Sera Of Cervical Cancer Patients Research Articles

Abstract 3672: Development of liquid biopsy HPV (Human Papilloma Virus) cell free DNA (cfDNA) based approach for prognosis of cervical cancer

Abstract Introduction: Recent progress in the analysis of blood samples for circulating tumor cells or cell-free circulating tumor DNA (ctDNA) has shown that liquid biopsies may have potential applications in the detection and monitoring of cancer. Human papillomavirus (HPV) circulating cell-free (ccf) DNA may act as a unique marker for the prognosis of high-risk HPV-related cancers. However, detection of circulating markers for cervical cancer (CC) requires extremely sensitive techniques that can measure circulating human papillomavirus DNA. Here, we used the sensitive droplet digital PCR (dd PCR) based approach for the detection and quantification of circulating human papillomavirus DNA in plasma from patients diagnosed with cervical cancer at baseline and subsequent follow up with intention of validating its utility as a prognostic marker in CC. Method: Blood sera form 34 Patients diagnosed with cervical cancer (Stage 1-1V) at AIIMS Delhi was collected at baseline and after 3 months of the treatment. 10 healthy controls were also recruited in the study after due approval from AIIMS IEC. Plasma samples were stored at -80°C. ccf DNA was isolated from 1 ml of plasma and further processed to detect the presence of circulating high risk Human papilloma virus (HPV) i.e., HPV-16 and HPV18 cf DNA using dd PCR. Result: The median cf DNA concentration was 9.3 ng/uL (range 3-26.6) for all patients while median cf DNA concentration was 7.2 ng/uL (Range 3-57.3) for healthy controls (n=10). Furthermore, the levels of cfDNA decreases post 3 months of treatment with average concentration of 8.07 ng/uL (Range 4.6-19.3). We further processed our samples for dd PCR based screening to detect low copy no HPV cf DNA and ddPCR detection yielded a positive rate of 51% (18/35) for circulating HPV18 DNA while a positivity of 88.5% (31/35) was seen for the circulating HPV16 DNA in the sera of CC patients. By comparing HPV16 and HPV18 genotypes, we observed detection of circulating HPV 16 DNA more frequently than circulating HPV18 DNA in CC patients coming at our center. Furthermore, we found that copies of circulating viral DNA (HPV18 and HPV16) decreased post induction therapy in CC patients indicating progression ability of thid marker. Conclusion: In the current studies we provide evidence that cell free DNA concentration in CC patient can predict tumor burden and detection of low copy no of circulating HPV DNA could be a promising indicator for prognosis in cervical cancer patients and can predict recurrence of the disease. Citation Format: Gunjan Dagar, Ashna Gupta, Ravi Chauhan, Abhishek Shankar, Daya Nand Sharma, Muzafar A. Macha, Ajaz A. Bhat, Rajeev Goyal, Vaishali Suri, Mayank Singh. Development of liquid biopsy HPV (Human Papilloma Virus) cell free DNA (cfDNA) based approach for prognosis of cervical cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3672.

Viral-Cellular DNA Junctions as Molecular Markers for Assessing Intra-Tumor Heterogeneity in Cervical Cancer and for the Detection of Circulating Tumor DNA.

The development of cervical cancer is frequently accompanied by the integration of human papillomaviruses (HPV) DNA into the host genome. Viral-cellular junction sequences, which arise in consequence, are highly tumor specific. By using these fragments as markers for tumor cell origin, we examined cervical cancer clonality in the context of intra-tumor heterogeneity. Moreover, we assessed the potential of these fragments as molecular tumor markers and analyzed their suitability for the detection of circulating tumor DNA in sera of cervical cancer patients. For intra-tumor heterogeneity analyses tumors of 8 patients with up to 5 integration sites per tumor were included. Tumor islands were micro-dissected from cryosections of several tissue blocks representing different regions of the tumor. Each micro-dissected tumor area served as template for a single junction-specific PCR. For the detection of circulating tumor-DNA (ctDNA) junction-specific PCR-assays were applied to sera of 21 patients. Samples were collected preoperatively and during the course of disease. In 7 of 8 tumors the integration site(s) were shown to be homogenously distributed throughout different tumor regions. Only one tumor displayed intra-tumor heterogeneity. In 5 of 21 analyzed preoperative serum samples we specifically detected junction fragments. Junction-based detection of ctDNA was significantly associated with reduced recurrence-free survival. Our study provides evidence that HPV-DNA integration is as an early step in cervical carcinogenesis. Clonality with respect to HPV integration opens new perspectives for the application of viral-cellular junction sites as molecular biomarkers in a clinical setting such as disease monitoring.

Identification of a serum-induced transcriptional signature associated with metastatic cervical cancer.

ObjectiveTumor cells that escape local tissue control can convert inflammatory cells from tumor suppressors to tumor promoters. Moreover, soluble immune-modulating factors secreted from the tumor environment can be difficult to identify in patient serum due to their low abundance. We used an alternative strategy to infer a metastatic signature induced by sera of cervical cancer patients.MethodsSera from patients with local and metastatic cervical cancer were used to induce a disease-specific transcriptional signature in cultured, healthy peripheral blood mononuclear cells (PBMCs). An empirical Bayesian method, EBarrays, was used to identify differentially expressed (DE) genes with a target false discovery rate of <5%. Ingenuity Pathway Analysis (IPA) software was used to detect the top molecular and cellular functions associated with the DE genes. IPA and in silco analysis was used to pinpoint candidate upstream regulators, including cancer-related microRNAs (miRNAs).ResultsWe identified enriched pathways in the metastatic cervical group related to immune surveillance functions, such as downregulation of engulfment, accumulation, and phagocytosis of hematopoietic cells. The predicted top upstream genes were IL-10 and immunoglobulins. In silco analysis identified miRNAs predicted to drive the transcriptional signature. Two of the 4 miRNAs (miR-23a-3p and miR-944) were validated in a cohort of women with local and metastatic cervical cancer.ConclusionsThis study supports the use of a cell-based assay that uses PBMC “reporters” to predict biologically relevant factors in patient serum. Further, disease-specific transcriptional signatures induced by patient sera have the potential to differentiate patients with local versus metastatic disease.

A high level of circulating HOTAIR is associated with progression and poor prognosis of cervical cancer.

We recently found that HOTAIR (HOX antisense intergenic RNA) promotes development and induces radioresistance in cervical cancer. In the present study, we investigated the circulating HOTAIR expression and determined its relationships with the clinicopathological parameters in cervical cancer. The sera samples were obtained from 118 pathological diagnosed cervical cancer patients and 100 normal age-matched women. The expression of HOTAIR was measured by quantitative real time PCR. Patients' information were collected and analyzed by the SPSS 17.0 software. Compared with normal control, the expression of HOTAIR was significantly upregulated in the sera of cervical cancer patients (P < 0.0001). In addition, elevated HOTAIR was associated with advanced tumor stages (P < 0.0001), adenocarcinoma (P < 0.0001), lymphatic vascular space invasion (P = 0.0065), and lymphatic node metastasis (P = 0.0259). In addition, our follow-up data showed that high HOTAIR was notably correlated with tumor recurrence (P = 0.013) and short overall survival (P = 0.009). Circulating HOTAIR was commonly upregulated and a potent prognostic marker in cervical cancer.

Relevance of activated leukocyte cell adhesion molecule (ALCAM) in tumor tissue and sera of cervical cancer patients

BackgroundAn altered expression of the activated leukocyte cell adhesion molecule (ALCAM) is associated with cancer progression in various cancer types. In some cancers ALCAM has a prognostic value or is predictive for the benefit of therapeutic interventions. To date there are no data on the role of ALCAM in cervical cancer available.MethodsIn this study, ALCAM expression was analysed by immunohistochemistry (IHC) in tissue samples of 233 patients with cervical cancer, among them 178 with complete follow-up information. In addition, soluble (s-)ALCAM was measured in sera of a subset of the included patients (n = 55) by enzyme-linked immunosorbent assay (ELISA).ResultsALCAM overexpression was detected (immunoreactive score (IRS) 2-12) in 58.4% of the cervical cancer samples. The normal ectocervical or endocervical epithelium showed no ALCAM reactivity. In untreated patients, ALCAM overexpression in tumor tissue tended to be associated with shorter cancer-specific survival (CSS) and disease-free survival (DFS). Patients, whose tumor samples showed ALCAM overexpression receiving a cytotoxic therapy like radiotherapy or chemoradiation, however, had a favourable prognosis compared to those patients, whose cancers showed no or minimal ALCAM staining. This effect was particularly apparent in patients receiving chemoradiation where the CSS was significantly longer in patients with ALCAM-positive tumors (p = 0.038; cumulative incidence rates at 96 months 8%, 95% CI 0%-23%, and 26%, CI 3%-43% in ALCAM-positive and ALCAM-negative cases, respectively).Median preoperative s-ALCAM concentration in sera from tumor patients was 27.6 ng/ml (range 17.5-55.1 ng/ml, mean 28.9 ng/ml), serum levels did not correlate with intratumoral ALCAM expression.ConclusionsThe data of our retrospective study suggest that the prognostic value of ALCAM expression in cervical carcinoma might be therapy-dependent, and that ALCAM might function as a predictive marker for the response to chemoradiation. This should be confirmed in further, prospective studies.

Abstract 2344: Soluble MICA in cervical cancer is in a deglycosylated form: Implication for soluble MICA detection

Abstract Background: Major histocompatibility complex class I chain related A (MICA/PERB11.1) molecules are expressed as transmembrane proteins. They are ligands recognized by the natural killer cell receptor group (NKG)2D, an activating receptor on NK cells, gamma delta T cells and activated CD8 T cells. Up-regulated MICA expression was reported in many tumors. Shedding of MICA from tumor cells as soluble MICA (sMICA) is a mechanism for immune evasion. MICA proteins could be presented in various sizes from 43 to 75 kDa depending on degree of glycosylation. Eight potential N-glycosylation sites in a MICA molecule were reported. This study aimed to investigate the size of sMICA in sera of cervical cancer patients and women blood donors by western blot technique. Methods: The sMICA*009 plasmid DNA was transfected to COS7 cells for recombinant sMICA protein production in culture supernatant. The recombinant sMICA protein was harvested and validated with mouse monoclonal anti-MICA antibody (WW6B7) by indirect ELISA and immunobloting. Molecular weight of deglycosylated recombinant sMICA protein treated with N-glycosidase F enzyme and sMICA in cervical cancer and healthy sera was determined. Results: The recombinant sMICA and serum sMICA showed 62 kDa and 45 kDa in molecular weight, respectively. The molecular weight of recombinant sMICA treated with N-glycosidase F enzyme was reduced to 45 kDa which equal to serum sMICA indicating that sMICA in sera of patients and blood donor controls was in a deglycosylated form. Conclusion: Recombinant sMICA produced in COS7 had a molecular weight of 62 kDa. In contrast, sMICA in sera was in a deglycosylated form and had a molecular weight of 45 kDa equal to in vitro deglycosylation with N-glycosidase F enzyme of produced recombinant sMICA. The mechanism and kinetic of deglycosylation of sMICA in sera await further investigation. It would be interesting to see whether this form of sMICA would affect sMICA detection in sera. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2344.

DNA methylation in serum and tumors of cervical cancer patients.

Promoter hypermethylation has been recognized to play an important role in carcinogenesis. Numerous studies have demonstrated tumor-specific alterations, such as aberrant promoter hypermethylation, in DNA recovered from plasma or serum of patients with various malignancies. The aim of this study was to investigate the methylation status of various genes in cervical cancer patients and their association with clinicopathological characteristics and outcome of the disease. The methylation status of CALCA, hTERT, MYOD1, PGR (progesterone receptor), and TIMP3 was investigated in serum samples from 93 cervical cancer patients and 19 corresponding tissue samples using the MethyLight technique. Aberrant promoter hypermethylation was detected in any of these genes in 87% (81 of 93) of the serum samples studied. Methylation of MYOD1 was detected more frequently in advanced stage. All of the genes found to be methylated in serum samples were also methylated in the corresponding tissue sample, except in one patient. Patients with unmethylated MYOD1 serum DNA had significantly better disease-free (P = 0.04) and overall survival (P = 0.02) in comparison with patients with methylated MYOD1. To the best of our knowledge, this is, thus far, the largest study investigating aberrant promoter hypermethylation in serum samples from cancer patients and the first study investigating methylation patterns in sera of cervical cancer patients. Our results suggest that serological detection of MYOD1 promoter hypermethylation may be of potential use as a prognostic marker for discriminating cervical cancer patients at high risk for lymph node metastasis or relapse. Additional studies, including a panel of additional genes, are necessary to elucidate the role of aberrant methylation in serum as a tool for surveillance of cervical cancer.

Human papillomavirus DNA in sera of cervical cancer patients as tumor marker

Persistent infection with human papillomavirus (HPV) has been widely recognized to induce carcinoma of the uterine cervix. Viral DNA has been documented to occur as tumor DNA in the circulation of patients with primary tumors caused by viral infection. The aim of this study was to evaluate the utility of serum HPV DNA as tumor marker in cervical cancer patients. Sera taken from 94 cervical cancer patients at the date of diagnosis and follow-up serum samples from 24 patients were examined for HPV DNA using PCR enzyme immunoassay. Serum samples taken at the date of diagnosis were HPV DNA positive in 45% of all investigated samples. Sera which were HPV DNA positive at the time of diagnosis became and also remained negative after primary treatment in patients without recurrence or persistent disease. HPV DNA was positive up to 423 days before the time of clinical diagnosis of recurrence in 10 out of 13 cases (median 72 days, range 0–423 days). Serum HPV DNA seems to reflect biological activity of the tumor. Our results indicate that serum HPV DNA might be a useful additional marker for early detection of recurrence in cervical cancer patients.

Low Incidence of HPV DNA in Sera of Pretreatment Cervical Cancer Patients

Objective. The aim of this study was to investigate the feasibility of using DNA in the circulation as a diagnostic tool for cervical cancer.Methods. We used PCR followed by Southern hybridization to detect human papillomavirus (HPV) DNA in serum samples taken from patients of cervical cancer before treatment.Results. A total of 60 samples were analyzed. In a set of 40 samples, without knowledge of the HPV DNA status in the corresponding cervical carcinomas, we could detect 8 (20%) positive samples, of which 7 (17.5%) were HPV 16 and 1 (2.5%) was HPV 18. In another set of 20 samples, known to be HPV 16 infected in the corresponding cervical carcinomas, we detected only 4 (20%) HPV-16-positive samples. The occurrence of HPV DNA in sera of cervical cancer patients seems sporadic.Conclusion. The low incidence indicates that serum HPV DNA has limited application in the diagnosis of cervical cancer.

Antibodies prevalence against HPV-6b and -16 recombinant fusion proteins in Korean patients with cervical neoplasia.

This study was to investigate the adjunctive diagnostic utility of HPV fusion proteins from the sera in the patients with cervical neoplasia. Immunologic researches on host factors in HPV infection could contribute to better understanding of patho-physiologic mechanisms of cervical carcinogenesis, and provide valuable information for HPV vaccine development. Prevalence of antibodies against human papillomavirus (HPV) in sera of the patients with carcinoma (n = 81), precancers (n = 25) of uterine cervix and normal controls (n = 40) in Korean women were investigated by Western blot immunoassay using partially purified TrpE fusion recombinant proteins for HPV-6b and HPV-16. In 81 patients with cervical cancer, 15 (19%) and 54 (67%) patients were positive for antibodies to at least one of the tested recombinant proteins related to HPV-6b or HPV-16, respectively. And in 25 patients with HPV-related squamous intraepithelial lesion (SIL) of cervix, 10 were positive for at least one of the tested antibodies for HPV-6b gene products (40%) and 11 were positive for HPV-16 related antibodies (44%). The prevalence rates of antibodies for HPV-6b in the sera of SIL patients were significantly higher than those from the sera of cervical cancer patients (p < 0.05). There was an inverse correlation between increasing the positivity against HPV-16 E6 protein and progression of cervical lesions, but there was no significant correlation between the positivities of the other antibodies to HPV-16 and the severity of disease. These results suggest that the recombinant proteins related to HPV-6b and HPV-16 could be useful in evaluation of the patients with HPV-related cervical lesions and these might play an adjunctive role in diagnosis and management of cervical neoplasia.

Antibodies against linear and conformational epitopes of the human papillomavirus (HPV) type 16 E6 and E7 oncoproteins in sera of cervical cancer patients.

Sera obtained from 137 cervical cancer patients were analysed for the presence of antibodies to the human papillomavirus (HPV) type 16 proteins E6 and E7 by the aid of different assays, i.e. ELISA using as antigen either synthetic peptides or the complete E7 protein and radio-immunoprecipitation (RIPA) which uses the viral protein made by in vitro transcription/translation. In agreement with previous reports, reactivity to the E7 protein was found more frequently than to the E6 protein (31.4% vs. 16.8%) when the sera were assayed by peptide-based ELISA. In contrast, when RIPA was employed, reactivity to either protein was obtained at similar frequency (38.7% vs 46.7%). When the protein was denatured prior to immuno-precipitation the reactivity was lost in all sera tested for E6-specific antibodies but only in a few samples in the E7-RIPA. Therefore it was concluded that the increased sensitivity of the E6-RIPA as compared to the E6 peptide-ELISA is due to the detection of antibodies to conformational epitopes which are presented by the in vitro product but not by the synthetic peptides. Eighty-two sera from healthy donors were tested by HPV 16E6- and E7-RIPA and also by ELISA using the HPV 16E7 protein which was produced in the fission yeast Schizosaccharomyces pombe. One sample reacted each in the E6- and E7-RIPA indicating a high specificity of these assays. The E7 protein-ELISA proved to be less sensitive for the detection of antibodies in cervical cancer patients' sera (22.6% positive) as compared to peptide-based ELISA or RIPA.