HPV DNA integration site as proof of the origin of ovarian metastasis from endocervical adenocarcinoma: three case reports.

BACKGROUND: Most endocervical adenocarcinomas are human papillomavirus (HPV)-related cancers associated with p16 immunostaining. Ovarian metastasis from cervical cancer is a rare phenomenon, the mechanism of dissemination remains unclear. The diagnosis of metastasis may be difficult to establish when the ovarian neoplasm presents features consistent with primary tumor. Immunohistochemical expression of p16 in ovarian tumors can guide the diagnosis of metastasis from HPV-related cervical cancer, but p16 positivity is nonspecific. Identical HPV genotype in the paired endocervical and ovarian tumors is a better marker for cervical origin, which may also be confirmed by identical HPV integration site. CASE PRESENTATION: Two women presented with HPV18 cervical adenocarcinoma. No signs of disease were visible on MRI after treatment. After several years of follow-up, mucinous ovarian tumors were discovered in both patients. Molecular analyses showed that the ovarian lesions were HPV18-positive; indicating a primary cervical origin. A third woman was diagnosed with grade 1 ovarian endometrioid carcinoma with no peritoneal carcinomatosis. Final histological examination and HPV genotyping revealed HPV18-related in situ endometrioid adenocarcinoma in the endocervix and HPV18-related invasive endometrioid adenocarcinoma in the endometrium and both ovaries. Additional molecular analyses performed in two patients identified the same HPV integration sites in both the ovarian and cervical tumors, confirming that the ovarian mass was a metastasis from the cervical adenocarcinoma. CONCLUSION: We report three new cases of ovarian neoplasia in which the diagnosis of metastasis from cervical cancer was supported by the same HPV genotype and the same integration site in the paired cervical and ovarian tumors. To our knowledge, this is the first report of molecular evidence of the cervical origin of an ovarian metastasis. HPV screening should be performed in ovarian tumors for all patients with history of cervical neoplasia.

Phase Ib/II trial of tipapkinogene sovacivec, a therapeutic human papillomavirus16-vaccine, in combination with avelumab in patients with advanced human papillomavirus16-positive cancers.

PURPOSE: To evaluate tipapkinogene sovacivec (TG4001), a viral immunotherapeutic vaccine expressing human papillomavirus (HPV)16 E6/E7 non-oncogenic proteins and IL-2, in combination with avelumab in HPV16+ cancer patients. PATIENTS AND METHODS: In this open-label, phase Ib/II, multicenter study, HPV16+ advanced cancer patients received subcutaneous TG4001 at two dose levels (DL) in phase Ib and at the recommended phase II dose (RP2D) in phase II weekly for 6 weeks, then every 2 weeks (q2Wk) until 6 months, thereafter every 12 weeks, in combination with avelumab q2Wk starting from day 8. Exploratory end-points included immunomonitoring from sequential tumour and blood samples. RESULTS: Forty-three patients, mainly heavily pretreated (88% ≥ 1 previous line), were included in the safety analysis, with a majority of anal cancer (44%). No dose-limiting toxicities were reported, and DL2 (5 × 107 Plaque forming units (PFU)) was selected as the RP2D. Treatment-related adverse events to TG4001 occurred in 93% of patients, mostly grade 1/2, with grade 3 anaemia in one patient and no grade 4/5. Overall response rate (ORR) was 22% (8/36) and 32% (8/25) in all and patients without liver metastases, respectively. Median progression-free survival (PFS) and Overall Survival (OS) were 2.8 months (95% CI: 1.4-5.6) and 11.0 months (95% CI:7.5-16.7) in the total population and 5.6 months (95% CI:1.6-9.6) and 13.3 months (95% CI:8.7-32.7) in patients without liver metastases. Antigen-specific T-cell response was identified in 7/11 patients by IFNγ ELISpot. CONCLUSIONS: TG4001 in combination with avelumab is safe, demonstrated antitumour activity in heavily pre-treated HPV16+ cancer patients, and is currently being evaluated in a randomised phase II trial in patients with incurable anogenital cancer and limited hepatic involvement. GOV IDENTIFIER: NCT03260023.

Importance of pre-analytical steps for transcriptome and RT-qPCR analyses in the context of the phase II randomised multicentre trial REMAGUS02 of neoadjuvant chemotherapy in breast cancer patients.

BACKGROUND: Identification of predictive markers of response to treatment is a major objective in breast cancer. A major problem in clinical sampling is the variability of RNA templates, requiring accurate management of tumour material and subsequent analyses for future translation in clinical practice. Our aim was to establish the feasibility and reliability of high throughput RNA analysis in a prospective trial. METHODS: This study was conducted on RNA from initial biopsies, in a prospective trial of neoadjuvant chemotherapy in 327 patients with inoperable breast cancer. Four independent centres included patients and samples. Human U133 GeneChips plus 2.0 arrays for transcriptome analysis and quantitative RT-qPCR of 45 target genes and 6 reference genes were analysed on total RNA. RESULTS: Thirty seven samples were excluded because i) they contained less than 30% malignant cells, or ii) they provided RNA Integrity Number (RIN) of poor quality. Among the 290 remaining cases, taking into account strict quality control criteria initially defined to ensure good quality of sampling, 78% and 82% samples were eligible for transcriptome and RT-qPCR analyses, respectively. For RT-qPCR, efficiency was corrected by using standard curves for each gene and each plate. It was greater than 90% for all genes. Clustering analysis highlighted relevant breast cancer phenotypes for both techniques (ER+, PR+, HER2+, triple negative). Interestingly, clustering on trancriptome data also demonstrated a "centre effect", probably due to the sampling or extraction methods used in on of the centres. Conversely, the calibration of RT-qPCR analysis led to the centre effect withdrawing, allowing multicentre analysis of gene transcripts with high accuracy. CONCLUSIONS: Our data showed that strict quality criteria for RNA integrity assessment and well calibrated and standardized RT-qPCR allows multicentre analysis of genes transcripts with high accuracy in the clinical context. More stringent criteria are needed for transcriptome analysis for clinical applications.

Circulating HPV DNA as a Marker for Early Detection of Relapse in Patients with Cervical Cancer.

PURPOSE: Almost all cervical cancers are caused by human papillomavirus (HPV) and patients with advanced stage are at high risk for relapse. Circulating HPV DNA (HPV ctDNA) may serve as a residual tumor marker at the end of chemoradiation or to predict relapse during the follow-up period. EXPERIMENTAL DESIGN: We analyzed serum samples from 94 HPV16- or HPV18-related CCs from the BioRAIDs prospective cohort. Samples were collected before and after treatment and during an 18-month follow-up period. Using digital droplet PCR (ddPCR), we assessed the relevance of circulating HPV E7 gene as a marker for residual disease compared to HPV integration site and PIK3CA mutations. Finally, the prognostic impact of circulating HPV E7 gene was assessed with its prediction value of relapse. RESULTS: HPV E7 gene was the most sensitive tumor marker, superior to both HPV integration sites and PIK3CA mutations in serum. Circulating HPV DNA (HPV ctDNA) was detected in 63% (59/94) of patients, before treatment. HPV ctDNA detection in serum sample was associated with high FIGO stage (P = 0.02) and para-aortic lymph node involvement (P = 0.01). The level of HPV ctDNA was positively correlated with HPV copy number in the tumor (R = 0.39, P < 0.001). Complete clearance of HPV ctDNA by the end of treatment was significantly associated with a longer PFS (P < 0.0001). Patients with persistent HPV ctDNA in serum relapsed with a median time of 10 months (range, 2-15) from HPV ctDNA detection. CONCLUSIONS: HPV ctDNA detection is a useful marker to predict relapse in cervical cancer.See related commentary by Wentzensen and Clarke, p. 5733.

Tumor aromatase expression as a prognostic factor for local control in young breast cancer patients after breast-conserving treatment.

INTRODUCTION: We sought to determine whether the levels of expression of 17 candidate genes were associated with locoregional control after breast-conserving treatments of early-stage breast cancers in young, premenopausal women. METHODS: Gene expression was measured by using RT-PCR in the breast tumors of a series of 53 young (younger than 40 years), premenopausal patients. All treatments consisted of primary breast-conserving surgery followed by whole-breast radiotherapy (+/- regional lymph nodes) with or without systemic treatments (chemotherapy +/- hormone therapy). The median follow-up was 10 years. RESULTS: The 10-year locoregional control rate was 70% (95% CI, 57% to 87%). In univariate analysis, no clinical/pathologic prognostic factors were found to be significantly associated with decreased locoregional control. Expression of three genes was found to be significantly associated with an increased locoregional recurrence rate: low estrogen-receptor beta, low aromatase, and high GATA3. Two others were associated with only a trend (P < 0.10): low HER1 and SKP2. In multivariate analysis, only the absence of aromatase was significantly associated with an increased locoregional recurrence rate (P = 0.003; relative risk = 0.49; 95% CI 0.29 to 0.82). CONCLUSIONS: Recent data give credit to the fact that breast cancer in young women is a distinct biologic entity driven by special oncogenic pathways. Our results highlight the role of estrogen-signaling pathways (mainly CYP19/aromatase, GATA3, and ER-beta) in the risk of locoregional recurrence of breast cancer in young women. Confirmation in larger prospective studies is needed.

Expression of progesterone and estradiol receptors in normal adrenal cortex, adrenocortical tumors, and primary pigmented nodular adrenocortical disease.

Adrenal tumors occur more frequently in women and are the leading cause of Cushing's syndrome during pregnancy. We aimed to evaluate the potential role of sex steroids in the susceptibility of women to adrenocortical tumors. We evaluated the presence of the progesterone receptor (PR), estradiol receptors (ERs), and aromatase in 5 patients with primary pigmented nodular adrenal disease (PPNAD), 15 adrenocortical adenomas (ACAs) and adjacent normal tissues, 12 adrenocortical carcinomas (ACCs), and 3 normal adrenal glands (NA). The expression of PR and ERalpha was evaluated by enzyme immunoassays, real-time RT-PCR, immunohistochemistry, and cytosol-based ligand-binding assays. ERbeta and aromatase levels were evaluated by real-time RT-PCR. ERalpha concentrations were low in NA, in adrenal tissues adjacent to ACA (51+/-33), in ACC (53+/-78), and lower in ACA (11+/-11 fmol/mg DNA). Conversely, PR concentrations were high in NA and adrenal tissues adjacent to ACA, at 307+/-216 fmol/mg DNA, and were even higher in tumors - 726+/-706 fmol/mg DNA in ACA and 1154+/-1586 fmol/mg DNA in ACC - and in isolated PPNAD nodules. Binding study results in four tumors were compatible with binding to a steroid receptor. In patients with PPNAD, a strong positive immunohistochemical signal was associated with the sole isolated nodular regions. ERbeta transcript levels were very high in all samples except those for two ACCs, whereas aromatase levels were low. PR and ERbeta are clearly present in normal adrenal glands and adrenal tumors. Further studies may shed light on the possible pathogenic role of these receptors in adrenal proliferation.

A new model of patient tumor-derived breast cancer xenografts for preclinical assays.

PURPOSE: To establish a panel of human breast cancer (HBC) xenografts in immunodeficient mice suitable for pharmacologic preclinical assays. EXPERIMENTAL DESIGN: 200 samples of HBCs were grafted into Swiss nude mice. Twenty-five transplantable xenografts were established (12.5%). Their characterization included histology, p53 status, genetic analysis by array comparative genomic hybridization, gene expression by Western blotting, and quantitative reverse transcription-PCR. Biological profiles of nine xenografts were compared with those of the corresponding patient's tumor. Chemosensitivities of 17 xenografts to a combination of Adriamycin and cyclophosphamide (AC), docetaxel, trastuzumab, and Degarelix were evaluated. RESULTS: Almost all patient tumors established as xenografts displayed an aggressive phenotype, i.e., high-grade, triple-negative status. The histology of the xenografts recapitulated the features of the original tumors. Mutation of p53 and inactivation of Rb and PTEN proteins were found in 83%, 30%, and 42% of HBC xenografts, respectively. Two HBCx had an ERBB2 (HER2) amplification. Large variations were observed in the expression of HER family receptors and in genomic profiles. Genomic alterations were close to those of original samples in paired tumors. Three xenografts formed lung metastases. A total of 15 of the 17 HBCx (88%) responded to AC, and 8 (47%) responded to docetaxel. One ERBB2-amplified xenograft responded to trastuzumab, whereas the other did not. The drug response of HBC xenografts was concordant with that of the patient's tumor in five of seven analyzable cases. CONCLUSIONS: This panel of breast cancer xenografts includes 15 triple-negative, one ER positive and 2 ERBB2 positive. This panel represents a useful preclinical tool for testing new agents and protocols and for further exploration of the biological basis of drug responses.

Quantitation of estradiol receptors alpha and beta and progesterone receptors in human breast tumors by real-time reverse transcription-polymerase chain reaction. Correlation with protein assays.

Hormone receptor content is the most useful parameter for predicting hormone response therapy in breast cancer. The high frequency of primary and secondary resistance to treatment makes it necessary to find out other parameters in order to improve the prediction of response to treatment. The newly described estrogen receptor beta (ERbeta) is a potential candidate. Using real-time quantitative RT-PCR, we evaluated estrogen receptor alpha (ERalpha), ERbeta, and progesterone receptors (PR) in comparison with ERalpha and PR protein content measured with the enzyme immunoassay (EIA), in a retrospective series of 98 breast tumors. We obtained a highly significant correlation between mRNA and EIA assays for ERalpha and PR (r=0.79 and r=0.71, respectively; P<0.001). We confirmed the low level of ERbeta mRNA transcripts in comparison to ERalpha in breast tumors. We did not find any statistically significant correlation between the absolute ERbeta mRNA level and ERalpha or PR mRNA level, and ERalpha or PR-EIA. We found a significant correlation between ERalpha mRNA and PR mRNA expressions. We did not find any correlation between ERbeta mRNA and clinical features of the patients (age, menopausal status, tumor size, and nodal status), nor with the histological type of the tumor. In conclusion, the accuracy of the present quantitative RT-PCR assay makes it suitable for a routine clinical use. In addition, the present results suggest that, ERbeta mRNA expression is independent of the classical parameters.

Role of chemotherapy resistance genes in outcome of neuroblastoma.

BACKGROUND: Neuroblastoma is a heterogeneous pediatric disease. Most patients with localized disease usually have a favorable prognosis, but patients with advanced disease have a poor prognosis despite combination chemotherapy. Treatment failure may be attributable to resistance to cytotoxic drugs. PROCEDURE: Using quantitative RT-PCR, we investigated the clinical significance of the level of mRNA expression of multidrug resistance genes (MDR1, MRP1, MRP5, LRP) in a series of 29 advanced neuroblastoma samples. RESULTS: At the end of induction chemotherapy, 48% of patients achieved a clinical complete response, 28% achieved a partial response or stable disease, and 24% presented progressive disease. MDR1 mRNA overexpression (i.e., mRNA level >2 copies of MDR1 gene) was observed in 74% of samples, and MRP1, MRP5, LRP overexpression was observed less frequently (30, 33, and 33% of samples, respectively). None of these parameters were predictive of response, relapse, or survival. However, clinical response to treatment was highly predictive of relapse-free survival and overall survival. CONCLUSIONS: High expression of these multidrug resistance genes in advanced neuroblastoma is not the main parameter of response to cytotoxic drugs; clinical response to treatment remains the most important parameter in predicting the prognosis of patients with advanced neuroblastoma, until other relevant laboratory parameters have been identified.

Gefitinib and chemotherapy combination studies in five novel human non small cell lung cancer xenografts. Evidence linking EGFR signaling to gefitinib antitumor response.

The epidermal growth factor receptor (EGFR) signaling pathway is often activated in NSCLC, and thus represents a promising therapeutic target. We studied the antitumor activity of gefitinib (Iressa), an orally active EGFR-tyrosine kinase inhibitor, alone and in combination with standard chemotherapy in 5 recently established human NSCLC xenografts with wild-type EGFR. Mice were treated with 2 protocols of chemotherapy based on cisplatin (CDDP) combined with either gemcitabine (GEM) or vinorelbine (VNR). Gefitinib alone significantly inhibited tumor growth (TGI) in 4 of the 5 tumor xenografts (mean TGI of 58%, range: 25-70%). CDDP+VNR alone failed to achieve any significant responses, while CDDP+GEM achieved significant responses in 2 xenografts (TGI of 93 and 47%). Addition of gefitinib to CDDP+GEM potentialized chemotherapy in the 3 CDDP+GEM-resistant xenografts, but did not potentialize the CDDP+VNR combination. The effect of gefitinib treatment on the activity of extra cellular-regulated kinase (Erk), Akt, JNK and p38 kinases was assessed in IC9LC11 and IC1LC131, two NSCLC xenografts selected for their sensitivity and resistance to gefitinib, respectively. In IC9LC11, gefitinib strongly inhibited Erk, Akt and Jnk phosphorylation, but P38 remained active. Inversely, in IC1LC131, Erk and Akt pathways remained active, while Jnk and P38 pathways were inhibited by gefitinib. The data indicate that the antitumor activity of gefitinib in NSCLC, alone or in combination with chemotherapy, is tumor-dependent and is influenced by downstream signaling events independent of EGFR status.

In vivo efficacy of STI571 in xenografted human small cell lung cancer alone or combined with chemotherapy.

STI571, or imatinib, selectively inhibits BCR/ABL, PDGFR and c-kit kinase activity. It has been reported that a large proportion of small cell lung cancer (SCLC) cell lines and tumors express c-kit and that STI571 inhibits tumor cell growth. We therefore investigated the therapeutic efficacy of STI571, alone or combined with chemotherapy, in human SCLC cells or tumors xenografted into nude mice. The level of c-kit mRNA expression was variable in SCLC tumors (positive for 2 of 4 xenografts), and c-kit protein was not detected by immunohistochemistry. On the 4 xenografted tumors, PDGFRalpha and PDGFRbeta were not detected by immunohistochemistry. STI571 induced inhibition of proliferation of the SCLC6 cell line without inducing apoptosis; in contrast, in combination with etoposide or topotecan, the growth inhibition of SCLC6 cells induced by STI571 was increased, with apoptotic DNA fragmentation. Four human SCLC xenografts (SCLC6, SCLC61, SCLC74 and SCLC108) were transplanted into mice. After intraperitoneal injection of STI571, we observed 80%, 40% and 78% growth inhibition of SCLC6, SCLC61 and SCLC108 tumors, respectively, without any significant inhibition of SCLC74 tumor growth. In mice bearing responsive SCLC tumors, we observed an increase of growth inhibition induced by chemotherapy (etoposide + ifosfamide or topotecan) by concomitant and continuous administration of STI571, associated with an increase of toxic deaths. In SCLC6-bearing mice receiving sequential treatments, we observed a reduction of toxic deaths but a decrease of synergistic antitumor efficacy. In conclusion, the efficacy of STI571 alone in SCLC xenografted tumors was variable and did not depend on c-kit expression. Moreover, a significant increase of chemotherapy-induced growth inhibition was obtained by concomitant administration of STI571 that should be carefully investigated in SCLC patients.