Ud Bafna

Regulatory T cells in a spectrum of HPV-induced cervical lesions: cervicitis, cervical intraepithelial neoplasia and squamous cell carcinoma.

Problem  Thriving of tumors amidst rich immune infiltrates is an unexplained paradox.

ORIGINAL ARTICLE: Regulatory T Cells in a Spectrum of HPV-Induced Cervical Lesions: Cervicitis, Cervical Intraepithelial Neoplasia and Squamous Cell Carcinoma

Problem  Thriving of tumors amidst rich immune infiltrates is an unexplained paradox.

Functional tumor infiltrating TH1 and TH2 effectors in large early-stage cervical cancer are suppressed by regulatory T cells.

ObjectiveAnalysis of tumor-infiltrating lymphocytes (TILs) is one of the cornerstones for the understanding of immune responses prevailing in the tumor microenvironment. We studied TILs from squamous cell carcinoma of the cervix ex vivo without proliferating them in vitro before analysis. MethodsWhereas TILs were magnetic activated cell separation enriched and flow sorted into CD4+ CD25hi (regulatory T cells [Tregs]), CD4+ CD25int (effector T cells [Teffs]) were directly purified by flow cytometry, and both these subsets were characterized phenotypically and functionally. Tissue sections were probed for interleukin 4 (IL-4) and interferon &ggr;. ResultsEffector T cells constitutively expressed both interferon &ggr; and IL-4 prototypical cytokines of TH1 and TH2, respectively, and were able to proliferate and secrete higher quantities of both cytokines in response to anti-CD3/anti-CD28 and autologous tumor lysates. Only 53% of cervical cancer Tregs were FOXP3+, elaborated transforming growth factor &bgr;1, and IL-10 and were able to inhibit both T helper subsets. ConclusionsIntratumoral Teffs represented functionally active subsets of both TH1 and TH2 that were not anergic but were suppressed by multiple Treg subsets, which comprised FOXP3 + Tregs and Tregs secreting transforming growth factor &bgr;1 and IL-10. These results imply that the microenvironment of cervical carcinomas harbored both TH1 and TH2 subsets of CD4+ Teffs that were functionally active but were perhaps unable to perform because of the overpowering effect of Tregs.Objective Analysis of tumor-infiltrating lymphocytes (TILs) is one of the cornerstones for the understanding of immune responses prevailing in the tumor microenvironment. We studied TILs from squamous cell carcinoma of the cervix ex vivo without proliferating them in vitro before analysis. Methods Whereas TILs were magnetic activated cell separation enriched and flow sorted into CD4+ CD25hi (regulatory T cells [Tregs]), CD4+ CD25int (effector T cells [Teffs]) were directly purified by flow cytometry, and both these subsets were characterized phenotypically and functionally. Tissue sections were probed for interleukin 4 (IL-4) and interferon γ. Results Effector T cells constitutively expressed both interferon γ and IL-4 prototypical cytokines of TH1 and TH2, respectively, and were able to proliferate and secrete higher quantities of both cytokines in response to anti-CD3/anti-CD28 and autologous tumor lysates. Only 53% of cervical cancer Tregs were FOXP3+, elaborated transforming growth factor β1, and IL-10 and were able to inhibit both T helper subsets. Conclusions Intratumoral Teffs represented functionally active subsets of both TH1 and TH2 that were not anergic but were suppressed by multiple Treg subsets, which comprised FOXP3 + Tregs and Tregs secreting transforming growth factor β1 and IL-10. These results imply that the microenvironment of cervical carcinomas harbored both TH1 and TH2 subsets of CD4+ Teffs that were functionally active but were perhaps unable to perform because of the overpowering effect of Tregs.

Toll-like receptors 7, 8, and 9 expression and function in primary human cervical cancer Langerhans cells: evidence of anergy.

Objective Human papillomavirus oncoproteins E6 and E7 down modulate Toll-like receptor (TLR) 9 expression in infected keratinocytes. We explored the status of expression and function of TLR7, TLR8, and TLR9 in primary human Langerhans cells (LCs) isolated from cervical tumors. Methodology Single-cell suspensions were made from fresh tissues of squamous cell carcinoma (International Federation of Gynecology and Obstetrics stage IB2); myeloid dendritic cells were purified using CD1c magnetic activated cell separation kits. Langerhans cells were further flow sorted into CD1a+CD207+ cells. Acute monocytic leukemia cell line THP-1–derived LCs (moLCs) formed the controls. mRNA from flow-sorted LCs was reverse transcribed to cDNA and TLR7, TLR8, and TLR9 amplified. Monocyte-derived Langerhans cells and cervical tumor LCs were stimulated with TLR7, TLR8, and TLR9 ligands. Culture supernatants were assayed for interleukin (IL) 1β, IL-6, IL-10, IL-12p70, interferon (IFN) &agr;, interferon γ, and tumor necrosis factor (TNF) &agr; by Luminex multiplex bead array. Human papillomavirus was genotyped. Results We have for the first time demonstrated that the acute monocytic leukemia cell line THP-1 can be differentiated into LCs in vitro. Although these moLCs expressed all the 3 TLRs, tumor LCs expressed TLR7 and TLR8, but uniformly lacked TLR9. Also, moLCs secreted IL-6, IL-1β, and tumor necrosis factor &agr; to TLR8 ligand and interferon &agr; in response to TLR9 ligand; in contrast, tumor LCs did not express any cytokine to any of the 3 TLR ligands. Human papillomavirus type 16 was one of the common human papillomavirus types in all cases. Conclusions Cervical tumor LCs lacked TLR9 expression and were functionally anergic to all the 3: TLR7, TLR8, and TLR9 ligands, which may play a crucial role in immune tolerance. The exact location of block(s) in TLR7 and TLR8 signaling needs to be investigated, which would have important immunotherapeutic implications.

Carcinoma of the vulva: a retrospective review of 37 cases at a regional cancer centre in South India

A retrospective review of 37 cases of carcinoma of the vulva presenting between 1996 and 2000 has been carried out. Thirty-three cases were managed with curative intent and four cases with advanced loco-regional disease were managed with palliative intent. The surgical treatment consisted of wide excision in one case, radical vulvectomy (RV) in six cases, radical vulvectomy and bilateral groin node dissection (RV + BGND) in 25 cases and radical vulvectomy and unilateral groin node dissection in one case. Nine of these 33 women also received adjuvant chemotherapy preoperatively in the hope of achieving better tumour-free surgical margins. Eight cases had a partial response and one case achieved complete response; the surgical margins were free in all these patients. One case received neoadjuvant radiotherapy to the vulva and pelvis followed by RV + BGND, which revealed no residual tumour. Overall, 26/33 cases had groin/inguinal node dissection and 23 (88.4%) of them had groin wound dehiscence. Thirteen of these 26 patients (50%) had inguinal node metastases (Stage III, four patients; Stage IV, nine patients). All the patients with negative nodes were free of disease while three of four patients with Stage III and two of nine patients with Stage IV with nodal metastases remained free of disease. The only patient with Stage III disease plus inguinal node metastases who recurred had multiple positive nodes with extracapsular spread. It appears that although bilateral involvement of the inguinal lymph nodes carries a worse prognosis, unilateral involvement with or without vaginal involvement carries an excellent prognosis provided multiple nodes are not involved. The role of neoadjuvant chemotherapy as compared to neoadjuvant radiotherapy, in locally advanced tumours, needs to be explored further.

The role of surgery in locally advanced carcinoma of cervix after sub-optimal chemoradiation: Indian scenario

Background: Standard treatment of advanced cervical cancer is concurrent chemoradiation. Radical radiotherapy for carcinoma cervix includes pelvic external beam radiotherapy (EBRT) with the concomitant platinum based chemotherapy followed by intracavitary brachytherapy (ICBT) to boost central disease. Management of patients who are suboptimally treated, especially, after unsuccessful ICBT insertion is not well-defined. This study explores the role of hysterectomy in these patients. Materials and Methods: From January 2006 to December 2011, 38 patients with locally advanced cervical cancer, in whom ICBT insertion was unsuccessful, were analyzed retrospectively. Operable patients with no parametrial involvement underwent hysterectomy and outcomes (recurrence free and overall survival) were noted. Results: The major complications in post operative period were wound infection, paralytic ileus and bladder atony all of which were conservatively managed with no mortality. At median follow-up of 36 months (range 12-60 months) there was no recurrence in patients with stage 1B2 and stage IIA, 25 out of 38 (65.8%) were event free and the overall survival was 71%. Conclusion: Many patients in Indian scenario receive suboptimal therapy in locally advanced cervical cancer. EBRT with chemotherapy followed by type 1 extra-fascial hysterectomy can be a good alternative for these patients.

Oestrogen Receptor-α binds the FOXP3 promoter and modulates regulatory T-cell function in human cervical cancer

Oestrogen controls Foxp3 expression in regulatory T cells (Treg cells) via a mechanism thought to involve oestrogen receptor alpha (ERα), but the molecular basis and functional impact of ERα signalling in Treg cells remain unclear. We report that ERα ligand oestradiol (E2) is significantly increased in human cervical cancer (CxCa) tissues and tumour-infiltrating Treg cells (CD4+CD25hiCD127low), whereas blocking ERα with the antagonist ICI 182,780 abolishes FOXP3 expression and impairs the function of CxCa infiltrating Treg cells. Using a novel approach of co-immunoprecipitation with antibodies to E2 for capture, we identified binding of E2:ERα complexes to FOXP3 protein in CxCa-derived Treg cells. Chromatin immunoprecipitation analyses of male blood Treg cells revealed ERα occupancy at the FOXP3 promoter and conserved non-coding DNA elements 2 and 3. Accordingly, computational analyses of the enriched regions uncovered eight putative oestrogen response elements predicted to form a loop that can activate the FOXP3 promoter. Together, these data suggest that E2-mediated ERα signalling is critical for the sustenance of FOXP3 expression and Treg cell function in human CxCa via direct interaction of ERα with FOXP3 promoter. Overall, our work gives a molecular insight into ERα signalling and highlights a fundamental role of E2 in controlling human Treg cell physiology.

Relationship between lymph node metastases and histopathological parameters in carcinoma cervix: A multivariate analysis

A retrospective review of 360 cases of carcinoma of the cervix with clinical stage IB and IIA who had undergone radical hysterectomy and pelvic node dissection between 2000 and 2005 was carried out. Lymph node metastasis was present in 79/360 patients (21.9%). LVSI positivity, full thickness stromal invasion, involvement of the uterine isthmus, positive parametrium, positive vaginal margins, involvement of uterine corpus was seen in: 25.3% and 9.2% (p < 0.001); 63% and 32% (p < 0.001); 32.9% and 13.8% (p < 0.001);15.2% and 5% (p < 0.004); 24% and 14.2% (p < 0.005); 17.7% and 13.8% (p = 0.11) of the patients, with and without lymph node metastasis, respectively. The tumour size was <4 cm in 50.6% and 58.3% and >4 cm in 49.3% and 41.6% of the patients, with and without lymph node metastasis, respectively (p = 0.22), which was statistically not significant. In the majority of patients, the histopathology type was squamous cell carcinoma in both the groups. In patients with lymph node metastases 79.7% had grade III tumour as compared with 69.5% in patients without lymph node metastases (p = 0.19). Multiple logistic regression indicated that only lymphovascular space involvement and full thickness stromal invasion were statistically significant (p < 0.001 and p < 0.002, respectively) for lymph node metastasis.

A review of squamous cell carcinoma arising in mature cystic teratoma of the ovary.

INTRODUCTION Germ cell tumors account for 20-25% of ovarian neoplasms. Mature cystic teratoma (MCT) is the most common ovarian germ cell tumor. Malignancy in MCT is seen in 1-2% of the cases. Squamous cell carcinoma (SCC) accounts for 80% of the cases and carries a poor prognosis. AIM To study the clinicopathological factors, management protocols and its outcome. MATERIALS AND METHODS Case records reviewed from August 2006 to August 2011 at our institute identified 10 women with SCC in ovarian MCT. Staging was done according to FIGO 2009 guidelines. Primary surgery followed by adjuvant treatment with platinum based chemotherapy was given. RESULTS Median age was 53.5 years. Six out of 10 patients were postmenopausal and aged above 50 years. Abdominal pain and abdominal mass were the most common presenting symptoms. According to FIGO: Two in stage 1, five in stage 2, two in stage 3 and one in stage 4. Among six optimally cytoreduced patients, five (83%) had no evidence of disease with a median follow up of 10 months. Whereas all four (100%) suboptimally cytoreduced patients had progressive disease within 3 to 4 months of primary surgery despite chemotherapy. CONCLUSION Squamous cell carcinoma in MCT of ovary is a rarity. It carries a poor prognosis, especially in advanced stages and suboptimally cytoreduced patients. Platinum with or without taxane based chemotherapy may be useful as adjuvant treatment. However, further studies and standardization of treatment protocols are required for any recommendations.

Malignant mixed Mullerian tumour of the uterus

Background: A malignant mixed Mullerian tumour (MMMT) of the uterine corpus is an extremely rare and aggressive malignancy. There are very few studies regarding the outcome of MMMT patients in India. Hence, we conducted the present study to analyse the outcome of MMMTs at our institute. Objective: To study the clinical profile, prognostic features, and treatment outcome of MMMT with multimodal therapy. Method: A five-year retrospective study of the MMMT cases diagnosed and treated at our centre was conducted. Twenty patients with pathological proven diagnosis of MMMT treated at our institute from January 2007 to May 2012 were analysed. These patients underwent comprehensive surgical staging followed by adjuvant therapy in the form of chemotherapy alone or chemoradiotherapy. These patients were analysed for event-free survival (EFS), and their outcomes were correlated with histology, therapy, myometrial invasion, and the stage of disease. Results: A majority of these patients presented with postmenopausal bleeding. Endometrial biopsy was diagnostic in only 20% of the patients. Of the 20 patients who underwent surgery, 18 patients received adjuvant therapy. At median follow-up of 16 months (range 3–30 months), the EFS was 30%. No difference in outcome was noted based on tumour histology (heterologous versus homologous). Concurrent chemoradiation improves local control and may delay recurrence but has shown little survival advantage. Conclusion: MMMT is an aggressive tumour of the uterine corpus. A negative endometrial biopsy does not rule out the diagnosis. Poor outcome is noted in patients with advanced-stage disease and myometrial invasion. The optimal adjuvant treatment for this uncommon disease is yet to be established, highlighting the need for larger multicentric studies on MMMTs.