Tumor Progression Identified by Dual-Color Microarray-Based Gene Expression Analysis

Background Children with ependymoma may experience a relapse in up to 50% of cases depending on the extent of resection. Key biological events associated with recurrence are unknown. Methodology/Principal Findings To discover the biology behind the recurrence of ependymomas, we performed CGHarray and a dual-color gene expression microarray analysis of 17 tumors at diagnosis co-hybridized with the corresponding 27 first or subsequent relapses from the same patient. As treatment and location had only limited influence on specific gene expression changes at relapse, we established a common signature for relapse. Eighty-seven genes showed an absolute fold change ≥2 in at least 50% of relapses and were defined as the gene expression signature of ependymoma recurrence. The most frequently upregulated genes are involved in the kinetochore (ASPM, KIF11) or in neural development (CD133, Wnt and Notch pathways). Metallothionein (MT) genes were downregulated in up to 80% of the recurrences. Quantitative PCR for ASPM, KIF11 and MT3 plus immunohistochemistry for ASPM and MT3 confirmed the microarray results. Immunohistochemistry on an independent series of 24 tumor pairs at diagnosis and at relapse confirmed the decrease of MT3 expression at recurrence in 17/24 tumor pairs (p = 0.002). Conversely, ASPM expression was more frequently positive at relapse (87.5% vs 37.5%, p = 0.03). Loss or deletion of the MT genes cluster was never observed at relapse. Promoter sequencing after bisulfite treatment of DNA from primary tumors and recurrences as well as treatment of short-term ependymoma cells cultures with a demethylating agent showed that methylation was not involved in MT3 downregulation. However, in vitro treatment with a histone deacetylase inhibitor or zinc restored MT3 expression. Conclusions/Significance The most frequent molecular events associated with ependymoma recurrence were over-expression of kinetochore proteins and down-regulation of metallothioneins. Metallothionein-3 expression is epigenetically controlled and can be restored in vitro by histone deacetylase inhibitors.

Circulating Tumor Cells in Lung Cancer

Circulating tumor cells (CTCs) have emerged as potential biomarkers in several cancers such as colon, prostate, and breast carcinomas, with a correlation between CTC number and patient prognosis being established by independent research groups. The detection and enumeration of CTCs, however, is still a developing field, with no universal method of detection suitable for all types of cancer. CTC detection in lung cancer in particular has proven difficult to perform, as CTCs in this type of cancer often present with nonepithelial characteristics. Moreover, as many detection methods rely on the use of epithelial markers to identify CTCs, the loss of these markers during epithelial-to-mesenchymal transition in certain metastatic cancers can render these methods ineffective. The development of personalized medicine has led to an increase in the advancement of molecular characterization of CTCs. The application of techniques such as FISH and RT-PCR to detect EGFR, HER2, and KRAS abnormalities in lung, breast, and colon cancer, for example, could be used to characterize CTCs in real time. The use of CTCs as a ‘liquid biopsy’ is therefore an exciting possibility providing information on patient prognosis and treatment efficacy. This review summarizes the state of CTC detection today, with particular emphasis on lung cancer, and discusses the future applications of CTCs in helping the clinician to develop new strategies in patient treatment.

Diagnosis of HPV-driven head and neck cancer with a single test in routine clinical practice.

Accurate screening of HPV-driven head and neck squamous cell carcinoma is a critical issue. Although there are commercial direct and indirect assays for HPV-related head and neck squamous cell carcinoma, none are ideal. Recently, a novel RNA in situ hybridization test (the RNAscope HPV-test) has been developed for the detection of high-risk HPV E6/E7 mRNA in formalin-fixed paraffin-embedded tissue. However, validation of this assay against the ‘gold standard’ (identification of high-risk HPV E6/E7 mRNA in fresh-frozen tissue by quantitative real-time (qRT)–PCR) has only been reported by one team. Formalin-fixed paraffin-embedded samples from 50 patients with tonsil or tongue base carcinoma were tested using the RNAscope HPV-test, p16 immunohistochemistry, and chromogenic in situ hybridization for high-risk HPV-DNA. The results were compared with those of qRT–PCR on matched fresh-frozen samples. Compared with the reference test, the sensitivity, specificity, positive, and negative predictive values of the RNAscope HPV-test and of p16 immunohistochemistry were 93%, 94%, 96%, 88% and 96%, 93%, 96%, and 93%, respectively. Five cases were discrepant between the RNAscope HPV-test and p16-immunohistochemisrty. The RNAscope HPV-test demonstrated excellent analytical performance against the ‘gold standard’ and is easier to interpret than chromogenic in situ hybridization. p16-immunohistochemistry also performed very well, however its main weakness is that it is an indirect marker of the presence of HPV. These data suggest that the RNAscope HPV-test is a promising test that could be developed as a clinical standard for the precise identification of HPV-driven oropharyngeal squamous cell carcinoma.

Human papillomavirus prevalence and prognostic implication in oropharyngeal squamous cell carcinomas

Human papillomavirus (HPV)‐related oropharyngeal squamous cell carcinoma (SCC) is associated with favorable survival. The purpose of this study was to evaluate the prevalence and prognostic significance of the HPV infection through both the p16 expression status and the oncogenic HPV DNA viral load.

Diagnosis of HPV driven oropharyngeal cancers: Comparing p16 based algorithms with the RNAscope HPV-test

BACKGROUND Accurate identification of HPV-driven oropharyngeal cancer (OPC) is a major issue and none of the current diagnostic approaches is ideal. An in situ hybridization (ISH) assay that detects high-risk HPV E6/E7 mRNA, called the RNAscope HPV-test, has been recently developed. Studies have suggested that this assay may become a standard to define HPV-status. METHODS To further assess this test, we compared its performance against the strategies that are used in routine clinical practice: p16 immunohistochemistry (IHC) as a single test and algorithms combining p16-IHC with HPV-DNA identification by PCR (algorithm-1) or ISH (algorithm-2). RESULTS 105 OPC specimens were analyzed. The prevalence of HPV-positive samples varied considerably: 67% for p16-IHC, 54% for algorithm-1, 61% for algorithm-2 and 59% for the RNAscope HPV-test. Discrepancies between the RNAscope HPV-test and p16-IHC, algorithm-1 and 2 were noted in respectively 13.3%, 13.1%, and 8.6%. The 4 diagnostic strategies were able to identify 2 groups with different prognosis according to HPV-status, as expected. However, the greater survival differential was observed with the RNAscope HPV-test [HR: 0.19, 95% confidence interval (CI), 0.07-0.51, p=0.001] closely followed by algorithm-1 (HR: 0.23, 95% CI, 0.08-0.66, p=0.006) and algorithm-2 (HR: 0.26, 95% CI, 0.1-0.65, p=0.004). In contrast, a weaker association was found when p16-IHC was used as a single test (HR: 0.33, 95% CI, 0.13-0.81, p=0.02). CONCLUSIONS Our findings suggest that the RNAscope HPV-test and p16-based algorithms perform better that p16 alone to identify OPC that are truly driven by HPV-infection. The RNAscope HPV-test has the advantage of being a single test.

Expression of EPHRIN-A1, SCINDERIN and MHC class I molecules in head and neck cancers and relationship with the prognostic value of intratumoral CD8+T cells

BackgroundOur group has previously shown that EPHRIN-A1 and SCINDERIN expression by tumor cells rendered them resistant to cytotoxic T lymphocyte-mediated lysis. Whereas the prognostic value of EPHRIN-A1 expression in cancer has already been studied, the role of SCINDERIN presence remains to be established. In the present work, we investigated the prognosis value of EPHRIN-A1 and SCINDERIN expression in head and neck carcinomas. In addition, we monitored the HLA-class I expression by tumor cells and the presence of tumor-infiltrating CD8+ T cells to evaluate a putative correlation between these factors and the survival prognosis by themselves or related to EPHRIN-A1 and SCINDERIN expression.MethodsTumor tissue sections of 83 patients with head and neck cancer were assessed by immunohistochemistry for the expression of EPHRIN-A1, SCINDERIN, HLA class I molecules and the presence of CD8+ T cells.ResultsNo significant prognosis value could be attributed to these factors independently, despite a tendency of association between EPHRIN-A1 and a worse clinical outcome. No prognostic value could be observed when CD8+ T cell tumor infiltration was analyzed combined with EPHRIN-A1, SCINDERIN or HLA class I expression.ConclusionThese results highlight that molecules involved in cancer cell resistance to cytotoxic T lymphocytes by themselves are not a sufficient criteria for prognosis determination in cancer patients. Other intrinsic or tumor microenvironmental features should be considered in prognostic evaluation.

Oropharyngeal cancers: Significance of HPV16 detection in neck lymph nodes

BACKGROUND An increasing proportion of oropharyngeal squamous cell carcinomas (OPSCCs) is associated with human papillomavirus (HPV) type 16 infection. Several authors have suggested that HR-HPV DNA could be used as a marker of metastases in cervical cancers. Although HPV16 DNA has been detected in neck lymph node (LN) metastases of HPV16-positive OPSCC, its significance remains controversial. Does this presence correlate to metastatic involvement or is it just the consequence of LN filter function? OBJECTIVES This study aims to analyse the relationship between HPV16 detection in neck LNs of HPV16-positive OPSCC and their pathological status. STUDY DESIGN HP16-viral load (VL) was quantified by real-time-polymerase-chain reaction in primary tumours and neck LNs, in 11 patients with HPV16-positive OPSCC and in three patients with HPV16-negative OPSCC. HPV16 in situ hybridisation and p16 immunohistochemistry were performed in all LNs. RESULTS A total of 45 LN levels were assessed. HPV16 DNA was not identified in HPV16-negative OPSCC LNs. All metastatic LNs from HPV16-positive OPSCC had a high VL and the viral DNA was located within tumoural cells. Among 27 pathologically tumour-free LN (PTFLN) levels 16/27 had no detectable VL, whereas the VL was low or medium (<10(5)copies/million cells) in 8/27 and high (>10(5)copies/million cells) in 3/27 PTFLN. In the latter group, no metastatic cell was identified and the viral DNA was located in immune cells. CONCLUSION HPV16 detection in LN is explained by its presence within either metastatic cells or immune cells. HPV16 detection in PTFLN is not necessarily correlated to occult LN metastases.

Refinement of high-risk endometrial cancer classification using DNA damage response biomarkers: a TransPORTEC initiative

The TransPORTEC consortium previouslclassified high-risk endometrial cancer including poor-risk histologies such as clear cells, into four molecular subtypes “POLE mutated,” “microsatellite unstable,” “TP53 mutated,” and “no specific molecular profile.” We evaluated whether DNA damage response biomarkers could further refine this high-risk tumors classification, in particular the heterogeneous “no specific molecular profile” and “TP53 mutated” subsets recently qualified as poor prognosis in high-risk endometrial cancer. DNA damage response biomarkers including proteins involved in DNA damage (δ-H2AX), homologous recombination (RAD51), regulators of error-prone Non Homologous End-Joining (DNA-pk, FANCD2), and PARP-1 were evaluated in 116 high-risk tumors by immunohistochemistry. CD8 and PD-1 expression by immunochemistry and mutation analyses were performed previously. Survival outcome were calculated using Kaplan-Meier and Log-rank test. None of the DNA damage response biomarkers alone were prognostic. However markers were informative within molecular subsets. Among the “no specific molecular profile” subset, δ-H2AX+ was significantly predictive of poor disease free survival (Hazard Ratio = 2.56; p = 0.026), and among “TP53 mutated,” a DNA-pk+/FANCD2- profile (favouring error-prone Non Homologous End-Joining) predicted worst disease free survival (Hazard Ratio = 4.95; p = 0.009) resulting in five distinct prognostic subgroups from best to worst prognosis: group1 “POLE mutated/Microsatellite unstable” > group2 “no specific molecular profile with no DNA damage” > group3 “TP53 mutated/Non Homologous End-Joining negative” > group4 “no specific molecular profile with high DNA damage” > group5 “TP53 mutated/Non Homologous End-Joining positive”; p = 0.0002). Actionable targets were also different among subsets. Group3 had significantly higher infiltration of PD-1+ immune cells (p = 0.003), segregating with group1. Group2 had frequent PI3K pathway mutations and ER positivity. While group5, with the worst prognosis, had high DNA damage and PARP-1 expression providing a rationale for PARP inhibition. Our findings have refined the TransPORTEC prognostic classification of high-risk endometrial cancer into five distinct subgroups by integrating DNA damage response biomarkers and identified molecular subtype-specific therapeutic strategies.

Abstract B02: DNA repair landscape in High Grade Ovarian Cancer (HGOC) and evolution with neo-adjuvant chemotherapy

Background: HGOC is frequently diagnosed at an advanced stage where NeoAdjuvant Chemotherapy (NACT) provides an essential component of the treatment strategy. HGOC are initially very chemosensitive, putatively due to DNA repair defects. While BRCA mutations explain impaired homologous recombination in 12% to 15% of HGOC, loss of other DNA repair proteins may also be relevant. Unfortunately despite initial response rates of 80%, most invariably relapse. Chemo-resistance mechanisms are poorly understood and previous studies have only focused on the characterization at diagnosis. However given their chemosensitivity, profiling the post-NACT tumor (cleared of sensitive cells and enriched for chemoresistant clones) may be more relevant to uncover mechanisms of platinum resistance. We aimed to evaluate the evaluate the expression of key DNA repair biomarkers in a large series of HGOC tumors obtained at baseline, Post-NACT and at relapse. Methods: TMA was constructed using sequential FFPE tumor samples of high-grade ovarian cancer collected at diagnosis (pre-NACT) (N=143), post-NACT (N=139) and at relapse (N=36) in 170 patients, encompassing 115 patients with sequential tumor samples. Expression of 53BP1, PAR, PARP-1 and ATM were scored by immunohistochemistry using an H-score (0-300) with biomarker negative defined as H-score = Results: At diagnosis, a significant proportion of tumors showed loss of DNA repair proteins: 61% were PAR-negative, and 24%, 15% and 3% negative for ATM, 53BP1 and PARP-1 respectively and a fifth of tumors showed concomitant loss in 2 or more DNA proteins. There was an overall decrease in DNA repair protein expression with NACT, which was significant for PAR (pre-NACT vs post-NACT H-score= 45 vs 19, p=0.0025, N=201) and PARP-1(H-score=208 vs 190; p=0.0149, N=205). In addition paired samples analysis of matched pre- and post-NACT samples showed complete loss of biomarker expression after treatment in a subset (24%, 16%, 6% and 3% for PAR, ATM, TP53BP1 and PARP-1). When comparing post-NACT to relapsed samples, PAR expression increased significantly (mean H-score=19 vs 54, p=0,025). The prognostic value of loss of each DNA repair at diagnosis and after NACT was evaluated. At diagnosis, 53BP1-negative tumors were associated with a significantly worst PFS (HR=3.484; p: 0.0025). Post-NACT, ATM-negative tumors had a significantly worst PFS (HR=1.690; p: 0.0374). As PAR, TP53BP1 and ATM were shown not to be correlated in individual tumors (Pearson correlation), the impact of combined biomarker loss on PFS was evaluated. At diagnosis, double TP53BP1/PAR-negative tumors predicted shorter PFS (PFS: median=17.65 vs 23.23 mths, p:0.0372, HR=0.2237, N=58), while post-NACT double ATM/PAR-negativity was significantly predictive of poor outcome in terms of PFS and OS (PFS: median=17.35 vs 23.77 mths, p:0.0198, HR=0.4759; OS: median=35 vs 50 mths, p:0.0120, HR=0.3602; N=80). Conclusions: We present the first study of change in DNA repair protein expression with NACT. At diagnosis, HGOC is associated with loss of key DNA repair proteins in a significant proportion of patients and NACT can induce significant loss of DNA repair proteins. Combined loss of DNA repair proteins was significantly predictive of survival. Work in ongoing to extend this analysis to a greater panel of DNA repair biomarkers. Citation Format: Aurelie Auguste, Soizick Mesnage, Audrey Le Formal, Elena Cojocaru, Francoise Drusch, Julien Adam, Sebastien Gouy, Enrica Bentivegna, Catherine Lhomme, Patricia Pautier, Catherine Genestie, Alexandra Leary. DNA repair landscape in High Grade Ovarian Cancer (HGOC) and evolution with neo-adjuvant chemotherapy [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr B02.

Abstract 4286: Gene expression profiles in adipose tissue of cancer-bearing breasts differ from that of tumor-free breasts

Introduction Adipose tissue - long believed to be no more than an energy storage organ - is metabolically active and consists of a variety of cell types. Adipocytes, fibroblasts and macrophages reside in adipose tissue and may have tumor-promoting properties through the release of cytokines and other growth stimulating molecules. Further, adipose tissue has the capacity of storing pollutants and carcinogens. Breast tumors are embedded in adipose tissue and the direct interface between tumor and adipose tissue in the breast defines a micro-environment potentially fostering tumor initiation and/or progression. The aim of this study was to investigate the transcriptome of adipose tissue from cancer-bearing breasts, localized either close to or distant from the tumor, in addition to comparing it to adipose tissue from tumor-free breasts. Methods We collected adipose tissue from n = 33 tumor-bearing breasts (a) close to ( 5cm) the tumor and from n = 5 tumor-free breasts to investigate gene expression profiles in these three series of tissues. Gene expression was measured from RNA isolated from fresh frozen adipose tissue using Illumina HT12 bead arrays. Quantile normalized expression values were analyzed between (a) and (b) as well as between (a)+(b) and (c) using t-test. Data was filtered by standard variation of 0.95. P-values ≤0.001 were considered significant. Results We did not observe any significant differences in gene expression when comparing adipose tissue close to or distant from the tumor. By contrast, expression profiles in adipose tissue of tumor-free breasts clearly differed from that of cancer-bearing breasts. We observed 81 genes significantly differentially expressed between the two groups. Among the overexpressed genes were the previously identified genes MARCO and VSIG4, which were 1.5-fold upregulated in adipose tissue from diseased patients (both p-value = 0.001). Both genes are involved in processes of immunity and inflammation, promoting immune-tolerance in macrophages and T-cells. Other differentially expressed genes also relate to these pathways including CD163, CCL13 and C3. This striking role of inflammatory and immune-modulatory processes was further supported by pathway analyses. Conclusions Breast adipose tissues of cancer-bearing breasts show distinct gene expression profiles from that of tumor-free breasts, whereas tumor-distant and tumor-close adipose tissues are similar. However the selected distance of ≤2cm from the tumor may by insufficient to capture the tumor micro-environment. Nevertheless, this rather provocative result raises issues related to the status of breast adipose tissue that may define individually determined cancer fields. Differentially expressed genes are, to a large proportion, involved in immunity-related and inflammatory processes, emphasizing that adipose tissue is an important contributor to one of the hallmarks of cancer. Citation Format: Dominique Scherer, Isabelle Miran, Chafika Mazouni, Benjamin Sarfati, Marine Bernard, Julien Adam, Emilie Louvet, Francoise Drusch, Philippe Vielh, Khalid Alhazmi, Cornelia M. Ulrich, Suzette Delaloge, Jean Feunteun. Gene expression profiles in adipose tissue of cancer-bearing breasts differ from that of tumor-free breasts. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4286.