Nanoparticle Conjugation of Human Papillomavirus 16 E7-long Peptides Enhances Therapeutic Vaccine Efficacy against Solid Tumors in Mice.

Treatment of patients bearing human papillomavirus (HPV)-related cancers with synthetic long-peptide (SLP) therapeutic vaccines has shown promising results in clinical trials against premalignant lesions, whereas responses against later stage carcinomas have remained elusive. We show that conjugation of a well-documented HPV-E7 SLP to ultra-small polymeric nanoparticles (NP) enhances the antitumor efficacy of therapeutic vaccination in different mouse models of HPV+ cancers. Immunization of TC-1 tumor-bearing mice with a single dose of NP-conjugated E7LP (NP-E7LP) generated a larger pool of E7-specific CD8+ T cells with increased effector functions than unconjugated free E7LP. At the tumor site, NP-E7LP prompted a robust infiltration of CD8+ T cells that was not accompanied by concomitant accumulation of regulatory T cells (Tregs), resulting in a higher CD8+ T-cell to Treg ratio. Consequently, the amplified immune response elicited by the NP-E7LP formulation led to increased regression of large, well-established tumors, resulting in a significant percentage of complete responses that were not achievable by immunizing with the non-NP-conjugated long-peptide. The partial responses were characterized by distinct phases of regression, stable disease, and relapse to progressive growth, establishing a platform to investigate adaptive resistance mechanisms. The efficacy of NP-E7LP could be further improved by therapeutic activation of the costimulatory receptor 4-1BB. This NP-E7LP formulation illustrates a "solid-phase" antigen delivery strategy that is more effective than a conventional free-peptide ("liquid") vaccine, further highlighting the potential of using such formulations for therapeutic vaccination against solid tumors. Cancer Immunol Res; 6(11); 1301-13. ©2018 AACR.

Vulvar intraepithelial neoplasia: Risk factors for recurrence.

OBJECTIVE: We studied a large population of women with high-grade vulvar intraepithelial neoplasia (VIN) in order to identify patient and treatment-related risk factors for recurrence and progression to cancer. METHODS: For this retrospective cohort study of women with a histologic diagnosis of VIN within Southern California Permanente Medical Group between 1995 and 2007 medical records were reviewed; clinical, demographic and pathologic data were collected. Statistical analyses included Chi-squared and Student's t-tests, univariate and multivariate logistic regression, and cumulative incidence analysis. RESULTS: 914 patients with high-grade VIN were identified; 784 met inclusion criteria. We found 26.3% recurrences among treated women, with 2.2% progression to cancer (8.2% among those with recurrence). Risk factors for recurrence on multivariate analysis were: age >50years (OR, 1.44; 95%CI 1.01-2.07), immunosuppression (OR 2.08; 95%CI 1.42-3.06), metasynchronous VAIN or CIN (OR 1.76; 95%CI 1.08-2.88) in addition to margin status (OR 8.17; 95%CI 4.60-14.51) and adjacent LSA (OR 9.91; 95%CI 1.53-31.32) or HPV (OR 2.15; 95%CI 1.13-3.37) with excisional treatment. Recurrence rates did not differ significantly by smoking status and treatment modalities. Median time to recurrence was 16.9months; 25% had late recurrences (44-196months). Cumulative incidence analyses of time to recurrence shows a significantly higher risk among patients over age 50 (log-rank p=0.0031). CONCLUSION: We identified independent risk factors for recurrence including age >50years, immunosuppression, metasynchronous vaginal or intraepithelial neoplasia, positive excision margins, and adjacent lichen sclerosus or human papilloma-virus. Regardless of treatment modality, 25% of recurrences occurred late, highlighting the need for long-term surveillance in women treated for VIN.

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 AACRSee related commentary by Karaki et al., p. 2317.

Idiopathic CD4 lymphocytopenia manifesting as refractory genital dysplasia.

BACKGROUND: Idiopathic CD4 lymphocytopenia is an immunodeficiency disorder with low absolute CD4 T-lymphocyte count with no evidence of human immunodeficiency virus or other known cause. CASE: A 22-year-old woman presented with a high-grade Pap test result. Work-up demonstrated cervical intraepithelial neoplasia 3 and vaginal intraepithelial neoplasia 3 with extensive condyloma. She presented 6 months after her initial treatment with recurrent disease and was referred to the immunology department, where she was found to have profound lymphopenia. After further evaluation, idiopathic CD4 lymphocytopenia was diagnosed. CONCLUSION: Idiopathic CD4 lymphocytopenia is a rare acquired immunodeficiency. Although genital dysplasia is common in young women, this case demonstrates the importance of determining other etiologies of recurrent human papillomavirus infections and possible immunodeficiencies that may affect management and outcomes.

Spontaneous cervicovaginal lesions and immune cell infiltrates in nonhuman primates.

Nonhuman primates, particularly rhesus macaques (Macaca mulatta), provide important model systems for studying human reproductive infectious diseases such as human immunodeficiency virus, human papillomavirus, and Chlamydia spp. An understanding of the spectrum of spontaneous cervical disease provides essential context for interpreting experimental disease outcomes in the female reproductive tract. This retrospective study characterizes the incidence of inflammatory and/or proliferative cervicovaginal lesions seen over a 14-year period in a multispecies nonhuman primate colony, focusing on rhesus macaques. The most common observations included a spectrum of lymphocytic accumulation from within normal limits to lymphoplasmacytic cervicitis, and suppurative inflammation with occasional squamous metaplasia or polyp formation. These inflammatory spectra frequently occurred in the context of immunosuppression following experimental simian immunodeficiency virus (SIV) infection. Cervical neoplasias were uncommon and included leiomyomas and carcinomas. Cervical sections from 13 representative cases, with an emphasis on proliferative and dysplastic lesions, were surveyed for leukocyte infiltration, abnormal epithelial proliferation, and the presence of papillomavirus antigens. Proliferative lesions showed sporadic evidence of spontaneous papillomavirus infection and variable immune cell responses. These results underscore the importance of pre screening potential experimental animals for the presence of preexisting reproductive tract disease, and the consideration of normal variability within cycling reproductive tracts in interpretation of cervical lesions.

Human papillomavirus-related genital disease in the immunocompromised host: Part II.

Human papillomavirus is responsible for common condyloma acuminata and a number of premalignant and malignant anogenital lesions. The immunocompromised population is at particular risk because of a higher incidence of malignant transformation. Lesions in this population may prove refractory to standard treatment. This is part II of a two-part review that will discuss the treatment of condyloma acuminata and vaginal, vulvar, penile, and anal intraepithelial neoplasias. This article will provide an updated review of available treatments, with a focus on recent advances and the challenges faced in successfully treating human papillomavirus lesions in the immunocompromised host.

Effect of human immunodeficiency virus infection on the prevalence and incidence of vaginal intraepithelial neoplasia.

OBJECTIVE: To estimate the prevalence, incidence, and clearance of abnormal vaginal cytology and vaginal intraepithelial neoplasia (VAIN) in human immunodeficiency virus (HIV)-seropositive women. METHODS: Pap tests were done semiannually for 335 HIV-seropositive and 75 HIV-seronegative women with prior hysterectomy in the prospective Women's Interagency HIV Study cohort. End points included abnormal Pap test results after hysterectomy and VAIN regardless of hysterectomy. RESULTS: Over a median of 5.6 years of follow-up, vaginal Pap test results were abnormal at 1,076 (29%; 95% confidence interval [CI] 25-33%) of 3,700 visits among HIV-seropositive compared with 31 (4%; 95% CI 2-8%) of 763 visits among HIV-seronegative women (P<.001). Abnormal Pap test results included 641 atypical squamous cells of undetermined significance, 425 low-grade squamous intraepithelial lesions, and 10 high-grade squamous intraepithelial lesions in HIV-seropositive women and 28 atypical squamous cells of undetermined significance and three low-grade squamous intraepithelial lesions in HIV-seronegative women. The incidence of abnormal Pap test results after hysterectomy was 14 per 100 person-years among HIV-seropositive and two per 100 person-years among HIV-seronegative women (P<.001) and remained stable across time. The 5-year clearance rate of abnormal Pap test results was 34 per 100 person-years for HIV-seropositive and 116 per 100 person-years for HIV-seronegative women (P<.001). In multivariate regression models, women with lower CD4 counts were more likely to have and less likely to clear abnormal cytology when it occurred. The incidence of VAIN 2 or worse was 0.2 and 0.01 per 100 person-years for HIV-seropositive and HIV-seronegative women (P=.001). Two HIV-seropositive women developed stage II cancers with remission after radiotherapy. CONCLUSION: Vaginal Pap test results are often abnormal in HIV-seropositive women. Although more common than in HIV-seronegative women, VAIN 2 or worse and especially vaginal cancers are infrequent.

Prophylactic HPV vaccines: new interventions for cancer control.

Human Papillomavirus (HPV) infection causes cervical cancer, a significant portion of anal, vulvar, vaginal, and oropharyngeal cancers, genital warts, and recurrent respiratory papillomatosis (RRP). HPV 16 and 18 cause 70-90% of HPV-related cancers whereas HPV 6 and 11 cause 90% of RRP and genital wart cases. Together these four types cause 30-50% of all cervical intraepithelial neoplasia such as those detected by Papinicalou screening. In June 2006, a quadrivalent HPV (6, 11, 16, 18) vaccine was licensed in the United States, and subsequently in the European Union (September 2006), both following expedited review. We describe the primary objectives of the quadrivalent HPV vaccine clinical trial program including studies in females aged 9-45 and males aged 9-26. Planned long-term efficacy and safety evaluations, as well as programs to evaluate vaccine impact on oropharyngeal cancer are also described.

Vaccinia-expressed human papillomavirus 16 and 18 e6 and e7 as a therapeutic vaccination for vulval and vaginal intraepithelial neoplasia.

PURPOSE: Anogenital intraepithelial neoplasia is a chronic disorder associated with infection by high-risk human papillomavirus (HPV) types. It is frequently multifocal and recurrence after conventional treatment is high. Boosting HPV-specific cell-mediated immune responses may reduce progression to carcinoma and could lead to disease clearance. We have tested the safety, immunogenicity, and efficacy of a recombinant vaccinia candidate vaccine (TA-HPV) in women with anogenital intraepithelial neoplasia. EXPERIMENTAL DESIGN: Twelve women, aged 42-54 years with high-grade HPV-positive vulval or vaginal intraepithelial neoplasia of up to 15 years duration, completed a Phase II study of TA-HPV, a live recombinant vaccinia virus, expressing modified versions of the E6 and E7 open reading frames from HPV-16 and HPV-18. RESULTS: The vaccine was well tolerated. Five of 12 (42%) patients showed at least a 50% reduction in total lesion diameter over 24 weeks with 1 patient showing complete regression of her lesion. Overall, 83% of women showed some improvement with an average decrease in lesion size of 40%. All cases showed an increased IgG titer and T-cell response to the vaccinia virus. An IFN-gamma enzyme-linked immunospot assay using pooled 22-mer peptides spanning HPV-16 E6 and E7 showed an increased specific T-cell response after vaccination in 6 of the 10 cases available for testing. There was no increase in specific cytotoxic response to selected individual HLA class I-restricted HPV-16 E6/7 peptides. CONCLUSIONS: The results suggest that the vaccine may have an effect on HPV-positive vulval intraepithelial neoplasia/vaginal intraepithelial neoplasia and that additional studies are warranted to develop an effective therapeutic vaccine.

Lymphoepithelioma-like carcinoma of the vagina.

This report describes a lymphoepithelioma-like carcinoma of the vagina. Although such tumours are well described in the cervix this is only the second report of such a neoplasm at this site. Histology showed a well circumscribed lesion composed of syncytial sheets of epithelioid tumour cells with an intense inflammatory infiltrate, largely consisting of T lymphoid cells. In situ hybridisation and immunohistochemistry for Epstein-Barr virus were negative. A review of the literature reveals that such neoplasms appear to be extremely rare within the female genital tract outside of the cervix.

Malignant fibrous histiocytoma of the vagina.

We describe here the case of an 82-year-old woman presenting with a hemorrhagic tumor on the anterior vaginal wall. She was diagnosed with malignant fibrous histiocytoma (MFH) from the findings of cytological analysis of biopsied surface tissue, histopathologic analysis of biopsied tissue, immunohistochemical staining, and electron microscopy. Cytological analysis of the biopsy sample harvested from the tumor surface showed multinucleated giant cells and large atypical cells with rough, granular, chromatin, as well as notable nucleoli. A storiform pattern was observed histopathologically, and immunohistochemical staining confirmed positive reactions to alpha 1-antichymotripsin (alpha 1-ACT), vimentin, and lysosome, but negative reactions to epithelial membrane antigen (EMA), cytokeratin, and alpha-smooth muscle action (alpha-SMA). Electron microscopy showed histiocyte-derived cells with a segmented nucleus with a large groove, pseudopodic cytoplasmic projections, and lysosome-like structures. However, intercellular adhesion factors were not notable, and microvilli were absent. Based on the above findings, a diagnosis of MFH originating from the vaginal wall was made. Because of the patient's advanced age, and, in accordance with her wishes, three courses of cancer chemotherapy, consisting of doxorubicin hydrochloride, fluorouracil, and cisplatin were carried out, without surgery. No reduction in the size of the tumor was seen at follow up, and despite the absence of metastasis and no exacerbation of her general condition, she died suddenly at home 2 years after being discharged.

The ultrastructural and immunohistochemical heterogeneity of CD-30-positive neoplasms: so-called anaplastic large cell Ki-1 lymphomas.

Anaplastic large cell lymphoma (ALCL), also referred to as Ki-1 lymphomas, was first recognized as an entity with characteristic light microscopic appearance in 1985. This tumor is composed of variably cohesive cells, often with large, markedly atypical, and multinucleated cellular forms. The recognition of ALCL resulted from the development of a monoclonal antibody in Kiel, Germany, named Ki-1, which was initially believed to be a putative marker for Reed-Sternberg cells. This antibody was later found to be specific against the epitope CD-30. Attempts to create strict criteria to preserve this neoplasm as a specific entity have undergone evolution. However, it is now clear that included in this group are a variety of pleomorphic neoplasms with CD-30 immunoreactivity. Some of these neoplasms are nonlymphoid and show marked heterogeneity in their immunohistochemical and ultrastructural profiles. This article aims to highlight the ultrastructural spectrum of neoplasms exhibiting CD-30 positivity that are within the spectrum of ALCL. It remains to be determined if there are subgroups of these CD-30-positive neoplasms that can be segregated on the basis of ultrastructural and immunohistochemical criteria with corresponding clinical correlates that may impact on their management, treatment, and prognosis. We review here the heterogeneity of CD-30-positive neoplasms (so-called anaplastic large cell Ki-1 lymphomas).

A phase I trial of a human papillomavirus (HPV) peptide vaccine for women with high-grade cervical and vulvar intraepithelial neoplasia who are HPV 16 positive.

Eighteen women with high-grade cervical or vulvar intraepithelial neoplasia who were positive for human papillomavirus (HPV) 16 and were HLA-A2 positive were treated with escalating doses of a vaccine consisting of a 9-amino acid peptide from amino acids 12-20 encoded by the E7 gene emulsified with incomplete Freund's adjuvant. Starting with the eleventh patient, an 8-amino acid peptide 86-93 linked to a helper T-cell epitope peptide with a covalently linked lipid tail was added. Patients with colposcopically and biopsy-proven cervical intraepithelial neoplasia/vulvar intraepithelial neoplasia II/III received four immunizations of increasing doses of the vaccine each 3 weeks apart, followed by a repeat colposcopy and definitive removal of dysplastic tissue 3 weeks after the fourth immunization. Patients were skin tested with the E7 12-20 peptide as well as control candida, mumps, and saline prior to and after the series of immunizations. Peripheral blood mononuclear cells were obtained by leucopheresis prior to and after the series of immunizations for analyses of CTL reactivity to the E7 12-20 and 86-93 epitope sequences. The presence of HPV 16 was assessed by DNA PCR on cervical scrapings and the biopsy specimens after vaccination. Pathology specimens were analyzed before and after vaccination for the presence of dysplasia, and the intralesional infiltrate of CD4/CD8 T-cells and dendritic cells was measured by immunohistochemical staining. Only 3 of 18 patients cleared their dysplasia after vaccine, but an increased S100+ dendritic cell infiltrate was observed in 6 of 6 patients tested. Cytokine release and cytolysis assays to measure E7-specific reactivity revealed increases in 10 of 16 patients tested. No positive delayed type hypersensitivity skin test reactivity was shown in any patient to HPV E7 12-20 before or after vaccinations. Virological assays showed that 12 of 18 patients cleared the virus from cervical scrapings by the fourth vaccine injection, but all biopsy samples were still positive by in situ RNA hybridization after vaccination. Six patients had partial colposcopically measured regression of their cervical intraepithelial neoplastic lesions in addition to the three complete responders. The data establish that a HPV-16 peptide vaccine may have important biological and clinical effects and suggest that future refinements of an HPV vaccine strategy to boost antigen-specific immunity should be explored.

Serum antibodies to the 27-kd heat shock protein in women with gynecologic cancers.

OBJECTIVES: Among women the association between heat shock protein immunity and cancer has been examined primarily for breast cancer. Autoantibodies to the 27-kd heat shock protein were detected in some patients with breast cancer but not in control subjects, and the presence of these antibodies was correlated with improved survival. We examined the relationship between autoimmunity to heat shock proteins and the diagnosis of malignancies of the female genital tract. STUDY DESIGN: Serum samples from women seen for possible gynecologic malignancies or returning for evaluation after surgery, radiation, chemotherapy, or a combination for gynecologic cancers were tested for immunoglobulin G antibodies to the 27-kd, 60-kd, 70-kd, and 90-kd heat shock proteins by enzyme-linked immunosorbent assay with the purified recombinant proteins bound to wells of a microtiter plate. Serum samples from women with no history of malignancies served as control preparations. RESULTS: Antibodies to the 27-kd heat shock protein were detected in only 1 of 29 healthy control subjects (3.4%) and 1 of 23 women whose lesions were benign (4.3%). In marked contrast, 39 of 96 women with gynecologic cancers (40.6%) had positive antibody detection (P =.0004 vs benign). The percentages of positive results seen for ovarian (17/34, 50%), endometrial (13/34, 38.2%), cervical and uterine (3/10, 30%), vaginal and vulvar (3/5, 60%), and other (3/13, 23.1%) cancers were not significantly different from each other. Similar prevalences of antibodies to the 27-kd heat shock protein were seen among patients with cancer who had untreated active disease and after treatment. Unlike the results with antibodies to the 27-kd heat shock protein there was no relationship between antibodies to the other heat shock proteins and any gynecologic cancer. CONCLUSION: Circulating autoantibodies to the 27-kd heat shock protein were found to be associated with malignancies of the female genital tract.

Cell-mediated immunological responses in cervical and vaginal cancer patients immunized with a lipidated epitope of human papillomavirus type 16 E7.

Human papillomavirus (HPV) infection has been causally associated with cervical cancer. We tested the effectiveness of an HLA-A*0201-restricted, HPV-16 E7 lipopeptide vaccine in eliciting cellular immune responses in vivo in women with refractory cervical cancer. In a nonrandomized Phase I clinical trial, 12 women expressing the HLA-A2 allele with refractory cervical or vaginal cancer were vaccinated with four E786-93 lipopeptide inoculations at 3-week intervals. HLA-A2 subtyping was also performed, and HPV typing was assessed on tumor specimens. Induction of epitope-specific CD8+ T-lymphocyte (CTL) responses was analyzed using peripheral blood leukapheresis specimens obtained before and after vaccination. CTL specificity was measured by IFN-gamma release assay using HLA-A*0201 matched target cells. Clinical responses were assessed by physical examination and radiographic images. All HLA-A*0201 patients were able to mount a cellular immune response to a control peptide. E786-93-specific CTLs were elicited in 4 of 10 evaluable HLA-A*0201 subjects before vaccination, 5 of 7 evaluable HLA-A*0201 patients after two vaccinations, and 2 of 3 evaluable HLA-A*0201 cultures after all four inoculations. Two of three evaluable patients' CTLs converted from unreactive to reactive after administration of all four inoculations. There were no clinical responses or treatment toxicities. The ability to generate specific cellular immune responses is retained in patients with advanced cervical cancer. Vaccination with a lipidated HPV peptide epitope appears capable of safely augmenting CTL reactivity. Although enhancements of cellular immune responses are needed to achieve therapeutic utility in advanced cervical cancer, this approach might prove useful in treating preinvasive disease.

Vaginal epithelioid angiosarcoma.

A case of epithelioid angiosarcoma of the vagina is described. Only five cases of angiosarcoma at this site have been reported, three of which followed radiotherapy for other gynaecological malignancies. None is described as an epithelioid angiosarcoma, an unusual and recently described variant which is readily confused with carcinoma. This is thought to be the first reported epithelioid angiosarcoma at this site and highlights the difficulties in diagnosis.

A case of malignant melanoma of the vagina during pregnancy: immunological problems.

BACKGROUND: A clinical case of malignant melanoma of the vagina diagnosed during late pregnancy is described. Prompt histological examination showed a malignant melanoma at an early stage. The treatment of choice for malignant melanoma is surgical exicision. At present we are trying to associate immunological and chemotherapeutic treatments with surgical ones. CASE: A 27-year-old primipara was diagnosed with malignant melanoma at 38 weeks' gestation. The patient underwent a cesarean section. Careful evaluation of the immunological characteristics of the patient induced us to treat the melanoma with human leukocyte alpha interferon. The result was an increase in T4 cells with respect to T8 cells, with an enhancement in the ratio of CD4 to CD8. CONCLUSION: The results suggest the immune system plays a major role in the modification and regulation of neoplasias of the genital tract.