Human papillomavirus genotyping predicts residual/recurrent disease after local treatment for cervical intraepithelial neoplasia better than viral DNA testing.

AIM: Type-specific persistent infection (TSPI) of human papillomavirus (HPV) is reportedly associated with a high risk of residual/recurrent disease after local treatment for cervical intraepithelial neoplasia (CIN). This study aimed to evaluate whether HPV genotyping is more accurate in detecting residual/recurrent disease than HPV DNA testing and identify which HPV genotype can predict a high risk of residual/recurrent disease. METHODS: We retrospectively reviewed patient outcomes and results of HPV DNA testing and genotyping at 6-12 months after local treatment for CIN2/3 for 439 women. We investigated residual/recurrent disease occurrence according to the TSPI and new infections. Sensitivity, specificity, and positive and negative predictive values (PPV and NPV, respectively) of the two testing methods for predicting residual/recurrent diseases were also evaluated. RESULTS: Eighty-five (19.4%) patients were positive for HPV DNA testing after treatment, of which 74 (87.1%) had TSPI. Residual/recurrent disease was identified in 34 (7.7%) patients, of which 30 were positive for HPV DNA testing and had TSPI of HPV16, 18, 31, 33, 52, and 58 (six HPV genotypes). The sensitivity and NPV of HPV DNA testing and TSPI were equal at 88.2% and 98.9%, respectively. The specificity and PPV of TSPI were higher than those of HPV DNA testing (89.1% vs. 86.4%, 40.5% vs. 35.2%, respectively). Furthermore, the TSPI of the six HPV genotypes further improved specificity (90.6%) and PPV (44.1%) with the same sensitivity and NPV. CONCLUSION: HPV genotyping is more useful than HPV DNA testing for determining TSPI, especially of the six HPV genotypes.

CD4/CD8 ratio is a prognostic factor in IgG nonresponders among peptide vaccine-treated ovarian cancer patients.

The identification of useful biomarkers is an urgent issue in cancer treatment, particularly for immunotherapy, as only some patients experience benefits from this treatment. The early induction of the IgG response has been reported as a useful biomarker of favorable prognosis for cancer patients treated with a personalized peptide vaccination, but a portion of these patients (IgG nonresponders) fail to achieve an early induction of IgG response yet experience long-term survival. It is thus necessary to identify other biomarkers of favorable prognosis among these patients. Here we report the usefulness of classical T-cell markers (ie, the CD8 content and the CD4/CD8 ratio in peripheral blood) in IgG nonresponders among advanced or recurrent ovarian cancer patients treated with a personalized peptide vaccination. Among IgG nonresponders (n = 25), the overall survival (OS) of the increased-CD8 group (n = 7) was significantly longer than that of the decreased-CD8 group (n = 18; P = .018), and the OS of the patients with a decreased CD4/CD8 ratio (n = 10) was significantly longer than that of the patients with an increased ratio (n = 15; P = .0055). Thus, an increased content of CD8 and a decreased CD4/CD8 ratio are each favorable prognosis markers in IgG nonresponders treated with a personalized peptide vaccination.

Integrity of plasma DNA is inversely correlated with vaccine-induced antitumor immunity in ovarian cancer patients.

Cancer immunotherapy including vaccine therapy is a promising modality for cancer treatment, but few patients show its clinical benefits currently. The identification of biomarkers that can identify patients who will benefit from cancer immunotherapy is thus important. Here, we investigated the potential utility of the circulating cell-free DNA (cfDNA) integrity-a ratio of necrotic cell-derived, longer DNA fragments versus apoptotic cell-derived shorter fragments of Alu gene-as a biomarker of vaccine therapy for patients with ovarian cancer. We analyzed plasma samples from 39 patients with advanced or recurrent ovarian cancer enrolled in clinical trials for personalized peptide vaccinations. We observed that (1) the cfDNA integrity was decreased after the first cycle of vaccination, and (2) the decreased levels of cfDNA integrity were correlated with vaccine-induced immune responses; i.e., decreased cfDNA integrity was observed in 91.7% and 59.3% of the IgG-positive and negative patients, respectively (p = 0.0445). Similarly, decreased cfDNA integrity was observed in 92.9% and 56.0% of CTL response-positive and negative patients, respectively (p = 0.0283). These results suggest that the circulating cfDNA integrity is a possible biomarker for cancer vaccine therapy.

A phase II trial of irinotecan in patients with advanced or recurrent endometrial cancer and correlation with biomarker analysis.

OBJECTIVE: Chemotherapy for advanced or recurrent endometrial cancer requires further development. Irinotecan hydrochloride (CPT-11) suppresses tumor growth in several endometrial cancer strains. The present study evaluated the anti-tumor activity and toxicity of CPT-11 in patients with advanced or recurrent endometrial cancer. METHODS: Enrolled patients had advanced endometrial cancer with measurable lesions and received 2 pretreatment regimens. A 90-minute intravenous infusion of CPT-11 (100 mg/m2) was given on days 1, 8, and 15 of a 4-week cycle, aiming for an effect with ≤2 cycles. Treatment was continued until the primary disease worsened or severe toxicity occurred. The primary endpoint was response rate, and the secondary endpoints were progression-free survival, overall survival, and adverse events. Antitumor effect and adverse events were evaluated according to RECIST version 1.1 and NCI-CTC AE version 3.0, respectively. RESULTS: Twenty-two patients were registered (11 endometrioid carcinomas and 11 serous carcinomas). The median duration of the treatment-free interval (TFI) was 7.5 months, and the median number of administered cycles per patient was 4. Response rate was 36.4% (complete response: 1 patient, partial response: 7 patients). Clinical benefit rate, including stable disease, was 77.3%. Median progression-free and overall survival was 4.4 and 18.4 months, respectively. Observed adverse events included grade 4 hematotoxicity (neutropenia and thrombocytopenia), and grade 2 or 3 non-hematotoxicity (diarrhea). All adverse events were manageable. Biomarker predictors of therapeutic effectiveness were not observed. CONCLUSION: As a single agent, CPT-11 has anti-tumor activity for advanced or recurrent endometrial cancer and has manageable adverse events.

Immunological evaluation of peptide vaccination for cancer patients with the HLA -A11+ or -A33+ allele.

The HLA-A11 or -A33 allele is found in approximately 18% or 10% of the Asian population, respectively, but each of which is a minor allele worldwide, and therefore no clinical trials were previously conducted. To develop a therapeutic peptide vaccine for each of them, we investigated immunological responses of advanced cancer patients with the HLA-A11+ /A11+ (n = 18) or -A33+ /A33+ (n = 13) allele to personalized peptide vaccine (PPV) regimens. The primary sites of HLA-A11+/A11+ or -A33+/A33+ patients were the colon (n = 4 or 2), stomach (2 or 3), breast (3 or 2), lung and pancreas (2 or 2), and so on. For PPV, a maximum of four peptides were selected from nine different peptides capable of binding to HLA-A11 and -A33 molecules based on the pre-existing peptide-specific IgG responses. There were no severe adverse events related to PPV. At the end of the first cycle, peptide-specific CTL responses were augmented in 4/12 or 2/9 of HLA-A11+ /A11+ or -A33+ /A33+ patients, while peptide-specific IgG responses were augmented in 6/14 or 4/10 patients, respectively. Clinical responses consisted of four stable diseases and 14 progressive diseases in HLA-A11+ /A11+ patients, versus seven and six in -A33+ /A33+ patients, respectively. Further clinical study of PPV could be recommended because of the safety and positive immunological responses.

Current status of tertiary debulking surgery and prognosis after secondary debulking surgery for recurrent Müllerian epithelial cancer in Japan: a retrospective analysis of 164 patients (KCOG-G1402).

BACKGROUND: This study aimed to evaluate the current status of secondary debulking surgery (SDS) and tertiary debulking surgery (TDS; performed for recurrence after SDS) and to assess the overall survival after recurrence of Müllerian epithelial cancer in Japan. We also evaluated the data of patients who underwent a fourth debulking surgery (i.e., quaternary debulking surgery (QDS)). METHODS: We conducted a retrospective study of 164 patients with recurrent Müllerian epithelial cancers (i.e., ovarian, tubal, and peritoneal cancers). The SDS was performed between January 2000 and September 2014 in 20 Japanese hospitals. Clinicopathological data were collected and analyzed. RESULTS: Of the 164 patients, 66 patients did not have a recurrence or died after SDS. Ninety-eight patients had a recurrence after SDS. Forty-three of the 98 patients underwent TDS; 55 of the 98 patients did not undergo TDS and were classified into the non-TDS group. The overall survival (OS) after SDS was significantly better in the TDS group than in the non-TDS group. The median OS after SDS was 123 and 42 months in the TDS group and non-TDS group, respectively. Of the 43 patients who received TDS, 11 patients were further treated with QDS. The median OS after SDS was 123 months for patients who underwent QDS. CONCLUSIONS: This multicenter study on the prognosis of post-SDS is apparently the first report on QDS in Japan. Patients undergoing TDS have a good prognosis, compared to patients in the non-TDS group. Novel drugs are being evaluated; however, debulking surgery remains a necessary treatment for recurrence.

Personalized peptide vaccination for cervical cancer patients who have received prior platinum-based chemotherapy.

A feasibility study was performed to evaluate the immunological efficacy and safety of a personalized peptide vaccine (PPV) for cervical cancer patients who have received platinum-based chemotherapy. A total of 24 patients with standard chemotherapy-resistant cervical cancer, including 18 recurrent cases, were enrolled in this study and received a maximum of 4 peptides based on HLA-A types and IgG levels to the vaccine candidate peptides in pre-vaccination plasma. The parental protein expression of most of the vaccine peptides was confirmed in the cervical cancer tissues. No vaccine-related systemic grade 3 or 4 adverse events were observed in any patients. Due to disease progression, 2 patients failed to complete the first cycle of vaccinations (sixth vaccination). Cytotoxic T-lymphocyte (CTL) or IgG responses specific for the peptides used for vaccination were augmented in half of cases after the first cycle. The median overall survival was 8.3 months. The clinical responses of the evaluable 18 cases consisted of 1 case with a partial response and 17 cases with disease progression; the remaining 6 cases were not evaluable. Performance status, injection site skin reaction and circulating PD-1(+) CD4(+) T-cells were significantly prognostic of overall survival, and multivariate analysis also indicated that the performance status and circulating PD-1(+) CD4(+) T-cells were prognostic. Because of the safety and immunological efficacy of PPV and the possible prolongation of overall survival, further clinical trials of PPV at a larger scale in advanced or recurrent cervical cancer patients who have received prior platinum-based chemotherapy are recommended.

Immunological evaluation of peptide vaccination for cancer patients with the HLA-A26 allele.

To develop a peptide vaccine for cancer patients with the HLA-A26 allele, which is a minor population worldwide, we investigated the immunological responses of HLA-A26(+) /A26(+) cancer patients to four different CTL epitope peptides under personalized peptide vaccine regimens. In personalized peptide vaccine regimens, two to four peptides showing positive peptide-specific IgG responses in pre-vaccination plasma were selected from the four peptide candidates applicable for HLA-A26(+) /A26(+) cancer patients and administered s.c. Peptide-specific CTL and IgG responses along with cytokine levels were measured before and after vaccination. Cell surface markers in PBMCs and plasma cytokine levels were also measured. In this study, 21 advanced cancer patients, including seven lung, three breast, two pancreas, and two colon cancer patients, were enrolled. Their HLA-A26 genotypes were HLA-A26:01 (n = 24), HLA-A26:03 (n = 10), and HLA-A26:02 (n = 8). One, 14, and 6 patients received two, three, and four peptides, respectively. Grade 1 or 2 skin reactions at the injection sites were observed in the majority of patients, but no severe adverse events related to the vaccination were observed. Peptide-specific CTL responses were augmented in 39% or 22% of patients after one or two cycles of vaccination, respectively. Notably, peptide-specific IgG were augmented in 63% or 100% of patients after one or two cycles of vaccination, respectively. Personalized peptide vaccines with these four CTL epitope peptides could be feasible for HLA-A26(+) advanced cancer patients because of their safety and higher rates of immunological responses.

Monitoring the impact of a national HPV vaccination program in Japan (MINT Study): rationale, design and methods.

We have developed a collaborative hospital-based approach to monitoring the impact of a human papillomavirus vaccine on cervical cancer, its precursor lesions and human papillomavirus type-specific prevalence in Japan. The monitoring will be conducted for a total period of 21 years on women aged <40 who are newly diagnosed with invasive cervical cancer, cervical intraepithelial neoplasia or adenocarcinoma in situ at 21 participating institutes. Women are monitored to determine their vaccine history and will be human papillomavirus-genotyped each year. The primary endpoint is the human papillomavirus16/human papillomavirus18-positive rate in women aged 16-25 who are diagnosed with invasive cervical cancer, cervical intraepithelial neoplasia grade 2/3 and adenocarcinoma in situ. The major secondary endpoints are the number of women aged <40 who are diagnosed with invasive cervical cancer, cervical intraepithelial neoplasia grade 2/3 and adenocarcinoma in situ, the human papillomavirus type-specific prevalence, and the number of deaths from invasive cervical cancer in women aged <40. Long-term surveillance for human papillomavirus-associated cervical diseases in young females is important for the development of future strategies for cervical cancer prevention in Japan.

Feasibility study of personalized peptide vaccination for recurrent ovarian cancer patients.

CONTEXT: To develop a personalized peptide vaccine (PPV) for recurrent ovarian cancer patients and evaluate its efficacy from the point of view of overall survival (OS), Phase II study of PPV was performed. PATIENTS AND METHODS: Forty-two patients, 17 with platinum-sensitive and 25 with platinum-resistant recurrent ovarian cancer, were enrolled in this study and received a maximum of four peptides based on HLA-A types and IgG responses to the peptides in pre-vaccination plasma. RESULTS: Expression of 13 of the 15 parental tumor-associated antigens encoding the vaccine peptides, with the two prostate-related antigens being the exceptions, was confirmed in the ovarian cancer tissues. No vaccine-related systemic severe adverse events were observed in any patients. Boosting of cytotoxic T lymphocytes or IgG responses specific for the peptides used for vaccination was observed in 18 or 13 of 42 cases at 6th vaccination, and 19 or 29 of 30 cases at 12th vaccination, respectively. The median survival time (MST) values of the platinum-sensitive- and platinum-resistant recurrent cases were 39.3 and 16.2 months, respectively. The MST of PPV monotherapy or PPV in combination with any chemotherapy during the 1st to 12th vaccination of platinum-sensitive cases was 39.3 or 32.2 months, and that of platinum-resistant cases was 16.8 or 16.1 months, respectively. Importantly, lymphocyte frequency and epitope spreading were significantly prognostic of OS. DISCUSSION AND CONCLUSION: Because of the safety and possible prolongation of OS, a clinical trial of PPV without chemotherapy during the 1st to 12th vaccination in recurrent ovarian cancer patients is merited.

Comprehensive Molecular Profiling and Clinicopathological Characteristics of Gastric-Type Mucinous Carcinoma of the Uterine Cervix in Japanese Women.

Gastric-type mucinous carcinoma (GAS) of the uterine cervix is the most common adenocarcinoma that develops independently of human papillomavirus infection; it is typically diagnosed at an advanced stage and has a poorer prognosis than usual-type endocervical adenocarcinoma. Few studies have examined the molecular profile of GAS, but genetic alterations in TP53 and STK11 have been repeatedly reported. We analyzed the clinicopathological characteristics and molecular profile of GAS. Fresh-frozen tissue specimens and formalin-fixed paraffin-embedded (FFPE) tissues from 13 patients with GAS treated between January 2000 and December 2020 were analyzed. We performed next-generation sequencing on eight fresh-frozen GAS specimens using the Cancer Hotspot Panel v2 (cases 1-8) and the FoundationOne companion diagnostic (F1CDx) assay on six FFPE samples (cases 8-13). Seventy-four genomic alterations were identified in 42 genes. In order of frequency, TP53, ATRX, CDKN2A, KRAS, APC, and STK11 were altered in at least three cases. Targetable genomic alterations were identified in all six patients' specimens analyzed using the F1CDx assay. GAS harbors various genomic alterations associated with sustained activation of signaling pathways or cell cycle regulation in addition to abnormalities in TP53, and precision medicine based on molecular profiling will be necessary to overcome GAS.

Identification of B cell epitopes reactive to human papillomavirus type-16L1- derived peptides.

BACKGROUND: Persistent infection of human papillomavirus (HPV) types 16 and 18 causes cervical cancer. To better understand immune responses to the prophylactic vaccine, HPV 16/18 L1 virus-like particles (HPV-VLPs), we investigated B cell epitopes of HPV16 L1-derived peptides. METHODS: Sera from mice immunized with HPV-16/18 L1 VLPs were analyzed for their IgG titers against 10 different HPV16 L1-derived peptides (20-mer) that contain human leukocyte antigen (HLA)-class I A-2, A-24 and class II DR. RESULTS: One 20-mer peptide at positions 300 to 319 was identified as a common B cell epitope in both Balb/c (H-2d) and C57BL/6 (H-2b) mice. Mapping analysis showed that the 10-amino-acid sequence at positions 304 to 313 was an immunogenic portion. It is of note that the binding capability of this 10-mer peptide to the HLA-A2 and HLA-A24 molecules was confirmed by the HLA class I stabilization assay. In addition, one unique 20-mer was determined as a B cell epitope in each strain. CONCLUSIONS: These results might provide new information for better understanding of immune responses to HPV 16 L1.

Integrity of plasma cell-free DNA as a prognostic factor for vaccine therapy in patients with endometrial cancer.

Endometrial cancer is the most prevalent gynecological cancer in developed countries. Although the prognosis of endometrial cancer is better than that of other gynecological cancers, the prognosis of advanced endometrial cancer is still poor and thus new therapeutic modalities, such as immune therapies, are urgently required. For the further development of new modalities, exploration of new biomarkers is important. The present study investigated the circulating cell-free DNA (cfDNA) integrity as a ratio of the necrotic tumor cell-derived long cfDNA fragments to the total dead cell-derived short cfDNA fragments from genomic Alu elements in patients with advanced endometrial cancer during peptide vaccination treatment. The results demonstrated that: i) The plasma cfDNA integrity was decreased during the first cycle of vaccination in patients with endometrial cancer treated with the personalized peptide vaccination, and ii) the post-vaccination cfDNA integrity levels were correlated with good prognosis. Some of these findings have been confirmed in other cancers, and thus cfDNA integrity might be an important marker for future cancer vaccine therapies in general, and might also be applicable for other immune therapies.

Subcategorization of atypical glandular cells is useful to identify lesion site.

BACKGROUND: In the subcategorization of atypical glandular cells (AGCs), origin of cells should be mentioned to estimate lesion sites for diagnosis. However, cases without subcategorization are often encountered due to limited reproducibility. We evaluated whether the subcategorization of AGC based on the Bethesda terminology can estimate lesion sites. METHODS: We retrospectively investigated cases whose cervical smears were interpreted as AGC and underwent pathological assessment at our institution between June 2009 and September 2017. AGC was subcategorized based on the Bethesda System. Not-otherwise-specified (NOS) was subcategorized into endocervical cells (NOS-EC), endometrial cells (NOS-EM), or glandular cells (NOS-G). Favor neoplastic (FN) was subcategorized into endocervical cells (FN-EC) or glandular cells (FN-G). FN-G was further subcategorized into endometrial cells (FN-EM) or unknown origin (FN-UO). Clinicopathological data were retrieved from the medical records. RESULTS: Of 88 AGC cases, there were 30 NOS-EC (34.1%), 2 NOS-EM (2.3%), 25 FN-EC (28.4%), 22 FN-EM (25.0%), and 9 FN-UO (10.2%). A significantly higher proportion of neoplastic lesions occurred in FN than in NOS (P <.001). The concordance of AGC subclass and lesion site was 88.0%, 70.7%, and 77.3% in FN-EC, FN-G, and FN-EM, respectively. The concordance of FN-EM and lesion site increased to 88.9% in patients aged >50 years. Of nine cases of FN-UO, six experienced nonendometrioid endometrial cancer and extrauterine malignancy. CONCLUSION: Subcategorization of NOS and FN would be useful in estimating neoplastic lesions. Further subcategorization into FN-EC, FN-EM, and FN-UO would similarly be beneficial in estimating the lesion site, especially for small endometrial and extrauterine lesions.

Achievement of Surgical Proficiency in Laparoscopic Surgery for Early Endometrial Cancer by Gynecologic Oncologists.

OBJECTIVE: Although studies have evaluated learning curves for laparoscopic surgery (LS), this issue has not yet been addressed in gynecologic oncologists. The present study aimed to evaluate LS proficiency for early-stage endometrial cancer among gynecologic oncologists. METHODS: We examined 25 cases in which LS with pelvic lymphadenectomy (PLA) for endometrial cancer was performed by two gynecologic oncologists undergoing training in LS. The LS duration, estimated blood loss (EBL), number of dissected pelvic lymph nodes (PLNs), and perioperative complications were assessed as measures of surgical proficiency. RESULTS: Operators A and B performed 10 and 15 cases, respectively, with median LS durations of 348.5 and 378 minutes, respectively. Although the LS duration and number of procedures did not exhibit a significant correlation, the regression lines for both operators showed decreasing trends. Both operators had a consistently low median EBL (A, 57 ml; B, 60 ml). Operators A and B dissected a median of 21.5 and 22 PLNs, respectively. Although both operators dissected a relatively large number of PLNs, with a median of over 20 per patient after the introduction of LS, this number decreased as the number of LS increased for Operator B (p=0.009). However, no correlation was observed between the LS duration and number of PLNs dissected by Operator B (ρ=0.353, p=0.197). A severe perioperative complication, specifically perforation of the sigmoid colon requiring emergent laparotomy, occurred in one case; however, the association with surgical manipulation was not definitive. CONCLUSION: The observed gynecologic oncologists were able to acquire early proficiency in LS for early-stage endometrial cancer.

Identification of biomarkers for personalized peptide vaccination in 2,588 cancer patients.

Peptide­based cancer vaccines have failed to provide sufficient clinical benefits in order to be approved in clinical trials since the 1990s. To understand the mechanisms underlying this failure, the present study investigated biomarkers associated with the lower overall survival (OS) among 2,588 patients receiving personalized peptide vaccination (PPV). Survival data were obtained from a database of 2,588 cancer patients including 399 patients with lung, 354 with prostate and 344 with colon cancer. They entered into phase II clinical trials of PPV in which 2 to 4 of 31 warehouse peptides were selected for vaccination on an individual patient basis based on human leukocyte antigen (HLA) class IA­types and pre­existing peptide­specific IgG levels. Higher pre­vaccination neutrophil, monocyte and platelet counts, and lower pre­vaccination lymphocyte and red blood cell counts were inversely associated with OS, with higher sensitivities in the proportions of neutrophils and lymphocytes, respectively. The most potent unfavorable and favorable factors for OS were the median percentage of neutrophils (≥64.8%) or percentage of lymphocytes (≥25.1%) with correlation coefficients (R2) of 0.98 and 0.92, respectively. Higher pre­vaccination levels of c­reactive protein and other inflammatory soluble factors were inversely associated with OS. Pre­vaccination peptide­specific immunity levels had no effect on OS, although lower immune boosting levels were inversely associated with OS. None of the 31 peptides was inversely associated with OS, although a few peptides were positively associated with it. On the whole, the findings of the present study suggested that pre­vaccination inflammatory signatures, but not those of post­vaccination immune induction, were associated with lower clinical benefits of PPV.

Distinct patient responses to activation of T-cells by free HER-2, G89 (777-789) and protected LRMK-linked HER-2, {AE-39 [p776 (Ava-774-788)], AE-47 [(Ava-776-788)] and AE-37[p776 (774-788)]} peptides could lead to development of personalized cancer vaccines.

The objective of this study was to find whether the peptide LRMK linked to the N-terminus of HER-2, 774-788 and shorter peptides create a T helper antigen, which can replace all functional HER-2 peptides in a cancer vaccine. Of the 6 LRMK-HER-2 peptides tested in the presence of IL-12, AE-37, AE-38 and AE-39 induced higher IFN-gamma in PBMCs from 4 healthy donors than the other peptides. AE-37 and AE-39 contained the immunogenic HER-2 peptide, p776 (774-788), while AE-38 contained the truncated HER-2 peptide G89 (777-788). The free, unprotected HER-2 peptide G89 (777-789) activated IFN-gamma production in PBMCs from another BRC patient. Responses to free p776 and F7 could not be tested. Three out of 11 patients responded strongly only to AE-37, while 3 of 10 patients responded strongly only to G89. One responded only to AE-39. All 3 patients diagnosed with DICS of mixed HER(hi), ER+ PR+ type responded to AE-37. Three out of 4 patients diagnosed with luminal cancer and one HERhi, ER- PR- BRC patient responded only to G89. AE-37, at low concentration, helped to expand more E75-TCR(Hi+Med) cells than the negative control AE-47, for IFN-gamma induction AE-47, but was a weaker helper than AE-47 at higher concentrations, and eliminated E75-TCR(Hi) cells. The strongest and most consistent effect of AE-37 was the elimination of CD4(Hi) CD25(Hi) cells. When LRMK is linked to AE-37, the side-chains of L(P-10) and R(P-9) are positioned away from MHC; LRMK forms a bi-strophynx (rotating-double-hook-like) structure when attached to AE-37 and the minimum HER-2 peptide in the peptide-binding groove (PBG). K(P-8) anchors the bi-strophynx to HLA-DR outside the PBG in a novel site DRalpha, 49-52. The HER-2 amino acids, Y(P-1) and R(P3) point towards TCR; R(P-9) and M(P-8) can contact the TCRValpha1. The positions of K(P-8) in AE-37 and Ava (epsilonV)(P-8) in AE-39 modulated immunogenicity of p776. It is unknown whether LRMK adds TCR contact points to the minimal HLA-DR-bound HER-2 peptide (780-788) or activates T-cells of other specificities to produce cytokines and die. Preferential activation of Th1 cells in distinct individuals by AE-37, G89 and AE-39 indicates that cancer vaccines will benefit from correct individual and disease-associated help. Emergence of distinct daughters of luminal and myoepithelial BRC stem cells during metastasis through "de-differentiation" and "reverse differentiation" of drug-resistant cancer requires personalized vaccines, which use an optimal-helper antigen.

Cap43/NDRG1/Drg-1 is a molecular target for angiogenesis and a prognostic indicator in cervical adenocarcinoma.

Cap43 is a nickel- and calcium-inducible gene that plays important roles in the primary growth of malignant tumors, as well as in invasion and metastasis, most likely through its ability to induce cellular differentiation. This study investigated associations of Cap43 expression with angiogenesis and other clinicopathological factors in cervical adenocarcinoma. The clinical records of 100 women who underwent surgery for cervical adenocarcinoma were reviewed retrospectively. Microvessel density and the expression of Cap43 and VEGF in the surgical specimens were evaluated immunohistochemically. The Cap43 expression level was significantly associated with angiogenesis, tumor diameter, stromal invasion, lymphovascular space invasion, lymph node metastasis, and histopathological differentiation. Kaplan-Meier analysis showed a significant association between the Cap43 expression level and survival: high Cap43 expression was related to poor survival. Our results suggest that increased expression of Cap43 is associated with angiogenesis and may be a poor prognostic indicator in women with cervical adenocarcinoma.

Functional idiotopes: tumor antigen-directed expression of CD8+ T-cell epitopes nested in unique NH2-terminal VH sequence of antiidiotypic antibodies?

Antiidiotypic antibodies have been and are being used for cancer immunotherapy based on the rationale that Ab2 carrying an "internal image" of the corresponding tumor antigen can induce tumor antigen-specific antibodies (i.e., Ab3 and inhibit tumor growth). Recent evidence indicates that Ab2 also induces cellular responses by CD4+ and CD8+ T cells. This finding has raised the question of where the short peptides, which express CD8+ T-cell-defined epitopes, are located and their relationship with the tumor antigen. We found that two of the four known Ab2 associated with tumor antigen, with known amino acid sequence, express unique NH2-terminal V(H) sequences which precede the framework regions. Both the unique and the shared NH2-terminal V(H) sequences are nested MHC class I antigen-binding peptides. These peptides were highly homologous with peptides from corresponding tumor antigen (carcinoembryonic antigen, CD55, and human high molecular weight melanoma-associated antigen) but differed from the tumor antigen peptides by the presence of the side chain known to mediate stronger forces of interaction with other atoms. The presence of candidate CTL epitopes in NH2-terminal V(H) of Ab2 homologous with tumor antigen may be important for the development of novel immunotherapeutic strategies for cancer.

Sensitivity of undifferentiated, high-TCR density CD8+ cells to methylene groups appended to tumor antigen determines their differentiation or death.

CD8(+) cells expressing high numbers of TCR per cell (TCR(hi)) are considered important mediators of antitumor effects. To understand the relationship between TCR density and antigen affinity for TCR in the outcome of stimulation with antigen and differentiation of CTL recognizing tumor antigen, we analyzed perforin induction in ovarian tumor-associated lymphocytes in response to the smallest possible changes in the atomic forces of interaction between antigen and TCR. Stimulating undifferentiated, apoptosis-resistant CD8(+) cells expressing high levels of E75-TCR (TCR(hi)) with variants of the CTL epitope E75, HER-2 (369-377), induced their stepwise differentiation, first to IFN-gamma(+) Perf(-) and to TCR(hi) IFN-gamma(+) Perf(+) cells. Blocking caspase-9 activation at antigen stimulation also enhanced the generation of TCR(hi) Perf(hi) cells, demonstrating that TCR density dictated the pathway of death activated by stimulation with the same agonist. Expansion and differentiation of TCR(hi) Perf(+) CTL required an agonist of optimal CH(2) side chain length, which in this study was equal to two CH(2) groups appended to E75 at the Gly(4) position. Side chains one CH(2) shorter or longer than optimal were either less stimulatory or induced death of TCR(hi) Perf(+) cells. Differentiation of TCR(hi) CD8(+) cells can be finely tuned by synthetic amino acids in the peptide, whose side chains induce small increments in the affinity of the antigen for TCR below the affinity which induce apoptosis.