Margin status revisited in vulvar squamous cell carcinoma.

OBJECTIVE: To determine the incidence of local recurrence of vulvar squamous cell carcinoma in relation to tumor- and/or precursor lesion free pathologic margins. METHODS: Consecutive patients with primary vulvar squamous cell carcinoma surgically treated in two Dutch expert centers between 2000 and 2010 were included. All pathology slides were independently reviewed by two expert gynecopathologists, and local recurrence was defined as any recurrent disease located on the vulva. Time to first local recurrence was compared for different subgroups using univariable and multivariable Cox-regression analyses. RESULTS: In total 287 patients with a median follow-up of 80months (range 0-204) were analyzed. The actuarial local recurrence rate ten years after treatment was 42.5%. Pathologic tumor free margin distance did not influence the risk on local recurrence (HR 1.03 (95% CI 0.99-1.06)), neither using a cutoff of eight, five, or three millimeters. Multivariable analyses showed a higher local recurrence rate in patients with dVIN and LS in the margin (HR 2.76 (95% CI 1.62-4.71)), in patients with dVIN in the margin (HR 2.14 (95% CI 1.11-4.12)), and a FIGO stage II or higher (HR 1.62 (95% CI 1.05-2.48)). CONCLUSIONS: Local recurrences frequently occur in patients with primary vulvar carcinoma and are associated with dVIN (with or without LS) in the pathologic margin rather than any tumor free margin distance. Our results should lead to increased awareness among physicians of an ongoing risk for local recurrence and need for life-long follow-up. Intensified follow-up and treatment protocols for patients with dVIN in the margin should be evaluated in future research.

Clinical consequences of upfront pathology review in the randomised PORTEC-3 trial for high-risk endometrial cancer.

Background: In the PORTEC-3 trial, women with high-risk endometrial cancer (HR-EC) were randomised to receive pelvic radiotherapy (RT) with or without concurrent and adjuvant chemotherapy (two cycles of cisplatin 50 mg/m2 in weeks 1 and 4 of RT, followed by four cycles of carboplatin AUC5 and paclitaxel 175 mg/m2). Pathology review was required before patient enrolment. The aim of this analysis was to evaluate the role of central pathology review before randomisation. Patients and methods: A total of 1295 cases underwent pathology review to confirm HR-EC in the Netherlands (n = 395) and the UK (n = 900), and for 1226/1295 (95%) matching review and original reports were available. In total, 329 of these patients were enrolled in the PORTEC-3 trial: 145 in the Netherlands and 184 in the UK, comprising 48% of the total PORTEC-3 cohort of 686 participants. Areas of discrepancies were evaluated, and inter-observer agreement between original and review opinion was evaluated by calculating the kappa value (κ). Results: In the 1226 pathology reviews, 6356 selected items were evaluable for both original and review pathology. In 43% of cases at least one pathology item changed after review. For 102 patients (8%), this discrepancy led to ineligibility for the PORTEC-3 trial, most frequently due to differences in the assessment of histological type (34%), endocervical stromal involvement (27%) and histological grade (19%). Lowest inter-observer agreement was found for histological type (κ = 0.72), lymph-vascular space invasion (κ = 0.72) and histological grade (κ = 0.70). Conclusion: Central pathology review by expert gynaeco-pathologists changed histological type, grade or other items in 43% of women with HR-EC, leading to ineligibility for the PORTEC-3 trial in 8%. Upfront pathology review is essential to ensure enrolment of the target trial-population, and to avoid over- or undertreatment, especially when treatment modalities with substantial toxicity are involved. This study is registered with ISRCTN (ISRCTN14387080, www.controlled-trials.com) and with ClinicalTrials.gov (NCT00411138).

Characteristics of Lynch syndrome associated ovarian cancer.

OBJECTIVE: To describe clinical characteristics of Lynch syndrome associated ovarian cancer and the efficacy of surveillance in the early detection of these ovarian cancers. METHODS: All Lynch syndrome associated ovarian cancer cases identified in either the Dutch Lynch syndrome registry (DLSR) between 1987 and 2016, and/or the cohort at the University Medical Center Groningen (UMCG) between 1993 and 2016 were included. Clinical data on age at diagnosis, mutation type, histological type, FIGO stage, treatment, follow-up and gynecological surveillance were collected. RESULTS: A total of 46/798 (6%) women in the DLSR and 7/80 (9%) in the UMCG cohort were identified as LS associated ovarian cancer patients. The median age at ovarian cancer diagnosis was 46.0 years (range 20-75 years). The most frequently reported histological type was endometrioid adenocarcinoma (40%; n = 21) and serous carcinoma (36%; n = 19). Most tumors (87%; n = 46) were detected at an early stage (FIGO I/II). Forty-one of 53 (77%) patients were diagnosed with ovarian cancer before LS was diagnosed. In the other 12/53 (23%) women, ovarian cancer developed after starting annual gynecological surveillance for LS; three ovarian cancers were screen-detected in asymptomatic women. Overall survival was 83%. CONCLUSION: Ovarian cancer in women with LS has a wide age-range of onset, is usually diagnosed at an early stage with predominantly endometrioid type histology and a good overall survival. The early stage at diagnosis could not be attributed to annual gynecological surveillance.

Prediction model for regional or distant recurrence in endometrial cancer based on classical pathological and immunological parameters.

BACKGROUND: Adjuvant therapy increases disease-free survival in endometrial cancer (EC), but has no impact on overall survival and negatively influences the quality of life. We investigated the discriminatory power of classical and immunological predictors of recurrence in a cohort of EC patients and confirmed the findings in an independent validation cohort. METHODS: We reanalysed the data from 355 EC patients and tested our findings in an independent validation cohort of 72 patients with EC. Predictors were selected and Harrell's C-index for concordance was used to determine discriminatory power for disease-free survival in the total group and stratified for histological subtype. RESULTS: Predictors for recurrence were FIGO stage, lymphovascular space invasion and numbers of cytotoxic and memory T-cells. For high risk cancer, cytotoxic or memory T-cells predicted recurrence as well as a combination of FIGO stage and lymphovascular space invasion (C-index 0.67 and 0.71 vs 0.70). Recurrence was best predicted when FIGO stage, lymphovascular space invasion and numbers of cytotoxic cells were used in combination (C-index 0.82). Findings were confirmed in the validation cohort. CONCLUSIONS: In high-risk EC, clinicopathological or immunological variables can predict regional or distant recurrence with equal accuracy, but the use of these variables in combination is more powerful.

Elevated serum CXCL16 is an independent predictor of poor survival in ovarian cancer and may reflect pro-metastatic ADAM protease activity.

BACKGROUND: In certain cancers, expression of CXCL16 and its receptor CXCR6 associate with lymphocyte infiltration, possibly aiding anti-tumour immune response. In other cancers, CXCL16 and CXCR6 associate with pro-metastatic activity. In the current study, we aimed to characterise the role of CXCL16, sCXCL16, and CXCR6 in ovarian cancer (OC). METHODS: CXCL16/CXCR6 expression was analysed on tissue microarray containing 306 OC patient samples. Pre-treatment serum sCXCL16 was determined in 118 patients using ELISA. In vitro, (primary) OC cells were treated with an ADAM-10/ADAM-17 inhibitor (TAPI-2) and an ADAM-10-specific inhibitor (GI254023x), whereupon CXCL16 levels were evaluated on the cell membrane (immunofluorescent analysis, western blots) and in culture supernatants (ELISA). In addition, cell migration was assessed using scratch assays. RESULTS: sCXCL16 independently predicted for poor survival (hazard ratio=2.28, 95% confidence interval=1.29-4.02, P=0.005), whereas neither CXCL16 nor CXCR6 expression correlated with survival. Further, CXCL16/CXCR6 expression and serum sCXCL16 levels did not associate with lymphocyte infiltration. In vitro inhibition of both ADAM-17 and ADAM-10, but especially the latter, decreased CXCL16 membrane shedding and strongly reduced cell migration of A2780 and cultured primary OC-derived malignant cells. CONCLUSIONS: High serum sCXCL16 is a prognostic marker for poor survival of OC patients, possibly reflecting ADAM-10 and ADAM-17 pro-metastatic activity. Therefore, serum sCXCL16 levels may be a pseudomarker that identifies patients with highly metastatic tumours.

Prognostic impact of clinicopathological features and expression of biomarkers related to (18)F-FDG uptake in esophageal cancer.

PURPOSE: To analyze the association between pretreatment 18-F-fluoro-deoxyglucose (FDG) uptake and characteristics of aggressive tumor biology in predicting outcome in esophageal cancer (EC). METHODS: Tumor FDG-uptake was measured by maximum standardized uptake values (SUVmax) in 47 patients undergoing esophagectomy with curative intent. ROC analyses were used to predict an optimal SUVmax cutoff for survival. Expression of hexokinase-II (HK-II), glucose transporter I (GLUT-I), hypoxia inducible factor-1α (HIF-Iα), vascular endothelial growth factor-C (VEGF-C), p53, and proliferative activity (Ki-67) were correlated with SUVmax values and clinicopathological characteristics. RESULTS: A SUVmax > 3.67 predicted a significantly lower disease-free survival (DFS) and distant recurrence-free survival (p = 0.022 and p = 0.005). High HK-II expression was correlated with reduced SUVmax values (p = 0.002) and was significantly higher in esophageal adenocarcinoma compared with squamous cell carcinoma (p = 0.005). Preoperative high FDG uptake in primary tumors was associated with nodal metastases (pN1; Spearman correlation 0.39, p = 0.01). We found no positive correlation between SUVmax and GLUT-1, HK-1, HIF-Iα 1, VEGF-C, p53, and Ki-67 expression. CONCLUSIONS: High preoperative FDG-uptake strongly predicts poor survival outcome and is associated with lymph node metastases in EC patients. HK-II expression was related to SUVmax and DFS.

Checkpoint kinase 2 (Chk2) supports sensitivity to platinum-based treatment in high grade serous ovarian cancer.

OBJECTIVE: Platinum-based chemotherapy is the standard treatment in advanced stage high grade serous ovarian cancer (HGSOC), but the majority of patients will relapse with drug-resistant disease. Platinum induces double-strand DNA breaks and subsequently activation of the DNA damage response (DDR). Drugs targeting DDR pathway components have gained major interest to be combined with chemotherapy as they could increase the therapeutic window. In the present study, we investigated the activation status of the Ataxia Telangiectasia Mutated (ATM) signaling axis within the DDR in a large, well-defined cohort of advanced stage HGSOC patients. METHODS: Pre-therapy activation status of the ATM signaling axis of the DDR was determined by immunohistochemistry in 125 chemo-naive advanced stage HGSOC patients. Ovarian cancer cell lines with stable checkpoint kinase 2 (Chk2) knock down were used to study cell cycle distribution and survival in long-term clonogenic survival assays. RESULTS: All ATM signaling axis components showed high expression levels. In two well-defined groups with the largest contrast in treatment response, high expression of Chk2 was related to good response (OR=0.132; P=0.014). Chk2 depletion abrogated the cisplatin-induced S-phase cell cycle arrest and caused increased resistance to cisplatin in long-term clonogenic survival assays. CONCLUSIONS: Chk2 is related to good response to platinum-based chemotherapy in advanced stage HGSOC patients. Chk2-depleted ovarian cancer cell lines have diminished platinum sensitivity, suggesting that Chk2 should not be considered a therapeutic target along with platinum-based treatment in HGSOC patients.

Nutlin-3 preferentially sensitises wild-type p53-expressing cancer cells to DR5-selective TRAIL over rhTRAIL.

BACKGROUND: Tumour cell-selective activation of apoptosis by recombinant human TNF-related apoptosis-inducing ligand (rhTRAIL) is enhanced through co-activation of p53 by chemotherapeutic drugs. The novel anticancer agent nutlin-3 provides a promising alternative for p53 activation by disrupting the interaction between p53 and its negative feedback regulator MDM2. METHODS: We examined whether nutlin-3 enhances apoptosis induction by rhTRAIL and the DR5-selective TRAIL variant D269H/E195R in wild-type p53-expressing ovarian, colon and lung cancer cell lines and in an ex vivo model of human ovarian cancer. RESULTS: Nutlin-3 enhanced p53, p21, MDM2 and DR5 surface expression. Although nutlin-3 did not induce apoptosis, it preferentially enhanced D269H/E195R-induced apoptosis over rhTRAIL. Combination treatment potentiated the cleavage of caspases 8, 9, 3 and PARP. P53 and MDM2 siRNA experiments showed that this enhanced apoptotic effect was mediated by wild-type p53. Indeed, nutlin-3 did not enhance rhTRAIL-induced apoptosis in OVCAR-3 cells harbouring mutant p53. Addition of the chemotherapeutic drug cisplatin to the combination further increased p53 and DR5 levels and rhTRAIL- and D269H/E195R-induced apoptosis. As a proof of concept, we show that the combination of D269H/E195R, nutlin-3 and cisplatin induced massive apoptosis in ex vivo tissue slices of primary human ovarian cancers. CONCLUSION: Nutlin-3 is a potent enhancer of D269H/E195R-induced apoptosis in wild-type p53-expressing cancer cells. Addition of DNA-damaging agents such as cisplatin further enhances DR5-mediated apoptosis.

The additional value of endometrial sampling in the early detection of endometrial cancer in women with Lynch syndrome.

OBJECTIVE: Based on previous studies, standard gynecological screening consisting of annual transvaginal ultrasonography (TVU) was added with endometrial sampling in women with Lynch syndrome (LS). The aim of this study was to evaluate the additional value of endometrial sampling in detecting (pre)malignancies of the endometrial tissue in women with LS or first-degree relatives. METHODS: All women above 30 years of age with LS or first-degree relatives at 50% risk of LS are offered annual gynecological screening in our family cancer clinic. Endometrial screening results from January 2003-December 2007 (period I: standard screening by transvaginal sonography and serum CA125) were compared with screening results from January 2008-June 2012 (period II: standard screening added with endometrial sampling). RESULTS: Seventy five women (300 patient years) were screened annually. There were 266 screening visits, 117 in period I and 149 in period II. In period I, four premalignant endometrial lesions were detected and one endometrial carcinoma (FIGO stage IB). In period II, two premalignancies were found. None of the lesions would have been missed without standard endometrial sampling. No interval endometrial cancers were detected in this study. CONCLUSION: In this study, annual endometrial screening seems an effective screening tool in the detection of premalignancies and early endometrial cancer in women with LS. Adding standard endometrial sampling to annual TVU has no additional value in the early detection of (pre)malignant endometrial lesions in women with LS in this study.

A role for MLH3 in hereditary nonpolyposis colorectal cancer.

We investigated a possible role of the mismatch-repair gene MLH3 in hereditary nonpolyposis colorectal cancer by scanning for mutations in 39 HNPCC families and in 288 patients suspected of having HNPCC. We identified ten different germline MLH3 variants, one frameshift and nine missense mutations, in 12 patients suspected of HNPCC. Three of the 12 also carried a mutation in MSH6.

Prognostic value of HPV-PCR, p16 and p53 immunohistochemical status on local recurrence rate and survival in patients with vulvar squamous cell carcinoma.

The primary aim of this study was to assess the association between human papilloma virus (HPV) and p53 expression and local recurrence (LR), disease specific survival (DSS), and overall survival (OS) in patients with vulvar squamous cell carcinoma (VSCC). Secondary, the accuracy of p16 immunohistochemistry for HPV status was assessed. The tumor tissue of 255 patients, surgically treated for primary unifocal VSCC between 2000 and 2010, was analyzed. HPV-PCR and P16 and p53 immunohistochemical stainings were performed. All histologic slides were independently reviewed by two expert gyneco-pathologists. Time to first LR, DSS, and OS for the variables p16, p53, and HPV-PCR were compared using univariable and multivariable Cox-regression analyses. In 211/255 (83.5%) patients, HPV-PCR was negative. The local recurrence rate was significantly lower in patients positive with HPV-PCR (10-year LR rate 24.6%) versus negative tumors (47.5%), p = 0.004. After multivariable analyses, this difference remained significant (HR 0.23 (95% CI 0.08-0.62) p = 0.004). There was no difference in LR rate correlated to the p53 expression. DSS and OS did not significantly differ after multivariable analyses for all different subgroups. Sensitivity and specificity of p16 staining for presence of HPV detected by HPV-PCR were 86.4% and 93.8%, respectively. In conclusion, patients with HPV-negative VSCCs have significantly more LR compared to patients with HPV-positive VSCCs, and p16 immunohistochemistry is a reliable surrogate marker for HPV status. No relevant subgroup for LR or survival based on HPV/p53 status could be identified. We advise to perform an HPV-PCR or p16 IHC staining in all patients with VSCC.

A four-gene methylation marker panel as triage test in high-risk human papillomavirus positive patients.

Cervical neoplasia-specific biomarkers, e.g. DNA methylation markers, with high sensitivity and specificity are urgently needed to improve current population-based screening on (pre)malignant cervical neoplasia. We aimed to identify new cervical neoplasia-specific DNA methylation markers and to design and validate a methylation marker panel for triage of high-risk human papillomavirus (hr-HPV) positive patients. First, high-throughput quantitative methylation-specific PCRs (QMSP) on a novel OpenArray™ platform, representing 424 primers of 213 cancer specific methylated genes, were performed on frozen tissue samples from 84 cervical cancer patients and 106 normal cervices. Second, the top 20 discriminating methylation markers were validated by LightCycler® MSP on frozen tissue from 27 cervical cancer patients and 20 normal cervices and ROCs and test characteristics were assessed. Three new methylation markers were identified (JAM3, EPB41L3 and TERT), which were subsequently combined with C13ORF18 in our four-gene methylation panel. In a third step, our methylation panel detected in cervical scrapings 94% (70/74) of cervical cancers, while in a fourth step 82% (32/39) cervical intraepithelial neoplasia grade 3 or higher (CIN3+) and 65% (44/68) CIN2+ were detected, with 21% positive cases for ≤CIN1 (16/75). Finally, hypothetical scenario analysis showed that primary hr-HPV testing combined with our four-gene methylation panel as a triage test resulted in a higher identification of CIN3 and cervical cancers and a higher percentage of correct referrals compared to hr-HPV testing in combination with conventional cytology. In conclusion, our four-gene methylation panel might provide an alternative triage test after primary hr-HPV testing.

A randomized phase II study investigating the addition of the specific COX-2 inhibitor celecoxib to docetaxel plus carboplatin as first-line chemotherapy for stage IC to IV epithelial ovarian cancer, Fallopian tube or primary peritoneal carcinomas: the DoCaCel study.

BACKGROUND: In ovarian cancer, cyclooxygenase-2 (COX-2) overexpression is prognostic for poor survival. We investigated the efficacy of celecoxib (C), a selective COX-2 inhibitor, added to docetaxel (Taxotere)/carboplatin (DC) in advanced ovarian cancer. PATIENTS AND METHODS: In a phase II, randomized study, 400 mg celecoxib b.i.d. was added to first-line DC treatment (DCC). Celecoxib was to be continued after DC termination up to 3 years. Study end points were tolerability, progression-free survival (PFS) and overall survival (OS). RESULTS: 151 of 196 eligible patients were diagnosed with stage IIIC/IV disease. Median follow-up for patients alive was 32.3 months. Celecoxib was used during a mean of 8.5 months. Twenty-three of 97 DCC patients stopped celecoxib prematurely, mainly due to skin reactions. Complete biochemical response was achieved in 51/78 DC patients (65%) versus 57/78 DCC patients (75%, not significant). In both study arms, median PFS was 14.3 months and median OS 34 months. COX-2 was expressed in 82% of 120 tumor samples retrospectively recovered. The PFS and OS of patients with intermediate/high COX-2 expression were similar to that in the other patients. CONCLUSION: Celecoxib did not influence PFS and OS, but interpretation of results is hampered by premature celecoxib discontinuation.

Status of cellular immunity lacks prognostic significance in vulvar squamous carcinoma.

OBJECTIVE: It is generally recognized that the immune system has an important role in regulating cancer development. Evidence indicating a prognostic role of the immune system in vulvar carcinoma is scarce. This study investigated the presence and prognostic significance of several aspects of the immune system in vulvar squamous carcinoma. METHODS: The number of intratumoral CD8(+) and Foxp3(+) T-lymphocytes, next to HLA class I (HLA-A, HLA-B/C and ß(2)-m) and indoleamine 2,3-dioxygenase (IDO) expression was determined by immunohistochemistry in a consecutively selected cohort of 286 vulvar squamous carcinoma patients, all treated in the University Medical Center Groningen, the Netherlands. Associations between immunohistochemistry expression and the influence on survival were determined. RESULTS: The number of tumor-infiltrating CD8(+) T-lymphocytes was significantly lower in tumors with loss of HLA-A (p=0.004), HLA-B/C (p=0.024) or ß(2)-m (p=0.025) expression compared with tumors with expression of HLA class I. No association was found between the number of intratumoral CD8(+) T-lymphocytes and Foxp3(+) T-lymphocytes, HLA class I and IDO expression and survival of vulvar squamous carcinoma patients. CONCLUSION: Our results indicate that the immune system does not seem to have a major influence on prognosis of patients with vulvar squamous carcinoma.

Identification of inguinofemoral lymph node metastases by methylation markers in vulvar cancer.

OBJECTIVE: Lymph node status in early-stage vulvar cancer can be accurately assessed by the sentinel-node (SN) procedure. Molecular techniques, such as DNA-methylation assay, might improve SN assessment. In this study, we selected methylation markers for vulvar cancer and determined if these methylation markers were suitable for lymph node assessment. METHODS: We performed methylation specific PCR on DNA isolated from primary tumors, metastatic lymph nodes, and negative lymph nodes from twenty vulvar cancer patients using the following genes: P16INK4a, MGMT, TWIST1, CADM1, TERT, and TFPI2. For P16INK4a and MGMT immunohistochemistry was performed on primary tumors and metastatic lymph nodes in order to explore intratumor heterogeneity in gene expression patterns. RESULTS: TERT was methylated in all vulvar cancers, P16INK4a in 13/20, TFPI2 in 12/20, CADM1 in 11/20, MGMT in 9/20, and TWIST1 in 7/20. A panel of three methylation markers (P16INK4a, TERT and TFPI2) reached a sensitivity of 67% and specificity of 100% for detection of metastatic lymph nodes. Immunohistochemistry showed intratumor heterogeneity for expression of P16INK4a and MGMT in respectively 55% and 45% of primary tumors. CONCLUSIONS: Our study shows methylation for one or more methylation markers in all vulvar cancers. Despite a specificity of 100% our panel of three methylation markers had only moderate sensitivity for metastatic lymph node detection, thereby limiting its applicability for lymph node assessment. Intratumor heterogeneity for expression of P16INK4a and MGMT may reflect intratumor heterogeneity for methylation patterns and thereby in general explain the moderate sensitivity of our marker panel for detection of metastases.

Prognostic image-based quantification of CD8CD103 T cell subsets in high-grade serous ovarian cancer patients.

CD103-positive tissue resident memory-like CD8+ T cells (CD8CD103 TRM) are associated with improved prognosis across malignancies, including high-grade serous ovarian cancer (HGSOC). However, whether quantification of CD8, CD103 or both is required to improve existing survival prediction and whether all HGSOC patients or only specific subgroups of patients benefit from infiltration, remains unclear. To address this question, we applied image-based quantification of CD8 and CD103 multiplex immunohistochemistry in the intratumoral and stromal compartments of 268 advanced-stage HGSOC patients from two independent clinical institutions. Infiltration of CD8CD103 immune cell subsets was independent of clinicopathological factors. Our results suggest CD8CD103 TRM quantification as a superior method for prognostication compared to single CD8 or CD103 quantification. A survival benefit of CD8CD103 TRM was observed only in patients treated with primary cytoreductive surgery. Moreover, survival benefit in this group was limited to patients with no macroscopic tumor lesions after surgery. This approach provides novel insights into prognostic stratification of HGSOC patients and may contribute to personalized treatment strategies in the future.

Identification of genes and pathways associated with cytotoxic T lymphocyte infiltration of serous ovarian cancer.

BACKGROUND: Tumour-infiltrating lymphocytes (TILs) are predictors of disease-specific survival (DSS) in ovarian cancer. It is largely unknown what factors contribute to lymphocyte recruitment. Our aim was to evaluate genes and pathways contributing to infiltration of cytotoxic T lymphocytes (CTLs) in advanced-stage serous ovarian cancer. METHODS: For this study global gene expression was compared between low TIL (n=25) and high TIL tumours (n=24). The differences in gene expression were evaluated using parametric T-testing. Selectively enriched biological pathways were identified with gene set enrichment analysis. Prognostic influence was validated in 157 late-stage serous ovarian cancer patients. Using immunohistochemistry, association of selected genes from identified pathways with CTL was validated. RESULTS: The presence of CTL was associated with 320 genes and 23 pathways (P<0.05). In addition, 54 genes and 8 pathways were also associated with DSS in our validation cohort. Immunohistochemical evaluation showed strong correlations between MHC class I and II membrane expression, parts of the antigen processing and presentation pathway, and CTL recruitment. CONCLUSION: Gene expression profiling and pathway analyses are valuable tools to obtain more understanding of tumour characteristics influencing lymphocyte recruitment in advanced-stage serous ovarian cancer. Identified genes and pathways need to be further investigated for suitability as therapeutic targets.

Presence of tumor-infiltrating lymphocytes is an independent prognostic factor in type I and II endometrial cancer.

OBJECTIVE: Presence of tumor-infiltrating lymphocytes (TIL) is of prognostic importance in a variety of malignancies. This study aims to determine the prognostic value of CD8(+) cytotoxic T-lymphocytes (CTL), FoxP3(+) regulatory T-lymphocytes (Treg) and CD45R0(+) memory T-lymphocytes in endometrial cancer. METHODS: The number of tumor-infiltrating CD8(+), FoxP3(+), and CD45R0(+) T-lymphocytes was determined by immunohistochemistry on tissue microarrays containing tumor material from 368 FIGO stage I-IV endometrial cancer patients. Results from immunohistochemistry were correlated with clinicopathological parameters and survival. RESULTS: High numbers of intra-tumoral CD8(+) T-lymphocytes, a high CD8(+)/FoxP3(+) ratio and the presence of CD45R0(+) T-lymphocytes were strongly associated with well-known favorable prognostic factors in endometrial cancer. Furthermore, high numbers of CD8(+) T-lymphocytes and a high CD8(+)/FoxP3(+) ratio were associated with a better disease free survival (DFS). High numbers of CD8(+) T-lymphocytes and the presence of CD45R0(+) T-lymphocytes were associated with a prolonged overall survival (OS). In multivariate analysis, high numbers of CD8(+) T-lymphocytes had an independent prognostic impact for overall survival in the entire cohort (HR 0.48, 95% C.I. 0.26-0.89, p=0.019) and in type II endometrial cancer (HR 0.17, 95% C.I. 0.08-0.36, p<0.001). A high CD8(+)/FoxP3(+) ratio was independently associated with improved survival in type I endometrial cancer (HR 0.44, 95% C.I. 0.23-0.84, p=0.013). CD45R0(+) lymphocytes were an independent factor for improved OS (HR 0.42, 95% C.I. 0.19-0.93, p=0.033). CONCLUSION: This study shows that the presence of TIL is an independent prognostic factor in endometrial cancer and indicates an important role for the immune system in endometrial cancer.

Routine follow-up intervals in patients with high-grade squamous intraepithelial lesions (HSIL) and free excision margins can safely be increased in the first two years after Large Loop Excision of the Transformation Zone (LLETZ).

OBJECTIVE: To assess the compliance of HSIL patients to the national Dutch routine follow-up protocol in the first 2 years after LLETZ and to determine if based on the status of excision margins, follow-up intervals could be modified. METHODS: A prospective cohort study was performed in patients, referred because of an abnormal Pap smear between 1996 and 2004 and treated for HSIL with LLETZ. The Dutch national routine follow-up protocol orders a Pap smear after 6, 12 and 24 months, respectively. Follow-up results were completed by using PALGA, the nationwide network and registry of histo- and cytopathology in the Netherlands. To assess compliance to the follow-up protocol, adequate follow-up was defined as three cervical smears taken after 6 (+/-3), 12 (+/-3) and 24 (+/-3) months, respectively. RESULTS: Compliance to the first 2 years follow-up protocol declined from 86.2% to 64.8% to 51.2% for first, second and third follow-up cervical smears, respectively. Patients with involved excision margins had a three times higher overall risk of developing a subsequent HSIL after LLETZ as compared to patients with free excision margins (HR: 3.2, 95% CI=1.3-7.9, p=0.01). Risk for diagnosing HSIL during the first 12 months of follow-up for patients with free excision margins was only 1%. CONCLUSIONS: Compliance to the Dutch national routine follow-up protocol in HSIL patients after LLETZ is only moderate. For HSIL patients with free excision margins after LLETZ the first cytological follow-up interval can safely be increased to 12 months.

The ErbB signalling pathway: protein expression and prognostic value in epithelial ovarian cancer.

Ovarian cancer is the most frequent cause of death from gynaecological cancer in the Western world. Current prognostic factors do not allow reliable prediction of response to chemotherapy and survival for individual ovarian cancer patients. Epidermal growth factor receptor (EGFR) and HER-2/neu are frequently expressed in ovarian cancer but their prognostic value remains unclear. In this study, we investigated the expression and prognostic value of EGFR, EGFR variant III (EGFRvIII), HER-2/neu and important downstream signalling components in a large series of epithelial ovarian cancer patients. Immunohistochemical staining of EGFR, pEGFR, EGFRvIII, Her-2/neu, PTEN (phosphatase and tensin homologue deleted on chromosome 10), total and phosphorylated AKT (pAKT) and phosphorylated ERK (pERK) was performed in 232 primary tumours using the tissue microarray platform and related to clinicopathological characteristics and survival. In addition, EGFRvIII expression was determined in 45 tumours by RT-PCR. Our results show that negative PTEN immunostaining was associated with stage I/II disease (P=0.006), non-serous tumour type (P=0.042) and in multivariate analysis with a longer progression-free survival (P=0.015). Negative PTEN staining also predicted improved progression-free survival in patients with grade III or undifferentiated serous carcinomas (P=0.011). Positive pAKT staining was associated with advanced-stage disease (P=0.006). Other proteins were expressed only at low levels, and were not associated with any clinicopathological parameter or survival. None of the tumours were positive for EGFRvIII. In conclusion, our results indicate that tumours showing negative PTEN staining could represent a subgroup of ovarian carcinomas with a relatively favourable prognosis.