Correlations between immune response and vascularization qRT-PCR gene expression clusters in squamous cervical cancer.

BACKGROUND: The tumour microenvironment comprises a network of immune response and vascularization factors. From this network, we identified immunological and vascularization gene expression clusters and the correlations between the clusters. We subsequently determined which factors were correlated with patient survival in cervical carcinoma. METHODS: The expression of 42 genes was investigated in 52 fresh frozen squamous cervical cancer samples by qRT-PCR. Weighted gene co-expression network analysis and mixed-model analyses were performed to identify gene expression clusters. Correlations and survival analyses were further studied at expression cluster and single gene level. RESULTS: We identified four immune response clusters: 'T cells' (CD3E/CD8A/TBX21/IFNG/FOXP3/IDO1), 'Macrophages' (CD4/CD14/CD163), 'Th2' (IL4/IL5/IL13/IL12) and 'Inflammation' (IL6/IL1B/IL8/IL23/IL10/ARG1) and two vascularization clusters: 'Angiogenesis' (VEGFA/FLT1/ANGPT2/ PGF/ICAM1) and 'Vessel maturation' (PECAM1/VCAM1/ANGPT1/SELE/KDR/LGALS9). The 'T cells' module was correlated with all modules except for 'Inflammation', while 'Inflammation' was most significantly correlated with 'Angiogenesis' (p < 0.001). High expression of the 'T cells' cluster was correlated with earlier TNM stage (p = 0.007). High CD3E expression was correlated with improved disease-specific survival (p = 0.022), while high VEGFA expression was correlated with poor disease-specific survival (p = 0.032). Independent predictors of poor disease-specific survival were IL6 (hazard ratio = 2.3, p = 0.011) and a high IL6/IL17 ratio combined with low IL5 expression (hazard ratio = 4.2, p = 0.010). CONCLUSIONS: 'Inflammation' marker IL6, especially in combination with low levels of IL5 and IL17, was correlated with poor survival. This suggests that IL6 promotes tumour growth, which may be suppressed by a Th17 and Th2 response. Measuring IL6, IL5 and IL17 expression may improve the accuracy of predicting prognosis in cervical cancer.

Intraepithelial macrophage infiltration is related to a high number of regulatory T cells and promotes a progressive course of HPV-induced vulvar neoplasia.

Human papilloma virus (HPV)-induced usual-type vulvar intraepithelial neoplasia (uVIN) is infiltrated by myeloid cells but the type and role of these cells is unclear. We used triple immunofluorescent confocal microscopy to locate, identify and quantify myeloid cells based on their staining pattern for CD14, CD33 and CD163 in a cohort of 43 primary and 20 recurrent uVIN lesions, 21 carcinomas and 26 normal vulvar tissues. The progressive course of uVIN is characterized by an increase in both intraepithelial and stromal mature M1 and M2 macrophages. While the M2 macrophages outnumber M1 macrophages in healthy controls and uVIN, they are matched in number by M1 macrophages in cancer. Importantly, uVIN patients with a dense intraepithelial infiltration with mature CD14+ macrophages (irrespective of M1 or M2 type) displayed approximately a six times higher risk to develop a recurrence and a high number of these cells constituted an independent prognostic factor for recurrence. In addition, a dense intraepithelial CD14+ cell infiltration was associated with high numbers of intraepithelial CD4+ Tregs and low numbers of stromal CD8+TIM3+ T cells. Patients with low numbers of intraepithelial CD14+ cells and high numbers of stromal CD8+TIM3+ cells showed the best recurrence-free survival. These data clearly show the importance of the local immune response in HPV-induced vulvar neoplasia and may be of help in predicting the prognosis of patients or their response to immunotherapy.

Expression of coinhibitory receptors on T cells in the microenvironment of usual vulvar intraepithelial neoplasia is related to proinflammatory effector T cells and an increased recurrence-free survival.

Human papillomavirus-induced usual-type vulvar intraepithelial neoplasia (uVIN) are infiltrated by immune cells but apparently not cleared. A potential explanation for this is an impaired T cell effector function by an immunesuppressive milieu, coinfiltrating regulatory T cells or the expression of coinhibitory molecules. Here, the role of these potential inhibitory mechanisms was evaluated by a detailed immunohistochemical analysis of T cell infiltration in the context of FoxP3, Tbet, indoleamine 2,3-dioxygenase, programmed cell death 1, T cell immunoglobulin mucin 3 (TIM3), natural killer cell lectin-like receptor A (NKG2A) and galectins-1, -3 and -9. Paraffin-embedded tissues of primary uVIN lesions (n=43), recurrent uVIN lesions (n=20), vulvar carcinoma (n=21) and healthy vulvar tissue (n=26) were studied. We show that the vulva constitutes an area intensely surveyed by CD8+, CD4+, Tbet+ and regulatory T cell populations, parts of which express the examined coinhibitory molecules. In uVIN especially, the number of regulatory T cells and TIM3+ T cells increased. The expression of the coinhibitory markers TIM3 and NKG2A probably reflected a higher degree of T cell activation as a dense infiltration with stromal CD8+TIM3+ T cells and CD3+NKG2A+ T cells was related to the absence of recurrences and/or a prolonged recurrence-free survival. A dense coinfiltrate with regulatory T cells was negatively associated with the time to recurrence, most dominantly when the stromal CD8+TIM3+ infiltration was limited. This notion was sustained in vulvar carcinoma's where the numbers of regulatory T cells progressively increased to outnumber coinfiltrating CD8+TIM3+ T cells and CD3+NKG2A+ T cells.

FoxP3(+) and IL-17(+) cells are correlated with improved prognosis in cervical adenocarcinoma.

Cervical adenocarcinoma comprises approximately 15 % of cervical cancer cases. This histological subtype has different characteristics than cervical squamous cell carcinoma, which may influence disease progression. To study whether the infiltration of T cell subpopulations was correlated with cervical adenocarcinoma patient survival, similar to squamous cell carcinoma, the tumor-infiltrating T cells, Tregs, Th17 cells and IL-17(+) cell frequencies were analyzed in a cohort of cervical adenocarcinoma patients (n = 67). Intraepithelial, stromal and total cell frequencies were scored using triple immunofluorescence. The majority of Tregs were present in the tumor stroma, while other T cells and IL-17(+) cells infiltrated the tumor epithelium three times more frequently. A high total number of Tregs were significantly correlated with improved disease-specific and disease-free survival (p = 0.010, p = 0.007). Within the tumor epithelium, a high T cell frequency was significantly correlated with improved disease-free survival (p = 0.034). In particular, a low number of both Tregs and IL-17(+) cells were correlated with poor disease-specific survival (p = 0.007). A low number of Tregs combined with Th17 cells present were also correlated with poor survival (p = 0.018). An increased number of IL-17(+) cells were significantly correlated with the absence of vaso-invasion (p = 0.001), smaller tumor size (p = 0.030) and less infiltration depth (p = 0.021). These results suggest that Tregs and IL-17(+) cells represent a beneficial immune response, whereas Th17 cells might represent a poor response in cervical adenocarcinoma. This contrasts with the correlations described in squamous cell carcinoma, suggesting that the local immune response in cervical adenocarcinoma contributes differently to tumor growth than in squamous cell carcinoma.

The long-term immune response after HPV16 peptide vaccination in women with low-grade pre-malignant disorders of the uterine cervix: a placebo-controlled phase II study.

The capacity of a low-dose HPV16 synthetic long-peptide vaccine (HPV16-SLP) to induce an HPV16-specific T-cell response as well as to establish long-term immunologic memory in patients with low-grade abnormalities of the cervix was determined in a placebo-controlled, double-blinded phase II study. In addition, the effect of a booster vaccination after 1 year was evaluated. Patients received either the HPV16-SLP or a placebo at the start of the study. After 1 year, the vaccinated patients were again randomized to receive the HPV16-SLP or a placebo. Patients were followed for 2 years. HPV16-specific T-cell responses were determined in pre- and post-vaccination blood samples by ELISPOT, proliferation assay and cytokine assays. We show that the HPV16-specific T-cell responses detected after vaccination are clearly due to vaccination and that reactivity was maintained for at least 2 years. Interestingly, a booster vaccination after 1 year especially augmented the HPV16-specific Th2 response. Furthermore, pre-existing immunity to HPV16 was associated with a stronger response to vaccination and with more side effects, reflected by flu-like symptoms. We conclude that two low-dose injections of HPV16-SLP can induce a strong and stable HPV16-specific T-cell response that lasts for at least 1 year. If booster vaccination is required, then polarizing adjuvant should be added to maintain the Th1 focus of the vaccine-induced T-cell response.

CDKN2A(p16) and HRAS are frequently mutated in vulvar squamous cell carcinoma.

BACKGROUND: Two etiologic pathways of vulvar cancer are known, a human papillomavirus (HPV)- and a TP53-associated route, respectively, but other genetic changes may also play a role. Studies on somatic mutations in vulvar cancer other than TP53 are limited in number and size. In this study, we investigated the prevalence of genetic mutations in 107 vulvar squamous cell carcinomas (VSCCs). METHODS: A total of 107 paraffin-embedded tissue samples of primarily surgically treated VSCCs were tested for HPV infection and screened for mutations in 14 genes (BRAF, CDKN2A(p16), CTNNB1, FBXW7, FGFR2, FGFR3, FOXL2, HRAS, KRAS, NRAS, PIK3CA, PPP2R1A, PTEN, and TP53) using Sanger sequencing and mass spectrometry. RESULTS: Mutations were detected in 7 genes. Of 107 VSCCs, 66 tumors (62%) contained at least one mutation (TP53=58, CDKN2A(p16)=14, HRAS=10, PIK3CA=7, PPP2R1A=3, KRAS=1, PTEN=1). Mutations occurred most frequently in HPV-negative samples. Five-year survival was significantly worse for patients with a mutation (47% vs 59%, P=.035), with a large effect from patients carrying HRAS-mutations. CONCLUSION: Somatic mutations were detected in 62% of VSCCs. As expected, HPV infection and TP53-mutations play a key role in the development of VSCC, but CDKN2A(p16), HRAS, and PIK3CA-mutations were also frequently seen in HPV-negative patients. Patients with somatic mutations, especially HRAS-mutations, have a significantly worse prognosis than patients lacking these changes, which could be of importance for the development of targeted therapy.

HPV16 synthetic long peptide (HPV16-SLP) vaccination therapy of patients with advanced or recurrent HPV16-induced gynecological carcinoma, a phase II trial.

BACKGROUND: Human papilloma virus type 16 (HPV16)-induced gynecological cancers, in particular cervical cancers, are found in many women worldwide. The HPV16 encoded oncoproteins E6 and E7 are tumor-specific targets for the adaptive immune system permitting the development of an HPV16-synthetic long peptide (SLP) vaccine with an excellent treatment profile in animal models. Here, we determined the toxicity, safety, immunogenicity and efficacy of the HPV16 SLP vaccine in patients with advanced or recurrent HPV16-induced gynecological carcinoma. METHODS: Patients with HPV16-positive advanced or recurrent gynecological carcinoma (n = 20) were subcutaneously vaccinated with an HPV16-SLP vaccine consisting of a mix of 13 HPV16 E6 and HPV16 E7 overlapping long peptides in Montanide ISA-51 adjuvant. The primary endpoints were safety, toxicity and tumor regression as determined by RECIST. In addition, the vaccine-induced T-cell response was assessed by proliferation and associated cytokine production as well as IFNγ-ELISPOT. RESULTS: No systemic toxicity beyond CTCAE grade II was observed. In a few patients transient flu-like symptoms were observed. In 9 out of 16 tested patients vaccine-induced HPV16-specific proliferative responses were detected which were associated with the production of IFNγ, TNFα, IL-5 and/or IL-10. ELISPOT analysis revealed a vaccine-induced immune response in 11 of the 13 tested patients. The capacity to respond to the vaccine was positively correlated to the patient's immune status as reflected by their response to common recall antigens at the start of the trial. Median survival was 12.6 ± 9.1 months. No regression of tumors was observed among the 12 evaluable patients. Nineteen patients died of progressive disease. CONCLUSIONS: The HPV16-SLP vaccine was well tolerated and induced a broad IFNγ-associated T-cell response in patients with advanced or recurrent HPV16-induced gynecological carcinoma but neither induced tumor regression nor prevented progressive disease. We, therefore, plan to use this vaccine in combination with chemotherapy and immunomodulation.

Success or failure of vaccination for HPV16-positive vulvar lesions correlates with kinetics and phenotype of induced T-cell responses.

One half of a group of 20 patients with human papillomavirus type 16 (HPV16)-induced vulvar intraepithelial neoplasia grade 3 displayed a complete regression (CR) after therapeutic vaccination with HPV16 E6/E7 synthetic long peptides. Patients with relatively larger lesions generally did not display a CR. To investigate immune correlates of treatment failure, patients were grouped according to median lesion size at study entry, and HPV16-specific immunity was analyzed at different time points by complementary immunological assays. The group of patients with smaller lesions displayed stronger and broader vaccine-prompted HPV16-specific proliferative responses with higher IFNgamma (P = 0.0003) and IL-5 (P < 0.0001) levels than patients with large lesions. Characteristically, this response was accompanied by a distinct peak in cytokine levels after the first vaccination. In contrast, the patient group with larger lesions mounted higher frequencies of HPV16-specific CD4(+)CD25(+)Foxp3(+) T cells (P = 0.005) and displayed a lower HPV16-specific IFNgamma/IL-10 ratio after vaccination (P < 0.01). No disparity in T memory immunity to control antigens was found, indicating that the differences in HPV-specific immunity did not reflect general immune failure. We observed a strong correlation between a defined set of vaccine-prompted specific immune responses and the clinical efficacy of therapeutic vaccination. Notably, a high ratio of HPV16-specific vaccine-prompted effector T cells to HPV16-specific CD4(+)CD25(+)Foxp3(+) T cells was predictive of clinical success. Foxp3(+) T cells have been associated previously with impaired immunity in malignancies. Here we demonstrate that the vaccine-prompted level of this population is associated with early treatment failure.

Vaccination against HPV-16 oncoproteins for vulvar intraepithelial neoplasia.

BACKGROUND: Vulvar intraepithelial neoplasia is a chronic disorder caused by high-risk types of human papillomavirus (HPV), most commonly HPV type 16 (HPV-16). Spontaneous regression occurs in less than 1.5% of patients, and the rate of recurrence after treatment is high. METHODS: We investigated the immunogenicity and efficacy of a synthetic long-peptide vaccine in women with HPV-16-positive, high-grade vulvar intraepithelial neoplasia. Twenty women with HPV-16-positive, grade 3 vulvar intraepithelial neoplasia were vaccinated three or four times with a mix of long peptides from the HPV-16 viral oncoproteins E6 and E7 in incomplete Freund's adjuvant. The end points were clinical and HPV-16-specific T-cell responses. RESULTS: The most common adverse events were local swelling in 100% of the patients and fever in 64% of the patients; none of these events exceeded grade 2 of the Common Terminology Criteria for Adverse Events of the National Cancer Institute. At 3 months after the last vaccination, 12 of 20 patients (60%; 95% confidence interval [CI], 36 to 81) had clinical responses and reported relief of symptoms. Five women had complete regression of the lesions, and HPV-16 was no longer detectable in four of them. At 12 months of follow-up, 15 of 19 patients had clinical responses (79%; 95% CI, 54 to 94), with a complete response in 9 of 19 patients (47%; 95% CI, 24 to 71). The complete-response rate was maintained at 24 months of follow-up. All patients had vaccine-induced T-cell responses, and post hoc analyses suggested that patients with a complete response at 3 months had a significantly stronger interferon-gamma-associated proliferative CD4+ T-cell response and a broad response of CD8+ interferon-gamma T cells than did patients without a complete response. CONCLUSIONS: Clinical responses in women with HPV-16-positive, grade 3 vulvar intraepithelial neoplasia can be achieved by vaccination with a synthetic long-peptide vaccine against the HPV-16 oncoproteins E6 and E7. Complete responses appear to be correlated with induction of HPV-16-specific immunity.

A placebo-controlled randomized HPV16 synthetic long-peptide vaccination study in women with high-grade cervical squamous intraepithelial lesions.

The aim of this study was to investigate the capacity of an HPV16 E6/E7 synthetic overlapping long-peptide vaccine to stimulate the HPV16-specific T-cell response, to enhance the infiltration of HPV16-specific type 1 T cells into the lesions of patients with HPV16+ high-grade cervical squamous intraepithelial lesion (HSIL) and HPV clearance. This was a placebo-controlled randomized phase II study in patients with HPV16-positive HSIL. HPV16-specific T-cell responses were determined pre- and post-vaccination by ELISPOT, proliferation assay and cytokine assays in PBMC and HSIL-infiltrating lymphocytes, and delayed-type hypersensitivity skin tests. Motivational problems of this patient group to postpone treatment of their premalignant lesions affected the inclusion rates and caused the study to stop prematurely. Of the accrued patients, 4 received a placebo and 5 received 1-2 vaccinations. Side effects mainly were flu-like symptoms and injection site reactions. A strong HPV-specific IFNγ-associated T-cell response was detected by ELISPOT in all vaccinated patients. The outcome of the skin tests correlated well with the ELISPOT analysis. The cytokine profile associated with HPV16-specific proliferation varied from robust type 1 to dominant type 2 responses. No conclusions could be drawn on vaccine-enhanced T-cell infiltration of the lesion, and there was no HPV clearance at the time of LEEP excision. Thus, vaccination of HSIL patients results in increased HPV16-specific T-cell immunity. Further development of this type of treatment relies on the ability to motivate patients and in the reduction in the side effects.

HLA-E expression in cervical adenocarcinomas: association with improved long-term survival.

BACKGROUND: Cervical cancer is the third most common cancer in women worldwide. The most common histopathological subtype is cervical squamous cell carcinoma (SCC, 75-80%), followed by adenocarcinoma (AC) and adenosquamous carcinoma (ASC; together 15-20%). Rising incidence rates of AC have been observed relative and absolute to SCC and evidence is accumulating that cervical AC is a distinct clinical entity. Cervical SCC, ASC, and AC are caused by a persistent infection with high-risk human papillomavirus (HPV) and failed control of the immune system plays a pivotal role in the carcinogenesis of all three histopathological subtypes. Human leukocyte antigen E (HLA-E), a non-classical HLA class Ib molecule, plays an important role in immune surveillance and immune escape of virally infected cells. In this study we investigated HLA-E expression in three well-defined cohorts of cervical AC, ASC, and SCC patients, and determined whether HLA-E expression was associated with histopathological parameters and patient survival. METHODS AND RESULTS: HLA-E expression was assessed by immunohistochemistry on formalin-fixed, paraffin-embedded tissue sections of 79 SCC, 38 ASC, and 75 AC patients. All patients included were International Federation of Gynaecology and Obstetrics stage I-II and underwent radical hysterectomy with lymphadenectomy as primary treatment. Significant differences between the histopathological subgroups were detected for age distribution, HPV positivity, HPV type distribution, tumour size, tumour infiltration depth, lymph-vascular space invasion, and adjuvant radiotherapy. High expression of HLA-E was found in 107/192 (56%) cervical carcinomas, with significantly more overexpression in cervical AC compared to SCC and ASC (37/79 SCC, 18/38 ASC, and 52/75 AC; P = 0.010). High HLA-E expression in cervical AC was associated with favourable long term disease-specific and recurrence-free survival (P = 0.005 and P = 0.001, respectively). CONCLUSION: High expression of HLA-E occurred in the majority of all histopathological subtypes of cervical cancer; especially in cervical AC. High HLA-E expression in cervical AC was associated with improved patient survival. This study also highlights the importance of careful evaluation of cervical carcinomas to distinguish histopathological subtypes. In the future, insight into the biological behaviour and distinct molecular carcinogenetic processes of the AC, ASC, and SCC subtypes may contribute to the development of more tumour-specific treatment strategies.

Cytomorphological analysis of uterine cervical pap smears in relation to human papillomavirus infection in Indonesian women.

OBJECTIVE: Cervical cancer is the number one cause of cancer-associated death in Indonesian women (30/100,000 annually), where no screening program is present. The Papanicolaou test is widely accepted as an effective screening method for cervical neoplasia detection and often shows certain cytological features associated with human papillomavirus (HPV) infection. Especially in developing countries, cytological investigation is still the method of choice as compared to the frequent use of HPV DNA testing in western countries. STUDY DESIGN: In the present study, we investigated the validity of the use of cytomorphological changes as a marker for HPV infection. A total of 140 smears collected in three different areas in Indonesia (Jakarta, Tasikmalaya and Bali) were analyzed. HPV DNA testing was performed using INNO-LiPA assays. RESULTS AND CONCLUSIONS: We found a highly significant association of classical koilocytosis, multinucleated cells, dyskeratosis-parakeratosis, nuclear membrane, enlarged nuclei, moderate/strong hyperchromasia and chromatin pattern with HPV positivity. Using classical and nonclassical cytomorphological parameters we found an overall sensitivity of 42% and a specificity of 90%. The combination of classical and nonclassical parameters led to a higher sensitivity of HPV positivity prediction. These results are of importance for cytologists in developing countries as molecular HPV testing still poses a major financial, logistic and expertise problem.

The detection of circulating human papillomavirus-specific T cells is associated with improved survival of patients with deeply infiltrating tumors.

A detailed analyses of HPV-specific immunity was performed in a large group of patients with HPV-induced cervical cancer (CxCa) in relation to HLA-types and prognostic factors. Patients were HLA-typed and HPV16/18-specific T-cell immunity was assessed by proliferation assay and cytometric bead array using freshly isolated PBMC and by phenotypic analysis of HPV-specific T cells. The results were analyzed in relation to known disease-related HLA-types (DR7, DR13, DR15/DQ06), invasion-depth and size of tumor, lymph node (LN) status and disease free survival. In total 119 HLA-typed patients with CxCa were analyzed. Patients expressing the HLA-DR13 haplotype were underrepresented as compared to the Dutch population (p = 0.014), whereas HLA-DR7 was overrepresented in patients with HPV16+ CxCa (p = 0.006). In 29 of 94 patients (31%) from whom blood could be tested, a proliferative response to HPV16/18 was detected, which was associated with increased numbers of HPV-specific CD4+CD25+ (activated) T cells (p = 0.03) and HPV-specific CD4+CD25+FoxP3-positive T cells (p = 0.04). The presence of both FoxP3-positive and negative HPV-specific CD4+CD25+ T cells was significantly correlated (p = 0.01). Interestingly, the detection of HPV-specific proliferation was associated with invasion depth (p = 0.020) but not with HLA type, tumor size nor LN status. Moreover, the detection of HPV-specific immunity was associated with an improved disease free survival (p = 0.04) in patients with deeply infiltrating tumors. In conclusion, HPV-specific proliferative T-cell response, comprising higher percentages of HPV-specific CD25+ and CD25+FoxP3-positive CD4+T cells, are more frequently detected in patients with deep infiltrating CxCa tumors and associated with an improved survival.

Molecular mechanisms of epidermal growth factor receptor overexpression in patients with cervical cancer.

The epidermal growth factor receptor is overexpressed in 70-90% of cervical cancers. Previously, we have shown that epidermal growth factor receptor overexpression independently predicts poor prognosis in cervical cancer patients, which makes it a potential therapeutic target. The aim of this study was to systematically analyze the molecular mechanism leading to epidermal growth factor receptor overexpression in cervical cancer. All experiments were performed on archival paraffin-embedded material. In 166 cervical cancer patients, cytoplasmic, membrane and phosphorylated epidermal growth factor receptor protein expression were studied in association with patient survival. Membrane epidermal growth factor receptor overexpression was associated with poor disease-specific survival (P=0.027). This association was particularly present in human papillomavirus 16-positive patients (P=0.029). We analyzed whether epidermal growth factor receptor overexpression was caused by gene amplification using fluorescence in situ hybridization. Epidermal growth factor receptor gene copy number was linked to chromosome 7 ploidy, as no gene amplification could be detected when corrected for chromosome 7 centromeric signals. Chromosome 7 aneuploidy was associated with membrane epidermal growth factor receptor overexpression (P=0.013). Additional mutation analysis was performed by sequencing pure, flow-sorted tumor cells, but no mutations were detected. Furthermore, human papillomavirus 16 E5 and E6 oncogene mRNA expression was measured, using quantitative real-time polymerase chain reaction, to determine the association between the human papillomavirus and epidermal growth factor receptor overexpression. High human papillomavirus 16 E5 and E6 mRNA expression were associated with decreased survival (P=0.045 and 0.047, respectively). High human papillomavirus 16 E6 mRNA expression was associated with membrane epidermal growth factor receptor overexpression (P=0.013). This is the first study performed on cancer patient material showing that chromosome 7 aneuploidy and high human papillomavirus 16 E6 mRNA expression lead to membrane epidermal growth factor receptor overexpression in cervical cancer.

Human leukocyte antigen class I, MHC class I chain-related molecule A, and CD8+/regulatory T-cell ratio: which variable determines survival of cervical cancer patients?

PURPOSE: To investigate the effect of intraepithelial tumor-infiltrating lymphocytes (ieTIL) and their ligands expressed by cervical tumor cells on the outcome of cervical cancer patients. EXPERIMENTAL DESIGN: The prognostic value of ieTILs was investigated in 115 cases of cervical cancer. T-cell subsets, CD57(+) cells, and regulatory T cells (Treg) were enumerated. The associations of these different ieTIL subtypes with human leukocyte antigen (HLA) class I and MHC class I chain-related molecule A (MICA) expression were determined in relation to clinical variables and patient survival. RESULTS: Survival analysis showed that a high number of intraepithelial Treg (FoxP3(+)), a low CD8(+)/regulatory T-cell ratio, and a weak HLA-A expression were all associated with worse survival (P=0.034, 0.025, and 0.033, respectively, log-rank test). Further stratification of patient groups based on HLA-A-MICA expression and HLA-A-MICA-CD8(+)/Treg ratio revealed an even poorer survival (P=0.005). In a multivariate Cox analysis, low CD8(+)/Treg ratio (P=0.047), weak HLA-A-MICA expression (P=0.003), and weak HLA-A-MICA expression combined with low CD8(+)/Treg ratio (P=0.002) were all found to be independent unfavorable prognostic predictors in cervical carcinoma (hazard ratios, 2.7, 4.0, and 4.9, respectively). CONCLUSION: Weak HLA-A-MICA expression combined with low CD8(+)/Treg ratio reveals a patient group with the poorest survival in cervical cancer. As a single variable, low CD8(+)/Treg ratio was a significant independent unfavorable prognostic factor.

Phase I immunotherapeutic trial with long peptides spanning the E6 and E7 sequences of high-risk human papillomavirus 16 in end-stage cervical cancer patients shows low toxicity and robust immunogenicity.

PURPOSE: To determine the toxicity, safety, and immunogenicity of a human papillomavirus 16 (HPV16) E6 and E7 long peptide vaccine administered to end-stage cervical cancer patients. EXPERIMENTAL DESIGN: Three groups of end-stage cervical cancer patients (in total n = 35) were s.c. vaccinated with HPV16 E6 combined with or separated from HPV16 E7 overlapping long peptides in Montanide ISA-51 adjuvant, four times at 3-week intervals. Group 1 received 300 microg/peptide at a single site and group 2 received 100 microg/peptide of the E6 peptides in one limb and 300 microg/peptide of the E7 peptides in a second limb. Group 3 received separate injections of E6 and E7 peptides, each at a dose of 50 microg/peptide. The primary end point was to determine safety and toxicity of the HPV16 long peptides vaccine. In addition, the vaccine-induced T-cell response was assessed by IFN gamma enzyme-linked immunospot. RESULTS: No toxicity beyond grade 2 was observed during and after four vaccinations. In a few patients, transient flu-like symptoms were observed. Enzyme-linked immunospot analysis of the vaccine-induced immune response revealed that coinjection of the E6 and E7 peptides resulted in a strong and broad T-cell response dominated by immunity against E6. Injection of the E6 and E7 peptides at two different sites increased the E7 response but did not affect the magnitude of the E6-induced immune response. CONCLUSIONS: The HPV16 E6 and E7 long peptide-based vaccine is well tolerated and capable of inducing a broad IFN gamma-associated T-cell response even in end-stage cervical cancer patients.

Induction of tumor-specific CD4+ and CD8+ T-cell immunity in cervical cancer patients by a human papillomavirus type 16 E6 and E7 long peptides vaccine.

PURPOSE: The study aims to evaluate the effect of a human papillomavirus type 16 (HPV16) E6 and E7 synthetic long peptides vaccine on the antigen-specific T-cell response in cervical cancer patients. EXPERIMENTAL DESIGN: Patients with resected HPV16-positive cervical cancer were vaccinated with an overlapping set of long peptides comprising the sequences of the HPV16 E6 and E7 oncoproteins emulsified in Montanide ISA-51. HPV16-specific T-cell immune responses were analyzed by evaluating the magnitude, breadth, type, and polarization by proliferation assays, IFN gamma-ELISPOT, and cytokine production and phenotyped by the T-cell markers CD4, CD8, CD25, and Foxp3. RESULTS: Vaccine-induced T-cell responses against HPV16 E6 and E7 were detected in six of six and five of six patients, respectively. These responses were broad, involved both CD4(+) and CD8(+) T cells, and could be detected up to 12 months after the last vaccination. The vaccine-induced responses were dominated by effector type CD4(+)CD25(+)Foxp3(-) type 1 cytokine IFN gamma-producing T cells but also included the expansion of T cells with a CD4(+)CD25(+)Foxp3(+) phenotype. CONCLUSIONS: The HPV16 E6 and E7 synthetic long peptides vaccine is highly immunogenic, in that it increases the number and activity of HPV16-specific CD4(+) and CD8(+) T cells to a broad array of epitopes in all patients. The expansion of CD4(+) and CD8(+) tumor-specific T cells, both considered to be important in the antitumor response, indicates the immunotherapeutic potential of this vaccine. Notably, part of the vaccine-induced T cells display a CD4(+)CD25(+)Foxp3(+) phenotype that is frequently associated with regulatory T-cell function, suggesting that strategies to disarm this subset of T cells should be considered as components of immunotherapeutic modalities against HPV-induced cancers.

Lack of TNFalpha mRNA expression in cervical cancer is not associated with loss of heterozygosity at 6p21.3, inactivating mutations or promoter methylation.

Infection with oncogenic human papillomavirus (HPV) is considered to be the major etiologic event for cervical cancer. Tumor necrosis factor alpha (TNFalpha), a proinflammatory cytokine, may be involved in orchestrating an antitumor immune response against human papillomavirus expressing cervical cancer cells. Hence, loss of TNFalpha could be advantageous for tumor cells to escape immune clearance. The aim of our study was to investigate TNFalpha gene expression and epigenetic characteristics associated with the loss of TNFalpha expression in cervical cancer. To this end, we examined TNFalpha expression, loss of heterozygosity (LOH) at 6p21.3, the locus of TNFalpha, mutational status of the TNFalpha locus, loss of the TNFalpha promoter variant 2 allele and CpG hypermethylation of the TNFalpha promoter. RNA in situ hybridization showed absence of TNFalpha expression in 45% of 63 tumors. LOH occurred in 57% of the tumors and was not concordant with absence of TNFalpha mRNA. No mutations in the TNFalpha gene were identified in 15 cases deficient in TNFalpha expression exhibiting LOH. Furthermore, lack of TNFalpha expression did not correlate with promoter methylation. In conclusion, TNFalpha mRNA expression is absent in nearly half of the cervical tumors analyzed. Neither promoter methylation nor genetic causes for lack of expression were evident.

High number of intraepithelial CD8+ tumor-infiltrating lymphocytes is associated with the absence of lymph node metastases in patients with large early-stage cervical cancer.

In a prospective study, we have examined the tumor-specific immune response in a group of 59 patients with human papillomavirus (HPV) 16-positive (HPV16(+))-induced or HPV18(+)-induced cervical cancer. Local antitumor immunity was analyzed by the enumeration of tumor-infiltrating dendritic cells and CD4+, CD8+, and regulatory T cells as well as by calculation of the ratio of CD8+/CD4+ T cells and CD8+/regulatory T cells. Systemic tumor-specific immunity was assessed by determination of the HPV E6- and/or E7-specific T-cell response in the blood of these patients. Finally, these variables were evaluated with respect to known histopathologic prognostic variables, including the absence (LN-) or presence (LN+) of lymph node metastases. Stratification according to the lymph node status of patients revealed a significantly stronger CD8+ T-cell tumor infiltration, a higher CD8+/CD4+ T-cell ratio, and higher CD8+/regulatory T-cell ratio in the group of patients in which the tumor failed to metastasize to the tumor-draining lymph node. Subdivision according to the presence (IR+) or absence (IR-) of circulating HPV-specific T cells disclosed that the highest number of tumor-infiltrating CD8+ T cells was found in the group of LN- patients displaying a concomitant systemic tumor-specific immune response (LN-IR+). CD8+ T-cell infiltration in LN-IR- patients was comparable with that of LN+ patients. In cervical cancer, the absence of lymph node metastases is strongly associated with a better prognosis. Our data indicate that, especially in a subgroup of LN- patients, a strong and effective interaction between immune system and tumor exists. This subgroup of cervical cancer patients may have the best prognosis.

Frequent mutations in the 3'-untranslated region of IFNGR1 lack functional impairment in microsatellite-unstable colorectal tumours.

Microsatellite repeats are frequently found to be mutated in microsatellite-instable colorectal tumours. This suggests that these mutations are important events during tumour development. We have observed frequent mutations in microsatellite-instable (MSI-H) tumours and cell lines of a conserved A14 repeat within the 3'-untranslated region of the interferon-gamma receptor 1 gene (IFNGR1). The repeat was mutated in 59% (41 of 70) of colon carcinomas and in all four MSI-H colon cancer cell lines tested. In-vitro analysis of these cell lines did not show a decreased responsiveness to standard IFNgamma concentrations when compared to microsatellite-stable colon cancer cell lines. A functional consequence of the frequently found microsatellite instability in IFNGR1 is therefore not evident.