Neoadjuvant chemotherapy alters the balance of effector to suppressor immune cells in advanced ovarian cancer.

BACKGROUND: At diagnosis, tumor-infiltrating lymphocytes (TILs) are prognostic in epithelial ovarian cancer (EOC). We recently demonstrated that neoadjuvant chemotherapy (NACT) significantly increased stromal TILs. Here, we investigated the impact of NACT on immune subpopulations with a particular focus on the balance of immune-reactive to tolerant subpopulations. MATERIALS AND METHODS: Tissue microarrays of EOC (145 pre-NACT, 139 post-NACT) were analyzed for CD3+, CD8+, FOXP3+, CD68+, and CD163+ by immunohistochemistry and CD4+ cells from deduction. Stromal TILs scored as percentage of stromal area, while intra-epithelial TILs scored as number of TILs in contact with tumor cells/HPF. Differences were evaluated by Wilcoxon or Chi square tests, Wilcoxon signed-rank for paired analyses, and cox model for PFS and OS. RESULTS: NACT significantly increased stromal CD3+ (p = 0.003) and CD8+ (p = 0.001) and intra-epithelial CD8+ (p = 0.022) and CD68+ (p = 0.0003) infiltration in unmatched samples and among paired samples for stromal CD3+ and CD8+. Neither CD3+, CD8+, CD4+, and CD68+ nor CD163+ expression correlated with outcome at diagnosis or post NACT. Using median value as a cut-off, high stromal CD8+/FOXP3+ ratio (HR = 0.59; p = 0.017) and high stromal CD3+/FOXP3+ ratio post NACT were associated with prolonged PFS (p = 0.0226). The more the balance shifted in favor of effector versus regulatory TILs, the better the survival. Similarly, high CD68+/CD163+ ratio post NACT improved PFS (p = 0.0445). CONCLUSION: NACT has a significant impact on the balance of immune-reactive to immune-tolerant subpopulations and a high ratio of CD8+/FOXP3+, CD3+/FOXP3+, and CD68+/CD163+ post NACT was significantly associated with improved outcomes. Whether this could select patients for immunotherapy in the post-operative setting should be investigated.

Adipose Tissue Properties in Tumor-Bearing Breasts.

The tissue stroma plays a major role in tumors' natural history. Most programs for tumor progression are not activated as cell-autonomous processes but under the conditions of cross-talks between tumor and stroma. Adipose tissue is a major component of breast stroma. This study compares adipose tissues in tumor-bearing breasts to those in tumor-free breasts with the intention of defining a signature that could translate into markers of cancer risk. In tumor-bearing breasts, we sampled adipose tissues adjacent to, or distant from the tumor. Parameters studied included: adipocytes size and density, immune cell infiltration, vascularization, secretome and gene expression. Adipose tissues from tumor-bearing breasts, whether adjacent to or distant from the tumor, do not differ from each other by any of these parameters. By contrast, adipose tissues from tumor-bearing breasts have the capacity to secrete twice as much interleukin 8 (IL-8) than those from tumor-free breasts and differentially express a set of 137 genes of which a significant fraction belongs to inflammation, integrin and wnt signaling pathways. These observations show that adipose tissues from tumor-bearing breasts have a distinct physiological status from those from tumor-free breasts. We propose that this constitutive status contributes as a non-cell autonomous process to determine permissiveness for tumor growth.

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.

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.

Whole exome sequencing of rare aggressive breast cancer histologies.

Little is known about mutational landscape of rare breast cancer (BC) subtypes. The aim of the study was to apply next generation sequencing to three different subtypes of rare BCs in order to identify new genes related to cancer progression. We performed whole exome and targeted sequencing of 29 micropapillary, 23 metaplastic, and 27 pleomorphic lobular BCs. Micropapillary BCs exhibit a profile comparable to common BCs: PIK3CA, TP53, GATA3, and MAP2K4 were the most frequently mutated genes. Metaplastic BCs presented a high frequency of TP53 (78 %) and PIK3CA (48 %) mutations and were recurrently mutated on KDM6A (13 %), a gene involved in histone demethylation. Pleomorphic lobular carcinoma exhibited high mutation rate of PIK3CA (30 %), TP53 (22 %), and CDH1 (41 %) and also presented mutations in PYGM, a gene involved in glycogen metabolism, in 8 out of 27 samples (30 %). Further analyses of publicly available datasets showed that PYGM is dramatically underexpressed in common cancers as compared to normal tissues and that low expression in tumors is correlated with poor relapse-free survival. Immunohistochemical staining on formalin-fixed paraffin-embedded tissues available in our cohort of patients confirmed higher PYGM expression in normal breast tissue compared to equivalent tumoral zone. Next generation sequencing methods applied on rare cancer subtypes can serve as a useful tool in order to uncover new potential therapeutic targets. Sequencing of pleomorphic lobular carcinoma identified a high rate of alterations in PYGM. These findings emphasize the role of glycogen metabolism in cancer progression.

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.

BACKGROUND: 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. METHODS: A retrospective chart review was conducted on all patients treated for oropharyngeal SCC between January 2007 and June 2009. P16 expression status by immunohistochemistry and HPV DNA viral load by quantitative polymerase chain reaction (qPCR) were evaluated on routine pretreatment tumor samples. RESULTS: One hundred thirty-three patients (94 men and 39 women) were included in the study. Mean age was 59 years. One hundred twenty-two lesions (92%) were localized to lymphoid areas. Sixty-seven patients (50%) were p16+, and 87 patients (65%) harbored HPV DNA. The p16+/HPV DNA+ profile (48%) was associated with the most favorable prognosis. HPV16 was responsible for the majority of the infections (89%). CONCLUSION: HPV is common among oropharyngeal SCC in France, and acts as an independent prognostic factor.

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.

BACKGROUND: Our 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. METHODS: Tumor 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. RESULTS: No 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. CONCLUSION: These 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.

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.

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.

Imaging of the distribution of (90)y-ibritumomab tiuxetan in bone marrow and comparison with pathology.

PURPOSE: Radioimmunotherapy with anti-CD20 antibodies (Abs) labeled with beta-emitters is now used in the treatment of non-Hodgkin's lymphoma (NHL). Because (90)Y is a pure beta-emitter, no direct image of its distribution can be obtained in humans. In this paper, we present in this study imaging data of (90)Y-Ab distribution in human-mantle-cell lymphoma within a mouse model. Describing the actual distribution of the radionuclide at the level of particles range may have important impact on patient dosimetry and therapy treatment planning. EXPERIMENTAL DESIGN: NOD/SCID mice were grafted with a human NHL cell line that involves the bone marrow. The mice were treated with (90)Y-ibritumomab tiuxetan (Zevalin); Schering AG, Germany) and sacrificed 2 hours after Zevalin administration. Tissue sections were then prepared and viewed under conventional microscopy. The distribution of the radioactivity in mouse femur was determined by using digital autoradiography and subsequently correlated with immunohistochemical results. RESULTS: Various extent of bone marrow infiltration was investigated and found to be reproducible. Zevalin uptake was heterogeneous within the bone marrow. However, unspecific mouse monoclonal uptake by accessory myeloid cells gave nonspecific background radioactivity. Treating mice with an irrelevant mouse IgG1 monoclonal antibody (mAb) before Zevalin injection controlled this unspecific uptake, and images were strongly correlated with bone marrow infiltration on histologic analysis. CONCLUSIONS: Our model was reproducible, and allows for the study of various bone marrow involvement with good sensitivity. We demonstrated that imaging of the beta-emitter was possible with good image quality and that (90)Y-Zevalin is distributed heterogeneously within bone marrow. These data suggest that detailed pharmacokinetics may be developed with this model.