Bart W. J. Hellebrekers

Use of fibrinolytic agents in the prevention of postoperative adhesion formation.

OBJECTIVE To review the events leading to the formation of adhesions, to describe the development of fibrinolytic agents, to review more than a century of research on the use of fibrinolytic agents in adhesion prevention, and to look at future aspects of adhesion prevention. RESULTS A better understanding of the pathogenesis of adhesion formation has resulted in the use of fibrinolytic agents in their prevention. Fibrinolytic agents promote fibrinolytic activity during the early period after peritoneal trauma during which an increased formation of fibrin is seen in combination with a deficiency of endogenous fibrinolytic activity. Initially, chemical attacks on fibrin (fibrolysin and hypertonic glucose), foreign digestive ferments (pepsin, trypsin, and papain), and stimulation of intraperitoneal leukocytosis (amniotic fluid) were used. Development of new thrombolytic agents was soon followed by experiments in animal adhesion models and clinical studies to examine their antiadhesion properties. Plasmin preparations (plasmin, actase, and fibrinolysin) and plasmin activators (streptokinase, urokinase, and tissue-type plasminogen activator) were found to be efficacious in preventing adhesion formation in the greater part of reviewed animal and clinical studies. CONCLUSION(S) From the current literature, it can be concluded that postoperative intraperitoneal administration of thrombolytic agents can significantly decrease adhesion formation. Given the large number of experimental studies in animals, future studies should focus on the clinical use of fibrinolytic agents in the prevention of postsurgical adhesion formation.

Human papilloma virus specific T cells infiltrating cervical cancer and draining lymph nodes show remarkably frequent use of HLA-DQ and -DP as a restriction element.

Human papillomavirus (HPV)‐induced malignancies are frequently infiltrated by lymphocytes. To comprehend the contribution of HPV‐specific T cells in this anti‐tumor response we developed a method that allowed the analysis of the presence and specificity of cervix‐infiltrating and draining lymph node resident T cells in a group of 74 patients with cervical malignancies, 54 of which were induced by HPV16 or HPV18. We detected the presence of HPV16 or HPV18‐specific T cells in at least 23 of the 54 HPV‐16 or ‐18 positive patients, and not in the 20 controls. Detailed studies resulted in the identification of 17 novel CD4+ and CD8+ T cell epitopes and their HLA‐restriction elements, and also revealed that the HPV‐specific immune response was aimed at both E6 and E7 and showed no preferential recognition of immunodominant regions. Unexpectedly, the vast majority of the CD4+ T cell epitopes were presented in the context of the less abundantly expressed HLA‐DQ and HLA‐DP molecules. Since the identified T cell epitopes constitute physiological targets in the immune response to HPV16 and HPV18 positive tumors they will be valuable for detailed studies on the interactions between the tumor and the immune system. This is crucial for the optimization of cancer immunotherapy in patients with pre‐existing tumor‐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.

Adjuvant radiotherapy following radical hysterectomy for patients with early-stage cervical carcinoma (1984-1996)

BACKGROUND AND PURPOSE The aim of this study was to assess the results of treatment (surgery alone or surgery and postoperative radiotherapy) for early-stage cervical carcinoma and to determine the morbidity associated with adjuvant radiotherapy. A subset of these patients (n = 10) was irradiated postoperatively for tumor related negative prognostic factors only and this retrospective analysis was also performed to determine if this decision was right and if the selection for this treatment was based on the right criteria. MATERIAL AND METHODS From 1984 to 1996, 233 women underwent radical hysterectomy as primary treatment of stage I or IIA cervical carcinoma. One hundred and fifty-six patients were treated with surgery alone (67%) and 77 patients (33%) received adjuvant radiotherapy for a, tumor related negative prognostic factors: the combination CLS(+), tumor size > or = 40 mm and poor differentiation grade or the combination tumor size > or = 40 mm and depth of invasion > or = 15 mm (n = 10), or b, positive surgical margins (n = 17), and/or c. lymphnode metastases (n = 42) and/ or d. parametrial involvement (n = 6). RESULTS For the entire group the most important prognostic factor for survival and disease free survival was node positivity. Additional factors were depth of invasion and positive surgical margins. Thirty-five patients recurred of which 12 after surgery alone. In all these cases the relapse was in the pelvis (100%). Of the 23 recurrences after surgery and adjuvant radiotherapy 13 were seen in the pelvis (56%) (P = 0.003). All patients with negative prognostic factors and N0, received adjuvant radiotherapy (n = 10) and none of these patients recurred. The incidence of severe gastrointestinal radiation related side effects was low (2%). The incidence of lymphedema of the leg was 11% which was similar in the surgery alone group. CONCLUSIONS The relatively low percentage of radiation related side effects together with 0% recurrence in a subgroup of node negative patients with high risk of recurrence, and a relatively low percentage of recurrence in the surgery alone group lead us to the conclusion that postoperative radiotherapy in special subsets of node negative patients is justified.

The case for completing the lymphadenectomy when positive lymph nodes are found during radical hysterectomy for cervical carcinoma

Background. In the present study we report on the results of a retrospective study on the effect on survival of the pelvic lymphadenectomy in a group of 294 patients with stage Ia2‐IIa cervical carcinoma treated by radical hysterectomy from 1984 through 1996 at the Leiden University Medical Center.

Vaccination against Oncoproteins of HPV16 for Noninvasive Vulvar/Vaginal Lesions: Lesion Clearance Is Related to the Strength of the T-Cell Response

Purpose: Therapeutic vaccination with human papillomavirus type 16 (HPV16) E6 and E7 synthetic long peptides (SLP) is effective against HPV16-induced high-grade vulvar and vaginal intraepithelial neoplasia (VIN/VaIN). However, clinical nonresponders displayed weak CD8+ T-cell reactivity. Here, we studied if imiquimod applied at the vaccine site could improve CD8+ T-cell reactivity, clinical efficacy, and safety of HPV16-SLP (ISA101). Experimental Design: A multicenter open-label, randomized controlled trial was conducted in patients with HPV16+ high-grade VIN/VaIN. Patients received ISA101 vaccination with or without application of 5% imiquimod at the vaccine site. The primary objective was the induction of a directly ex vivo detectable HPV16-specific CD8+ T-cell response. The secondary objectives were clinical responses (lesion size, histology, and virology) and their relation with the strength of vaccination-induced immune responses. Results: Forty-three patients were assigned to either ISA101 with imiquimod (n = 21) or ISA101 only (n = 22). Imiquimod did not improve the outcomes of vaccination. However, vaccine-induced clinical responses were observed in 18 of 34 (53%; 95% CI, 35.1–70.2) patients at 3 months and in 15 of 29 (52%; 95% CI, 32.5–70.6) patients, 8 of whom displayed a complete histologic response, at 12 months after the last vaccination. All patients displayed vaccine-induced T-cell responses, which were significantly stronger in patients with complete responses. Importantly, viral clearance occurred in all but one of the patients with complete histologic clearance. Conclusions: This new study confirms that clinical efficacy of ISA101 vaccination is related to the strength of vaccine-induced HPV16-specific T-cell immunity and is an effective therapy for HPV16-induced high-grade VIN/VaIN. Clin Cancer Res; 22(10); 2342–50. ©2016 AACR. See related commentary by Karaki et al., p. 2317

Preoperative predictors of postsurgical adhesion formation and the Prevention of Adhesions with Plasminogen Activator (PAPA-study): results of a clinical pilot study

OBJECTIVE To identify predictors of postsurgical adhesion formation in peritoneal fluid and plasma, and assess efficacy and safety of reteplase (recombinant plasminogen activator [r-PA]). DESIGN Prospective randomized study. SETTING University Medical Center. PATIENT(S) Twenty-six abdominal myomectomy patients with early second-look laparoscopy (ESL). INTERVENTION(S) Randomization to IP treatment with 1 mg reteplase in 300 mL Ringers lactate or 300 mL Ringers lactate only. Scoring of adhesions and collecting peritoneal fluid during both surgical procedures and collecting plasma samples at ten time points. MAIN OUTCOME MEASURE(S) Incidence, severity, and extent of adhesions at ESL. Concentrations of C-reactive protein (CRP), tissue-type plasminogen activator (tPA), plasminogen activator inhibitor 1 (PAI-1), and fibrin degradation products (FbDPs). RESULT(S) Significant correlation between the extent of uterine adhesion formation and preoperative plasma levels of CRP (r(s) = 0.558), PAI-1 (r(s) = 0.413), and the change in tPA concentration in peritoneal fluid from initial surgery to ESL (Delta+PA: r(s) = -0.636). No significant differences in adhesion scores between treatment and control groups. CONCLUSION(S) Our finding that preoperative plasma CRP and PAI-1-levels are significantly correlated with extent of adhesion formation points to a role of chronic inflammation in the disease process. Results are highly indicative for the paradigm that adhesions are caused by an insufficiency in peritoneal fibrinolytic capacity. For successful adhesion prevention therapy relatively high amounts of r-PA are required.

Surgery followed by Persistence of High-Grade Squamous Intraepithelial Lesions Is Associated with the Induction of a Dysfunctional HPV16-Specific T-Cell Response

Purpose: To characterize HPV16 E6- and E7-specific T-cell immunity in patients with high-grade squamous intraepithelial lesions (HSIL). Experimental Design: Peripheral blood mononuclear cells isolated from 38 patients with HPV16+ HSIL were used to determine the magnitude, breadth, and polarization of HPV16-specific T-cell responses by proliferation assays and cytokine assays. Furthermore, HSIL-infiltrating T cells isolated from 7 cases were analyzed for the presence of HPV16 E6- and/or E7-specific T cells, phenotyped, and tested for the specific production of IFN-γ and interleukin-10 as well as for their capacity to suppress immune responses. Results: HPV16-specific T-cell responses were absent in the circulation of the majority (∼60%) of patients who visit the clinic for treatment of a HPV16+ HSIL lesion. Notably, HPV16-specific T-cell reactivity was predominantly detected in patients returning to the clinic for repetitive treatment of a persistent or recurrent HPV16+ HSIL lesion after initial destructive treatment. The majority (>70%) of these HPV16-specific T-cell responses did not secrete proinflammatory cytokines, indicating that most of the subjects, although in principle able to mount a HPV16-specific immune response, fail to develop protective cellular immunity. This notion is sustained by our observation that only three HSIL-infiltrating T-cell cultures contained HPV16-specific T cells, one of which clearly consisted of HPV16 E7-specific regulatory T cells. Conclusions: The presence of HPV16-specific T cells with a non-Th1/Th2 cytokine and even suppressive signature in patients with HSIL may affect the outcome of vaccine approaches aiming at reinforcing human papillomavirus-specific immunity to attack human papillomavirus-induced lesions.

The long learning curve of gynaecological cancer surgery: an argument for centralisation

Objective To study the development of surgical performance of an unchanging surgical team over 13 years.

Alterations in classical and nonclassical HLA expression in recurrent and progressive HPV-induced usual vulvar intraepithelial neoplasia and implications for immunotherapy.

Immunotherapy of usual vulvar intraepithelial neoplasia (uVIN) is promising; however, many patients still fail to show clinical responses, which could be explained by an immune escape through alterations in human leukocyte antigen (HLA) expression. Therefore, we analyzed a cohort of patients with a primary (n = 43) and subsequent recurrent uVIN lesion (n = 20), vaccine‐treated uVIN patients (n = 12), patients with human papillomavirus (HPV)‐induced vulvar carcinoma (n = 21) and healthy controls (n = 26) for the expression of classical HLA‐class I/II and nonclassical HLA‐E/‐G and MHC class I chain‐related molecule A (MICA). HLA‐class I was downregulated in 70% of uVIN patients, including patients with a clinical response to immunotherapy. Downregulation of HLA‐class I is probably reversible, as only 15% of the uVIN cases displayed loss of heterozygosity (LOH) and HLA‐class I could be upregulated in uVIN keratinocyte cultures by interferon γ. HLA‐class I downregulation is more frequently associated with LOH in vulvar carcinomas (25–55.5%). HLA‐class II was found to be focally expressed in 65% of uVIN patients. Of the nonclassical molecules, MICA was downregulated in 80% of uVIN whereas HLA‐E and ‐G were expressed in a minority of cases. Their expression was more prominent in vulvar carcinoma. No differences were found between the alterations observed in paired primary and recurrent uVIN. Importantly, downregulation of HLA‐B/C in primary uVIN lesions was associated with the development of recurrences and progression to cancer. We conclude that downregulation of HLA is frequently observed in premalignant HPV‐induced lesions, including clinical responders to immunotherapy, and is associated with worse clinical outcome. However, in the majority of cases downregulation may still be reversible.