Proteomic alterations in early stage cervical cancer.

Laser capture microdissection (LCM) allows the capture of cell types or well-defined structures in tissue. We compared in a semi-quantitative way the proteomes from an equivalent of 8,000 tumor cells from patients with squamous cell cervical cancer (SCC, n = 22) with healthy epithelial and stromal cells obtained from normal cervical tissue (n = 13). Proteins were enzymatically digested into peptides which were measured by high-resolution mass spectrometry and analyzed by "all-or-nothing" analysis, Bonferroni, and Benjamini-Hochberg correction for multiple testing. By comparing LCM cell type preparations, 31 proteins were exclusively found in early stage cervical cancer (n = 11) when compared with healthy epithelium and stroma, based on criteria that address specificity in a restrictive "all-or-nothing" way. By Bonferroni correction for multiple testing, 30 proteins were significantly up-regulated between early stage cervical cancer and healthy control, including six members of the MCM protein family. MCM proteins are involved in DNA repair and expected to be participating in the early stage of cancer. After a less stringent Benjamini-Hochberg correction for multiple testing, we found that the abundances of 319 proteins were significantly different between early stage cervical cancer and healthy controls. Four proteins were confirmed in digests of whole tissue lysates by Parallel Reaction Monitoring (PRM). Ingenuity Pathway Analysis using correction for multiple testing by permutation resulted in two networks that were differentially regulated in early stage cervical cancer compared with healthy tissue. From these networks, we learned that specific tumor mechanisms become effective during the early stage of cervical cancer.

Integrative Kinome Profiling Identifies mTORC1/2 Inhibition as Treatment Strategy in Ovarian Clear Cell Carcinoma.

Purpose: Advanced-stage ovarian clear cell carcinoma (OCCC) is unresponsive to conventional platinum-based chemotherapy. Frequent alterations in OCCC include deleterious mutations in the tumor suppressor ARID1A and activating mutations in the PI3K subunit PIK3CA In this study, we aimed to identify currently unknown mutated kinases in patients with OCCC and test druggability of downstream affected pathways in OCCC models.Experimental Design: In a large set of patients with OCCC (n = 124), the human kinome (518 kinases) and additional cancer-related genes were sequenced, and copy-number alterations were determined. Genetically characterized OCCC cell lines (n = 17) and OCCC patient-derived xenografts (n = 3) were used for drug testing of ERBB tyrosine kinase inhibitors erlotinib and lapatinib, the PARP inhibitor olaparib, and the mTORC1/2 inhibitor AZD8055.Results: We identified several putative driver mutations in kinases at low frequency that were not previously annotated in OCCC. Combining mutations and copy-number alterations, 91% of all tumors are affected in the PI3K/AKT/mTOR pathway, the MAPK pathway, or the ERBB family of receptor tyrosine kinases, and 82% in the DNA repair pathway. Strong p-S6 staining in patients with OCCC suggests high mTORC1/2 activity. We consistently found that the majority of OCCC cell lines are especially sensitive to mTORC1/2 inhibition by AZD8055 and not toward drugs targeting ERBB family of receptor tyrosine kinases or DNA repair signaling. We subsequently demonstrated the efficacy of mTORC1/2 inhibition in all our unique OCCC patient-derived xenograft models.Conclusions: These results propose mTORC1/2 inhibition as an effective treatment strategy in OCCC. Clin Cancer Res; 24(16); 3928-40. ©2018 AACR.

Methylome analysis of extreme chemoresponsive patients identifies novel markers of platinum sensitivity in high-grade serous ovarian cancer.

BACKGROUND: Despite an early response to platinum-based chemotherapy in advanced stage high-grade serous ovarian cancer (HGSOC), the majority of patients will relapse with drug-resistant disease. Aberrant epigenetic alterations like DNA methylation are common in HGSOC. Differences in DNA methylation are associated with chemoresponse in these patients. The objective of this study was to identify and validate novel epigenetic markers of chemoresponse using genome-wide analysis of DNA methylation in extreme chemoresponsive HGSOC patients. METHODS: Genome-wide next-generation sequencing was performed on methylation-enriched tumor DNA of two HGSOC patient groups with residual disease, extreme responders (≥18 months progression-free survival (PFS), n = 8) and non-responders (≤6 months PFS, n = 10) to platinum-based chemotherapy. DNA methylation and expression data of the same patients were integrated to create a gene list. Genes were validated on an independent cohort of extreme responders (n = 21) and non-responders (n = 31) using pyrosequencing and qRT-PCR. In silico validation was performed using publicly available DNA methylation (n = 91) and expression (n = 208) datasets of unselected advanced stage HGSOC patients. Functional validation of FZD10 on chemosensitivity was carried out in ovarian cancer cell lines using siRNA-mediated silencing. RESULTS: Integrated genome-wide methylome and expression analysis identified 45 significantly differentially methylated and expressed genes between two chemoresponse groups. Four genes FZD10, FAM83A, MYO18B, and MKX were successfully validated in an external set of extreme chemoresponsive HGSOC patients. High FZD10 and MKX methylation were related with extreme responders and high FAM83A and MYO18B methylation with non-responders. In publicly available advanced stage HGSOC datasets, FZD10 and MKX methylation levels were associated with PFS. High FZD10 methylation was strongly associated with improved PFS in univariate analysis (hazard ratio (HR) = 0.43; 95% CI, 0.27-0.71; P = 0.001) and multivariate analysis (HR = 0.39; 95% CI, 0.23-0.65; P = 0.003). Consistently, low FZD10 expression was associated with improved PFS (HR = 1.36; 95% CI, 0.99-1.88; P = 0.058). FZD10 silencing caused significant sensitization towards cisplatin treatment in survival assays and apoptosis assays. CONCLUSIONS: By applying genome-wide integrated methylome analysis on extreme chemoresponsive HGSOC patients, we identified novel clinically relevant, epigenetically-regulated markers of platinum-sensitivity in HGSOC patients. The clinical potential of these markers in predictive and therapeutic approaches has to be further validated in prospective studies.

Genome-wide methylation profiling of ovarian cancer patient-derived xenografts treated with the demethylating agent decitabine identifies novel epigenetically regulated genes and pathways.

BACKGROUND: In high-grade serous ovarian cancer (HGSOC), intrinsic and/or acquired resistance against platinum-containing chemotherapy is a major obstacle for successful treatment. A low frequency of somatic mutations but frequent epigenetic alterations, including DNA methylation in HGSOC tumors, presents the cancer epigenome as a relevant target for innovative therapy. Patient-derived xenografts (PDXs) supposedly are good preclinical models for identifying novel drug targets. However, the representativeness of global methylation status of HGSOC PDXs compared to their original tumors has not been evaluated so far. Aims of this study were to explore how representative HGSOC PDXs are of their corresponding patient tumor methylome and to evaluate the effect of epigenetic therapy and cisplatin on putative epigenetically regulated genes and their related pathways in PDXs. METHODS: Genome-wide analysis of the DNA methylome of HGSOC patients with their corresponding PDXs, from different generations, was performed using Infinium 450 K methylation arrays. Furthermore, we analyzed global methylome changes after treatment of HGSOC PDXs with the FDA approved demethylating agent decitabine and cisplatin. Findings were validated by bisulfite pyrosequencing with subsequent pathway analysis. Publicly available datasets comprising HGSOC patients were used to analyze the prognostic value of the identified genes. RESULTS: Only 0.6-1.0 % of all analyzed CpGs (388,696 CpGs) changed significantly (p < 0.01) during propagation, showing that HGSOC PDXs were epigenetically stable. Treatment of F3 PDXs with decitabine caused a significant reduction in methylation in 10.6 % of CpG sites in comparison to untreated PDXs (p < 0.01, false discovery rate <10 %). Cisplatin treatment had a marginal effect on the PDX methylome. Pathway analysis of decitabine-treated PDX tumors revealed several putative epigenetically regulated pathways (e.g., the Src family kinase pathway). In particular, the C-terminal Src kinase (CSK) gene was successfully validated for epigenetic regulation in different PDX models and ovarian cancer cell lines. Low CSK methylation and high CSK expression were both significantly associated (p < 0.05) with improved progression-free survival and overall survival in HGSOC patients. CONCLUSIONS: HGSOC PDXs resemble the global epigenome of patients over many generations and can be modulated by epigenetic drugs. Novel epigenetically regulated genes such as CSK and related pathways were identified in HGSOC. Our observations encourage future application of PDXs for cancer epigenome studies.

Genome-wide methylome analysis using MethylCap-seq uncovers 4 hypermethylated markers with high sensitivity for both adeno- and squamous-cell cervical carcinoma.

BACKGROUND: Cytology-based screening methods for cervical adenocarcinoma (ADC) and to a lesser extent squamous-cell carcinoma (SCC) suffer from low sensitivity. DNA hypermethylation analysis in cervical scrapings may improve detection of SCC, but few methylation markers have been described for ADC. We aimed to identify novel methylation markers for the early detection of both ADC and SCC. RESULTS: Genome-wide methylation profiling for 20 normal cervices, 6 ADC and 6 SCC using MethylCap-seq yielded 53 candidate regions hypermethylated in both ADC and SCC. Verification and independent validation of the 15 most significant regions revealed 5 markers with differential methylation between 17 normals and 13 cancers. Quantitative methylation-specific PCR on cervical cancer scrapings resulted in detection rates ranging between 80% and 92% while between 94% and 99% of control scrapings tested negative. Four markers (SLC6A5, SOX1, SOX14 and TBX20) detected ADC and SCC with similar sensitivity. In scrapings from women referred with an abnormal smear (n=229), CIN3+ sensitivity was between 36% and 71%, while between 71% and 93% of adenocarcinoma in situ (AdCIS) were detected; and CIN0/1 specificity was between 88% and 98%. Compared to hrHPV, the combination SOX1/SOX14 showed a similar CIN3+ sensitivity (80% vs. 75%, respectively, P>0.2), while specificity improved (42% vs. 84%, respectively, P < 10-5). CONCLUSION: SOX1 and SOX14 are methylation biomarkers applicable for screening of all cervical cancer types.

CD103+ intraepithelial T cells in high-grade serous ovarian cancer are phenotypically diverse TCRαß+ CD8αß+ T cells that can be targeted for cancer immunotherapy.

CD103+ tumor-infiltrating lymphocytes (TIL) have been linked to specific epithelial infiltration and a prolonged survival in high-grade serous epithelial ovarian cancer (HGSC). However, whether these cells are induced as part of an ongoing anti-HGSC immune response or represent non-specifically expanded resident or mucosal lymphocytes remains largely unknown. In this study, we first confirmed that CD103+ TIL from HGSC were predominantly localized in the cancer epithelium and were strongly correlated with an improved prognosis. We further demonstrate that CD103+ TIL were almost exclusively CD3+ TCRαß+ CD8αß+ CD4- T cells, but heterogeneously expressed T cell memory and differentiation markers. Activation of peripheral T cells in the presence of HGSC was sufficient to trigger induction of CD103 in over 90% of all CD8+ cells in a T cell receptor (TCR)- and TGFßR1-dependent manner. Finally, CD103+ TIL isolated from primary HGSC showed signs of recent activation and dominantly co-expressed key immunotherapeutic targets PD-1 and CD27. Taken together, our data indicate CD103+ TIL in HGSC are formed as the result of an adaptive anti-tumor immune response that might be reactivated by (dual) checkpoint inhibition.

CD103 defines intraepithelial CD8+ PD1+ tumour-infiltrating lymphocytes of prognostic significance in endometrial adenocarcinoma.

INTRODUCTION: Intraepithelial CD8+ tumour-infiltrating T-lymphocytes (TIL) are associated with a prolonged survival in endometrial cancer (EC). By contrast, stromal infiltration of CD8+ TIL does not confer prognostic benefit. A single marker to discriminate these populations would therefore be of interest for rapid assessment of the tumour immune contexture, ex vivo analysis of intraepithelial and stromal T-cells on a functional level and/or adoptive T-cell transfer. Here we determined whether CD103, the αE subunit of the αEß7integrin, can be used to specifically discriminate the epithelial and stromal CD8+ TIL populations in EC. METHODS: CD103+ TIL were quantified in a cohort of 305 EC patients by immunohistochemistry. Localization of CD103+ cells and co-expression of CD103 with CD3, CD8, CD16 and FoxP3 were assessed by immunofluorescence. Further phenotyping of CD103+ cells was performed by flow cytometry on primary endometrial tumour digests. RESULTS: CD8+CD103+ cells were preferentially located in endometrial tumour epithelium, whereas CD8+CD103- cells were located in stroma. CD103+ lymphocytes were predominantly CD3+CD8+ T-cells and expressed PD1. The presence of a high CD103+ cell infiltration was associated with an improved prognosis in patients with endometrial adenocarcinoma (p = 0.035). Moreover, this beneficial effect was particularly evident in high-risk adenocarcinoma patients (p = 0.031). CONCLUSIONS: Because of the restricted expression on intraepithelial CD8+ T-cells, CD103 may be a suitable biomarker for rapid assessment of immune infiltration of epithelial cancers. Furthermore, this intraepithelial tumour-reactive subset might be an interesting T-cell subset for adoptive T-cell transfer and/or target for checkpoint inhibition therapy.

Treatment Regimen, Surgical Outcome, and T-cell Differentiation Influence Prognostic Benefit of Tumor-Infiltrating Lymphocytes in High-Grade Serous Ovarian Cancer.

PURPOSE: Tumor-infiltrating lymphocytes (TIL) are associated with a better prognosis in high-grade serous ovarian cancer (HGSC). However, it is largely unknown how this prognostic benefit of TIL relates to current standard treatment of surgical resection and (neo-)adjuvant chemotherapy. To address this outstanding issue, we compared TIL infiltration in a unique cohort of patients with advanced-stage HGSC primarily treated with either surgery or neoadjuvant chemotherapy. EXPERIMENTAL DESIGN: Tissue microarray slides containing samples of 171 patients were analyzed for CD8(+) TIL by IHC. Freshly isolated CD8(+) TIL subsets were characterized by flow cytometry based on differentiation, activation, and exhaustion markers. Relevant T-cell subsets (CD27(+)) were validated using IHC and immunofluorescence. RESULTS: A prognostic benefit for patients with high intratumoral CD8(+) TIL was observed if primary surgery had resulted in a complete cytoreduction (no residual tissue). By contrast, optimal (<1 cm of residual tumor) or incomplete cytoreduction fully abrogated the prognostic effect of CD8(+) TIL. Subsequent analysis of primary TIL by flow cytometry and immunofluorescence identified CD27 as a key marker for a less-differentiated, yet antigen-experienced and potentially tumor-reactive CD8(+) TIL subset. In line with this, CD27(+) TIL were associated with an improved prognosis even in incompletely cytoreduced patients. Neither CD8(+) nor CD27(+) cell infiltration was of prognostic benefit in patients treated with neoadjuvant chemotherapy. CONCLUSIONS: Our findings indicate that treatment regimen, surgical result, and the differentiation of TIL should all be taken into account when studying immune factors in HGSC or, by extension, selecting patients for immunotherapy trials.

Biobanking of patient and patient-derived xenograft ovarian tumour tissue: efficient preservation with low and high fetal calf serum based methods.

Using patient-derived xenografts (PDXs) for preclinical cancer research demands proper storage of tumour material to facilitate logistics and to reduce the number of animals needed. We successfully established 45 subcutaneous ovarian cancer PDXs, reflecting all histological subtypes, with an overall take rate of 68%. Corresponding cells from mouse replaced human tumour stromal and endothelial cells in second generation PDXs as demonstrated with mouse-specific vimentin and CD31 immunohistochemical staining. For biobanking purposes two cryopreservation methods, a fetal calf serum (FCS)-based (95%v/v) "FCS/DMSO" protocol and a low serum-based (10%v/v) "vitrification" protocol were tested. After primary cryopreservation, tumour take rates were 38% and 67% using either the vitrification or FCS/DMSO-based cryopreservation protocol, respectively. Cryopreserved tumour tissue of established PDXs achieved take rates of 67% and 94%, respectively compared to 91% using fresh PDX tumour tissue. Genotyping analysis showed that no changes in copy number alterations were introduced by any of the biobanking methods. Our results indicate that both protocols can be used for biobanking of ovarian tumour and PDX tissues. However, FCS/DMSO-based cryopreservation is more successful. Moreover, primary engraftment of fresh patient-derived tumours in mice followed by freezing tissue of successfully established PDXs is the preferred way of efficient ovarian cancer PDX biobanking.

CD20+ T cells have a predominantly Tc1 effector memory phenotype and are expanded in the ascites of patients with ovarian cancer.

Recently, a small subset of T cells that expresses the B cell marker CD20 has been identified in healthy volunteers and in patients with rheumatoid arthritis and multiple sclerosis. The origin of these CD20-positive T cells as well as their relevance in human disease remains unclear. Here, we identified that after functional B cell/T cell interaction CD20 molecules are transferred to the cell surface of T cells by trogocytosis together with the established trogocytosis marker HLA-DR. Further, the presence of CD20 on isolated CD20+ T cells remained stable for up to 48h of ex vivo culture. These CD20+ T cells almost exclusively produced IFNγ (∼70% vs. ∼20% in the CD20- T cell population) and were predominantly (CD8+) effector memory T cells (∼60-70%). This IFNγ producing and effector memory phenotype was also determined for CD20+ T cells as detected in the peripheral blood and ascitic fluids of ovarian cancer (OC) patients. In the latter, the percentage of CD20+ T cells was further strongly increased (from ∼6% in peripheral blood to 23% in ascitic fluid). Taken together, the data presented here indicate that CD20 is transferred to T cells upon intimate T cell/B cell interaction. Further, CD20+ T cells are of memory and IFNγ producing phenotype and are present in increased amounts in ascitic fluid of OC patients.

A panel of regulated proteins in serum from patients with cervical intraepithelial neoplasia and cervical cancer.

We developed a discovery-validation mass-spectrometry-based pipeline to identify a set of proteins that are regulated in serum of patients with cervical intraepithelial neoplasia (CIN) and squamous cell cervical cancer using iTRAQ, label-free shotgun, and targeted mass-spectrometric quantification. In the discovery stage we used a "pooling" strategy for the comparative analysis of immunodepleted serum and revealed 15 up- and 26 down-regulated proteins in patients with early- (CES) and late-stage (CLS) cervical cancer. The analysis of nondepleted serum samples from patients with CIN, CES, an CLS and healthy controls showed significant changes in abundance of alpha-1-acid glycoprotein 1, alpha-1-antitrypsin, serotransferrin, haptoglobin, alpha-2-HS-glycoprotein, and vitamin D-binding protein. We validated our findings using a fast UHPLC/MRM method in an independent set of serum samples from patients with cervical cancer or CIN and healthy controls as well as serum samples from patients with ovarian cancer (more than 400 samples in total). The panel of six proteins showed 67% sensitivity and 88% specificity for discrimination of patients with CIN from healthy controls, a stage of the disease where current protein-based biomarkers, for example, squamous cell carcinoma antigen (SCCA), fail to show any discrimination. Additionally, combining the six-protein panel with SCCA improves the discrimination of patients with CES and CLS from healthy controls.

Radical surgery in patients with residual disease after (chemo)radiation for cervical cancer.

OBJECTIVE: The aim of this study was to determine possible impact of routinely scheduled biopsies and more radical surgery for residual central disease in locally advanced cervical cancer after (chemo)radiation. METHODS/MATERIALS: Data were analyzed of a consecutive series of cervical cancer patients (The International Federation of Gynecology and Obstetrics stages IB1-IVA) treated with (chemo) radiation between 1994 and 2011. Patients underwent gynecologic examination with biopsies 8 to 10 weeks after treatment. Since 2001, larger biopsies by electric loop excision were taken, and more radical surgery (type III hysterectomy or exenteration) was performed for central residual disease. Primary outcome was locoregional recurrence. Secondary outcomes were treatment-associated morbidity and disease-specific survival. RESULTS: Primary (chemo)radiation was given to 491 cervical cancer patients; 345 patients had a posttreatment biopsy. Viable tumor cells were identified in 84 patients, and 61 patients were eligible for salvage surgery. Residual disease after (chemo)radiation was an independent poor prognostic factor (hazard ratio, 3.59; 95% confidence interval, 2.18-5.93; P < 0.001). After 2001, larger biopsies were more frequently taken (29% vs 76%, P < 0.001), and in patients without viable tumor cells, locoregional recurrence after 2001 decreased from 21% to 10% (P = 0.01). After 2001, more patients underwent more radical surgery (46% vs 90%) (P < 0.001). Locoregional recurrence after surgery before 2001 occurred in 6 (46%) of the 13 patients, comparable with 19 (40%) of the 48 (P = 0.67) after 2001. More radical surgery was not associated with improved disease-specific survival (HR, 0.84; 95% CI, 0.20-3.46; P = 0.81) but did result in significantly more severe morbidity. CONCLUSION: More radical surgery in patients with (minimal) central residual disease identified by routine biopsy 8 to 10 weeks after (chemo)radiation does not improve survival and should not be recommended.

Comparing the Cervista HPV HR test and Hybrid Capture 2 assay in a Dutch screening population: improved specificity of the Cervista HPV HR test by changing the cut-off.

The diagnostic performance of the widely-used Cervista HPV HR test was compared to the Hybrid Capture 2 (HC2) test in a Dutch population-based cervical cancer screening program. In 900 scrapings of women with normal cytomorphology, specificity was 90% (95%CI: 87.84-91.87) for the Cervista HPV HR test and 96% (95%CI: 94.76-97.37) for the HC2 test with 93% agreement between both tests (κ = 0.5, p<0.001). The sensitivity for CIN2+ using 65 scrapings of women with histological-confirmed CIN2+ was 91% (95%CI: 80.97-96.51) for the Cervista HPV HR test and 92% (95%CI: 82.94-97.43) for the HC2 test with 95% agreement between both tests (κ = 0.7, p<0.001). Fifty-seven of 60 HC2 negative/Cervista positive cases tested HPV-negative with PCR-based HPV assays; of these cases 56% were defined as Cervista triple-positive with FOZ values in all 3 mixes higher than the second cut-off of 1.93 (as set by manufacturer). By setting this cut-off at 5.0, specificity improved significantly without affecting sensitivity. External validation of this new cut-off at 5.0 in triple-positive scrapings of women selected from the SHENCCASTII database revealed that 22/24 histological normal cases now tested HPV-negative in the Cervista HPV HR test, while CIN2+ lesions remained HPV-positive. The intra-laboratory reproducibility of the Cervista HPV HR test (n = 510) showed a concordance of 92% and 93% for cut-off 1.93 and 5.0 (κ = 0.83 and κ = 0.84, p<0.001) and inter-laboratory agreement of the Cervista HPV HR test was 90% and 93% for cut-off 1.93 and 5.0 (κ = 0.80 and κ = 0.85, p<0.001). In conclusion, the specificity of the Cervista HPV HR test could be improved significantly by increasing the second cut-off from 1.93 to 5.0, without affecting the sensitivity of the test in a population-based screening setting.

Genome-wide methylation profiling identifies hypermethylated biomarkers in high-grade cervical intraepithelial neoplasia.

Epigenetic modifications, such as aberrant DNA promoter methylation, are frequently observed in cervical cancer. Identification of hypermethylated regions allowing discrimination between normal cervical epithelium and high-grade cervical intraepithelial neoplasia (CIN2/3), or worse, may improve current cervical cancer population-based screening programs. In this study, the DNA methylome of high-grade CIN lesions was studied using genome-wide DNA methylation screening to identify potential biomarkers for early diagnosis of cervical neoplasia. Methylated DNA Immunoprecipitation (MeDIP) combined with DNA microarray was used to compare DNA methylation profiles of epithelial cells derived from high-grade CIN lesions with normal cervical epithelium. Hypermethylated differentially methylated regions (DMRs) were identified. Validation of nine selected DMRs using BSP and MSP in cervical tissue revealed methylation in 63.2-94.7% high-grade CIN and in 59.3-100% cervical carcinomas. QMSP for the two most significant high-grade CIN-specific methylation markers was conducted exploring test performance in a large series of cervical scrapings. Frequency and relative level of methylation were significantly different between normal and cancer samples. Clinical validation of both markers in cervical scrapings from patients with an abnormal cervical smear confirmed that frequency and relative level of methylation were related with increasing severity of the underlying CIN lesion and that ROC analysis was discriminative. These markers represent the COL25A1 and KATNAL2 and their observed increased methylation upon progression could intimate the regulatory role in carcinogenesis. In conclusion, our newly identified hypermethylated DMRs represent specific DNA methylation patterns in high-grade CIN lesions and are candidate biomarkers for early detection.

Cell surface delivery of TRAIL strongly augments the tumoricidal activity of T cells.

PURPOSE: Adoptive T-cell therapy generally fails to induce meaningful anticancer responses in patients with solid tumors. Here, we present a novel strategy designed to selectively enhance the tumoricidal activity of T cells by targeted delivery of TNF-related apoptosis-inducing ligand (TRAIL) to the T-cell surface. EXPERIMENTAL DESIGN: We constructed two recombinant fusion proteins, anti-CD3:TRAIL and K12:TRAIL. Tumoricidal activity of T cells in the presence of these fusion proteins was assessed in solid tumor cell lines, primary patient-derived malignant cells, and in a murine xenograft model. RESULTS: When added to T cells, K12:TRAIL and anti-CD3:TRAIL selectively bind to the T-cell surface antigens CD3 and CD7, respectively, leading to cell surface accretion of TRAIL. Subsequently, anti-CD3:TRAIL and K12:TRAIL increased the tumoricidal activity of T cells toward cancer cell lines and primary patient-derived malignant cells by more than 500-fold. Furthermore, T-cell surface delivery of TRAIL strongly inhibited tumor growth and increased survival time of xenografted mice more than 6-fold. CONCLUSIONS: Targeted delivery of TRAIL to cell surface antigens of T cells potently enhances the tumoricidal activity of T cells. This approach may be generally applicable to enhance the efficacy of adoptive T-cell therapy.

Methylation markers for CCNA1 and C13ORF18 are strongly associated with high-grade cervical intraepithelial neoplasia and cervical cancer in cervical scrapings.

PURPOSE: Recently, we reported 13 possible cervical cancer-specific methylated biomarkers identified by pharmacologic unmasking microarray in combination with large-genome computational screening. The aim of the present study was to perform an in-depth analysis of the methylation patterns of these 13 candidate genes in cervical neoplasia and to determine their diagnostic relevance. EXPERIMENTAL DESIGN AND RESULTS: Five of the 13 gene promoters (C13ORF18, CCNA1, TFPI2, C1ORF166, and NPTX1) were found to be more frequently methylated in frozen cervical cancer compared with normal cervix specimens. Quantitative methylation analysis for these five markers revealed that both CCNA1 and C13ORF18 were methylated in 68 of 97 cervical scrapings from cervical cancer patients and in only 5 and 3 scrapings, respectively, from 103 healthy controls (P < 0.0005). In cervical scrapings from patients referred with an abnormal Pap smear, CCNA1 and C13ORF18 were methylated in 2 of 43 and 0 of 43 CIN 0 (no cervical intraepithelial neoplasia) and in 1 of 41 and 0 of 41 CIN I, respectively. Furthermore, 8 of 43 CIN II, 22 of 43 CIN III, and 3 of 3 microinvasive cancer patients were positive for both markers. Although sensitivity for CIN II or higher (for both markers 37%) was low, specificity (96% and 100%, respectively) and positive predictive value (92% and 100%, respectively) were high. CONCLUSION: Methylation of CCNA1 and C13ORF18 in cervical scrapings is strongly associated with CIN II or higher-grade lesions. Therefore, these markers might be used for direct referral to gynecologists for patients with a methylation-positive scraping.

Survival-related profile, pathways, and transcription factors in ovarian cancer.

BACKGROUND: Ovarian cancer has a poor prognosis due to advanced stage at presentation and either intrinsic or acquired resistance to classic cytotoxic drugs such as platinum and taxoids. Recent large clinical trials with different combinations and sequences of classic cytotoxic drugs indicate that further significant improvement in prognosis by this type of drugs is not to be expected. Currently a large number of drugs, targeting dysregulated molecular pathways in cancer cells have been developed and are introduced in the clinic. A major challenge is to identify those patients who will benefit from drugs targeting these specific dysregulated pathways.The aims of our study were (1) to develop a gene expression profile associated with overall survival in advanced stage serous ovarian cancer, (2) to assess the association of pathways and transcription factors with overall survival, and (3) to validate our identified profile and pathways/transcription factors in an independent set of ovarian cancers. METHODS AND FINDINGS: According to a randomized design, profiling of 157 advanced stage serous ovarian cancers was performed in duplicate using approximately 35,000 70-mer oligonucleotide microarrays. A continuous predictor of overall survival was built taking into account well-known issues in microarray analysis, such as multiple testing and overfitting. A functional class scoring analysis was utilized to assess pathways/transcription factors for their association with overall survival. The prognostic value of genes that constitute our overall survival profile was validated on a fully independent, publicly available dataset of 118 well-defined primary serous ovarian cancers. Furthermore, functional class scoring analysis was also performed on this independent dataset to assess the similarities with results from our own dataset. An 86-gene overall survival profile discriminated between patients with unfavorable and favorable prognosis (median survival, 19 versus 41 mo, respectively; permutation p-value of log-rank statistic = 0.015) and maintained its independent prognostic value in multivariate analysis. Genes that composed the overall survival profile were also able to discriminate between the two risk groups in the independent dataset. In our dataset 17/167 pathways and 13/111 transcription factors were associated with overall survival, of which 16 and 12, respectively, were confirmed in the independent dataset. CONCLUSIONS: Our study provides new clues to genes, pathways, and transcription factors that contribute to the clinical outcome of serous ovarian cancer and might be exploited in designing new treatment strategies.

P53-specific T cell responses in patients with malignant and benign ovarian tumors: implications for p53 based immunotherapy.

Despite intensive treatment, 70% of the ovarian cancer patients will develop recurrent disease, emphasizing the need for new approaches such as immunotherapy. A promising antigenic target for immunotherapy in ovarian cancer is the frequently overexpressed p53 protein. The aim of the study was to evaluate the nature and magnitude of the baseline anti-p53 immune response in ovarian cancer patients. P53-specific T cell responses were detected in both half of the ovarian cancer patients as in the group of control subjects, consisting of women with benign ovarian tumors and healthy controls. Importantly, while in the control group p53-specific immunity was detected among the CD45RA(+) naïve subset of T cells only, the p53-specific T-cell responses in ovarian cancer patients were also present in the CD45RO(+) memory T-cell subset, suggesting that in the cancer patients sufficient amounts of cancer-derived p53 was presented to induce the formation of a p53-specific memory T-cell response. Further characterization of the p53-specific memory T-cell responses revealed that in addition to the type 1 cytokine IFN-gamma also the type 2 cytokines IL-4 and IL-5, as well as the immunosuppressive cytokine IL-10 were produced. Notably, p53-specific T cells were not only detected in the peripheral blood, but also among tumor infiltrating lymphocytes and in tumor-draining lymph nodes. In conclusion, the existence of a weak mixed T-helper type 1 and 2 p53-specific T-cell repertoire supports the rationale of using p53 long peptides in vaccination strategies aiming at the induction of p53-specific Th1/CTL immunity.

Enhancement of human papilloma virus type 16 E7 specific T cell responses by local invasive procedures in patients with (pre)malignant cervical neoplasia.

It has been suggested that local invasive procedures may alter the natural course of (pre)malignant cervical disease. This could be due to partial excision of the lesions, or via induction of cellular immunity against human papillomavirus (HPV) by the local invasive procedures. We studied the influence of local invasive procedures on HPV-16 E7 specific immune responses in patients with different grades of cervical intra-epithelial neoplasia (CIN) and different stages of cervical cancer. Blood was obtained at intake and after invasive procedures from patients with CIN or cervical cancer. Antigen specific T-cell responses were measured by IFN-gamma ELISPOT analysis, after stimulation with recombinant HPV-16 E7 protein. As expected, HPV-16 E7 specific IFN-gamma T cell responses were more frequent in HPV-16 DNA positive patients compared with that in HPV-16 DNA negative patients (39/50 vs. 16/36, (p=0.006, chi2 test). After invasive procedures, a small number of HPV-16 DNA positive CIN patients, but a considerable proportion of HPV-16 DNA positive cervical cancer patients, showed an enhancement of T cell responses against HPV-16 E7. Induction of T cell reactivity was most pronounced in cervical cancer patients who had undergone previous invasive procedures. Both CD4+ and CD8+ T cells showed E7 specific IFN-gamma production upon in-vitro stimulation. Our study shows that invasive procedures may enhance HPV-specific cell-mediated immunity in a considerable number of patients with cervical cancer, but in only a minority of CIN patients. Our data indicate that invasive procedures should be considered as possible confounding factors when analyzing the effectiveness of therapeutic immunization studies, especially, when induction of HPV-specific immune responses is used as intermediate end-point.