Blood Of Cervical Cancer Patients Research Articles

The association between ambient PM2.5's constituents exposure and cervical cancer survival

Increasing evidence links exposure to ambient particulate matter with a diameter less than 2.5 μm (PM2.5) with reduced survival in cancer survivors, but little was known about the association between PM2.5 exposure and cervical cancer survival. We analyzed data from 5144 cervical cancer patients diagnosed between January 2014 and December 2020, who completed recommended treatments. Exposure levels were determined by the monthly average concentration of ambient PM2.5 and its five constituents, obtained from Tracking Air Pollution in China (TAP) based on individual residential addresses. Log-rank tests and multivariate Cox Proportional Hazardous regression were performed to examine the impacts of PM2.5 and its constituents on overall survival (OS) of cervical cancer patients. We observed that for every increase of 1 μg/m3 in average individual exposure, the hazard ratios (95%CI) for ambient PM2.5, sulfate (SO42−), ammonium (NH4+), and nitrates (NO3−) were 1.078(1.069–1.086), 6.755(5.707–7.996), 2.123(1.935–2.329), and 3.717(3.237–4.267), respectively. Subgroups with longer OS had larger HRs of PM2.5 and its constituents, which might attributed to more cumulative exposure. No evidence of a threshold for the hazardous effects of PM2.5 on the OS of cervical cancer patients was identified. Furthermore, long-term exposure to PM2.5 was negatively associated with pretreatment counts of monocytes, neutrophils, and lymphocytes in peripheral blood of cervical cancer patients. In conclusion, elevated levels of PM2.5 mass, SO42−, NH4+, and NO3− in ambient PM2.5 exposure were associated with reduced OS among cervical cancer patients. There may be no discernible threshold effect of PM2.5 on the risk for cervical cancer patients.

Changes in T lymphocyte subsets in peripheral blood of patients with middle-advanced cervical cancer before and after nimotuzumab combined with concurrent chemoradiotherapy

The current study sought to investigate the effect of nimotuzumab combined with concurrent chemoradiotherapy (CCRT + Nim) on T lymphocyte subsets in middle-advanced CC. Firstly, patients with middle-advanced CC were administered CCRT or CCRT + Nim. Next, levels of T lymphocytes in peripheral blood of CC patients pre- or post-treatment and healthy females were determined by flow cytometry. The short-term efficacy was evaluated, and overall survival (OS) and progression-free survival (PFS) of patients were recorded. In addition, the correlation of T lymphocyte subsets post-treatment with OS/PFS was assessed with Pearson analysis. CC patients exhibited decreased total T cells/T helper cells/CD4+/CD8+ ratio and increased T suppressor cells/Tregs in peripheral blood. Meanwhile, CCRT and CCRT + Nim improved T lymphocyte subset imbalance, with CCRT + Nim exhibiting better efficacy. CCRT + Nim exhibited better short-term efficacy and higher PFS than CCRT, with no evident difference in OS. The levels of total T cells/T helper cells/T suppressor cells/Tregs were not significantly-correlated with OS/PFS, and the CD4+/CD8+ ratio was correlated with PFS but not OS. Collectively, CCRT + nimotuzumab ameliorate the imbalance of T lymphocyte subsets in peripheral blood of middle-advanced CC patients, and the CD4+/CD8+ ratio after therapy is correlated with PFS. IMPACT STATEMENT What is already known on this subject? The utilisation of Nimotuzumab targeting epidermal growth factor receptor (EGFR) combined with concurrent chemoradiotherapy (CCRT) as an efficient treatment for middle-advanced cervical cancer (CC) has garnered the attention of numerous researchers over the years. T cells represent a major immune cell type in the tumour microenvironment and serve as the basis for maintaining cellular immune functions. What do the results of this study add? Our findings revealed that nimotuzumab combined with CCRT improves the abnormality of T lymphocyte subsets in peripheral blood of patients with middle-advanced CC, such that the CD4+/CD8+ ratio after treatment was significantly correlated with progression-free survival (PFS). What are the implications of these findings for clinical practice and/or further research? CCRT of CC may have a short-term negative impact on the peripheral T-cell immune micro-environment, and the combination of nimotuzumab, cisplatin-based chemotherapy, and radiotherapy enhances the frequency of Tregs in peripheral blood. Our findings illustrated that nimotuzumab combined with CCRT can improve the imbalance of T lymphocyte subsets in peripheral blood of patients with middle-advanced CC. A better understanding of the mechanisms of these therapies will optimise the selection of patients most likely to benefit from treatment, serving as a reference for further research on the relationship between EGFR-specific T cells and clinical benefit in patients treated with nimotuzumab in combination with CCRT.

Extracellular vesicles mediated proinflammatory macrophage phenotype induced by radiotherapy in cervical cancer

BackgroundRadiotherapy is a highly effective treatment for cervical cancer. Recent studies focused on the radiotherapy induced anti-tumor immunity. Whether tumor-derived extracellular vesicles (EVs) play roles in radiotherapy induced tumor associated macrophage (TAM) polarization remains unclear.Materials and MethodsThis study analysed the phenotype of macrophages in cancer tissue and peripheral blood of cervical cancer patients using flow cytometry analysis. The role of EVs from plasma of post-irradiated patients on M2-like transformed macrophages was assessed. The M1- and M2-like macrophages were assessed by expression of cell surface markers (CCR7, CD163) and intracellular cytokines (IL-10, TNFα and iNOS). The capacity of phagocytosis was assessed by PD-1 expression and phagocytosis of pHrodo Red E. coli bioparticles.ResultsOur results demonstrated that radiotherapy of cervical cancer induced an increase in the number of TAMs and a change in their subtype from the M2-like to the M1-like phenotype (increased expression of CCR7 and decreased expression of CD163). The EVs from plasma of post-irradiated patients facilitated the M2-like to the M1-like phenotype transition (increased expression of CCR7, TNFα and iNOS, and decreased expression of CD163 and IL-10) and increased capacity of phagocytosis (decreased PD-1 expression and increased phagocytosis of pHrodo Red E. coli bioparticles).ConclusionsOur data demonstrated that irradiation in cervical cancer patients facilitated a proinflammatory macrophage phenotype which could eventually able to mediate anti-tumor immune responses. Our findings highlight the importance of EV in the crosstalk of tumor cells and TAM upon irradiation, which potentially leading to an increased inflammatory response to cancer lesions.

Predicting 3D Structure, Cross Talks, and Prognostic Significance of KLF9 in Cervical Cancer.

Our study aimed to identify the new blood-based biomarkers for the diagnosis and prognosis of cervical cancer. Moreover, the three-dimensional (3D) structure of Kruppel-like factor 9 (KLF9) was also determined in order to better understand its function, and a signaling pathway was constructed to identity its upstream and downstream targets. In the current study, the co-expressions of tumor protein D52 (TPD52), KLF9, microRNA 223 (miR-223), and protein kinase C epsilon (PKCϵ) were evaluated in cervical cancer patients and a possible relation with disease outcome was revealed. The expressions of TPD52, KLF9, miR-223, and PKCϵ were studied in the blood of 100 cervical cancer patients and 100 healthy controls using real-time PCR. The 3D structure of KLF9 was determined through homology modeling via the SWISS-MODEL and assessed using the Ramachandran plot. The predicted 3D structure of KLF9 had a similarity index of 62% with its template (KLF4) with no bad bonds in it. In order to construct a genetic pathway, depicting the crosstalk between understudied genes, STRING analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG), and DAVID software were used. The constructed genetic pathway showed that all the understudied genes are linked to each other and involved in the PI3K/Akt signaling pathway. There was a 23-fold increase in TPD52 expression, a 2-fold increase in miR-223 expression, a 0.14-fold decrease in KLF9 expression, and a 0.05-fold decrease of PKCϵ expression in cervical cancer. In the present study, we observed an association of the expressions of TPD52, KLF9, miR-223, and PKCϵ with tumor stage, metastasis, and treatment status of cervical cancer patients. Elevated expressions of TPD52 and miR-223 and reduced expressions of KLF9 and PKCϵ in peripheral blood of cervical cancer patients may serve as predictors of disease diagnosis and prognosis. Nevertheless, further in vitro and tissue-level studies are required to strengthen their role as potential diagnostic and prognostic biomarkers.

Dynamics of oxidative blood status and activity of antioxidant system of erythrocytes in patients with cervical cancer receiving combination therapy with dendritic cell vaccine.

e17516 Background: The combination treatment with plasmapheresis and non-specific immunotherapy for locally advanced cervical cancer (CC) is shown to be effective. We studied the effect of immunotherapy with dendritic cell vaccine (DCV) as a part of combination therapy on the intensity of the lipid peroxidation (LPO) and activity of antioxidant enzymes. Methods: Plasma and erythrocyte content of malondialdehyde (MDA), the activity of superoxide dismutase (SOD), catalase, glutathione dependent enzymes and the content of reduced glutathione (GSH) in erythrocytes were determined in 24 patients with CC of T2a-2bN0-1M0 stages and in 25 women without cancer. The patients underwent neoadjuvant chemotherapy (CT) with cisplatin and bleomycin, followed by 2 cycles of DCV, for a total of 6 courses of CT and 12 cycles of DCV. Results: Initially, patients demonstrated intensification of free radical processes enhancing after PCT and the first two DCV: MDA in erythrocytes elevated by 52.2-111.7%, in the blood plasma by 2.2-3.5 times. Subsequent DCV cycles normalized the LPO. MDA levels after 4 DCV cycles were lower than the initial values in erythrocytes by 30.4% (p = 0.04), in plasma by 55.6% (p = 0.007), i.e. were similar to the levels in healthy women. The SOD activity was decreased by 16.7-27.3% (p < 0.005) after 4-6 DCV cycles and normalized after the 7th cycle. The catalase activity in erythrocytes was decreased before treatment and after PCT by 42.4% (p = 0.000000), during vaccine therapy by 20.5-37.7% (p < 0.0005). In reduced catalase activity, inactivation of hydrogen peroxide was realized, apparently, by glutathione peroxidase (GPO), and its increased activity (by 37-102.3%, p≤0.00005) was supported by a high GSH content - by 11.6-52.4% (p < 0.02) above the norm at all stages after the 2nd DCV cycle. And only with the complete normalization of the erythrocyte MDA content, the GPO activity after the 7th DCV cycle decreased by 46.7% (p = 0.000000) relative to the levels before the treatment and did not differ significantly from the values in donors. Conclusions: Vaccine therapy helped to enhance the activity of the glutathione system becoming the most important component of antioxidant protection in the blood of CC patients. Already 3-4 DCV cycles normalized the LPO sharply increased in CC patients during CT. Multi-course vaccine therapy allowed maintaining the optimal oxidative status of the blood.

Aldehyde dehydrogenase 1, a target of miR-222, is expressed at elevated levels in cervical cancer.

The aim of the present study was to investigate the expression of microRNA-222 (miR-222) and aldehyde dehydrogenase 1 (ALDH1) in tissues and peripheral blood of cervical cancer patients, and to elucidate their underlying mechanisms of action. Tumor tissues and tumor-adjacent tissues were obtained from 33 cervical cancer patients and peripheral blood was obtained from these patients and 28 healthy subjects. The expression of miR-222 and ALDH1 mRNA was evaluated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). To examine the levels of ALDH1 protein in tissues and blood, western blotting and ELISA were used. To confirm a direct interaction between miR-222 and ALDH1 mRNA, a dual luciferase reporter assay was performed. HeLA cells were transfected with agomiR-222 and expression of ALDH1 in the cells was measured by RT-qPCR and western blotting. MTT assay was preform to investigate the proliferation of HeLA cells. Expression of ALDH1 mRNA and protein was elevated in cervical cancer tissues and peripheral blood from patients compared with tumor-adjacent tissues and healthy controls, while the expression of miR-222 was reduced. Upregulation of miR-222 inhibited HeLA cell proliferation possibly due to a reduction in the expression of ALDH1. A dual luciferase reporter assay showed that miR-222 can bind with the 3′-untranslated seed region of ALDH1 mRNA to regulate its expression. miR-222 regulation of ALDH1 expression may play a role in the prevention of cervical cancer.

Human papilloma virus E1-specific T cell immune response is associated with the prognosis of cervical cancer patients with squamous cell carcinoma

BackgroundCervical cancer is attributable to human papilloma virus (HPV) infection in the majority cases. E1, an HPV derived-protein, plays an important role in the initiation and development of cervical cancer. Our study aims to investigate the HPV E1-specific T cell response in patients with cervical squamous cell carcinoma (CSCC).MethodsA total of 66 CSCC patients with FIGO stage IIB-IIIB and 60 healthy controls were enrolled. Enzyme-Linked ImmunoSpot (ELISPOT) assays was used to measure the HPV E1-specific T cell response in the peripheral blood of these patients before treatment. The patients were treated with chemotherapy and/or radiotherapy and followed up clinically for three years. The relationship between the T cell response, various clinical characteristics and the prognosis were studied with univariate analysis, multivariate analysis and survival curve analysis.ResultsThe frequency of HPV E1-specific T cell response in peripheral blood of cervical cancer patients was 59.09%, with mean response intensity 24.56 SFC/106 PBMCs. The frequency and intensity of HPV E1-specific T cell response in patients were higher than healthy controls(p < 0.001; p = 0.009). The intensity of HPV E1-specific T cell responses were higher in the stage IIB patients and patients with no pelvic lymph node metastasis (p = 0.038; p = 0.044). Univariate analysis showed that HPV E1 specific T cell response was associated with progression-free survival (PFS) and overall survival (OS) (PFS: p = 0.021; OS: p = 0.004). Multivariate analysis showed that HPV E1-specific T cell response was an independent prognostic factor influencing PFS and OS among all the factors included in our study (PFS: HR = 7.252, 95%CI = 1.690–31.126, p = 0.008; OS: HR = 7.499, 95%CI = 1.661–33.856, p = 0.009). The survival curves showed that the rate of PFS and OS in patients with HPV E1 specific T cell response was significantly higher than those who did not response.ConclusionsOur study demonstrated that the level of HPV E1-specific T cell response was correlated with the survival of advanced patients with CSCC. Patients who displayed no HPV E1-specific T cell response were more likely to be those with poor prognosis.

Let‑7a suppresses cell proliferation via the TGF‑β/SMAD signaling pathway in cervical cancer.

Cervical cancer is the second most commonly diagnosed type of cancer among women after breast cancer. Recent research has addressed the role of microRNAs in cervical cancer. In the present study, we aimed to determine the effect of let‑7a on the regulation of the cell proliferation of cervical cancer and the related signaling pathway. Real‑time RT‑PCR was used to detect the expression of let‑7a in the blood of cervical cancer patients and normal controls. The expression of let‑7a was also assessed in cervical cancer cell lines: HeLa, SiHa and normal human immortalized keratinocyes HaCaT. Cell proliferation was tested by MTT assay, and cell apoptosis and cell cycle were examined by flow cytometric analysis in HeLa cells. Moreover, bioinformatic analysis, dual‑luciferase reporter assay and western blotting were used to confirm the target gene for let‑7a. In addition, the expression of TGF‑β1, SMAD4 and p53 were assessed by western blotting and real‑time PCR. Our studies showed that the expression of let‑7a in cervical cancer was significantly reduced in cervical cancer patients compared with the expression in the normal control group. Cell proliferation of HeLa cells was inhibited by overexpression of let‑7a. The cell cycle analysis showed that an increased population was arrested in the G2phase in the let‑7a mimic group when compared with that in the mimic control and untreated groups. In addition, the cell cycle‑related factor p53 was increased in the let‑7a overexpression group compared with that in the control and untreated groups. Furthermore, TGFBR1 was confirmed to be a target of let‑7a. Moreover, the expression of TGF‑β1 and SMAD4 proteins was elevated in cervical squamous carcinoma and cervical adenocarcinoma tissues. However, the expression of TGF‑β1 and SMAD4 was decreased in the let‑7a‑overexpressing cervical cancer cell lines (HeLa, SiHa and CaSki). Our data suggest that let‑7a may play a role in the cell proliferation of cervical cancer by regulating the TGF‑β/SMAD pathway, and may participate in the regulation of the occurrence and development of cervical cancer.

Profiling analysis of circulating microRNA expression in cervical cancer.

MicroRNA (miRNA) expression is altered in cancer cells and is associated with the development and progression of various types of cancer. Accordingly, miRNAs may serve as diagnostic or prognostic biomarkers in cancer patients. In this study, we attempted to analyze circulating exosomal miRNA in patients with cervical cancer. Total RNA was extracted from the serum of healthy subjects, subjects with cervical intraepithelial neoplasia (CIN) and patients with cervical cancer. We first investigated miRNA expression profiles in 6 serum samples from healthy subjects and patients with cervical cancer using the miRCURY LNA microRNA array. miRNAs with significant differences in expression were validated in a larger sample set by quantitative reverse transcription-polymerase chain reaction, using TaqMan gene expression assays. The results of the miRCURY LNA microRNA array indicated that 6 of 1,223 miRNAs found in serum samples from cervical cancer patients and normal controls exhibited a >3.0-fold change in expression level in subjects with cervical cancer, with a P-value of <0.01. In a validation set (n=131) that investigated the expression of 4 of the 6 miRNAs (miR-483-5p, miR-1246, miR-1275 and miR-1290), miR-1290 was found to have significantly higher expression levels in cervical cancer samples (n=45) compared with control samples (n=31). We also found that the median levels of these miRNAs were significantly higher in subjects with cervical cancer (n=45) compared with those in subjects with CIN (n=55). Circulating miRNAs were not correlated with clinicopathological parameters. However, receiver operating characteristic curve analysis suggested that these serum miRNAs may be useful diagnostic markers in cervical cancer. The expression of circulating miR-1290 was significantly higher in the blood of cervical cancer patients compared with that in controls and may thus serve as a useful biomarker in cervical cancer diagnosis. However, larger studies are required to fully elucidate the role of circulating exosomal miRNAs in cervical cancer.

Elevated Expression of Programmed Death-1 and Programmed Death Ligand-1 Negatively Regulates Immune Response against Cervical Cancer Cells

The present study is to measure the expression of programmed death-1 (PD-1) and programmed death ligand-1 (PD-L1), as well as its clinical significance in cervical cancer patients. Our results showed that different T cell subsets in patients with cervical cancer had high expression of PD-1, and DCs had high expression of PD-L1. High expression of PD-1 on Treg cells in cervical cancer patients facilitated the production of TGF-β and IL-10 but inhibited the production of IFN-γ. Cervical cancer elevated the expression of PD-1 and PD-L1 in mRNA level. PD-1 expression in peripheral blood of cervical cancer patients was related with tumor differentiation, lymph node metastasis, and invasiveness. PD-1/PD-L1 pathway inhibited lymphocyte proliferation but enhanced the secretion of IL-10 and TGF-β in vitro. In summary, our findings demonstrate that elevated levels of PD-1/PD-L1, TGF-β, and IL-10 in peripheral blood of cervical cancer patients may negatively regulate immune response against cervical cancer cells and contribute to the progression of cervical cancer. Therefore, PD-1/PD-L1 pathway may become an immunotherapy target in the future.

The Th17/Treg balance and the expression of related cytokines in Uygur cervical cancer patients

BackgroundThe fine balance of Th17/Treg is crucial for maintenance of immune homeostasis. The objective of this study was to investigate the balance of Th17/Treg and the expression of related cytokines in Uighur cervical cancer patients.MethodsPeripheral blood was collected from 65 cases of cervical cancer patients, 42 cases of cervical CIN patients and 40 healthy people. Flow cytometry was used to detect the percentages of T cell subsets, including CD3+ T cells, CD4+ T cells, CD8+ T cells, Treg cells and Th17 cells. ELISA assay was conducted to detect expression levels of TGF-β, IL-6, IL-10, IL-17, IL-23 and IFN-γ.ResultsThere were no significant difference in the levels of CD3+ T cells, CD4+ T cells, CD8+ T cells, and the ratio of CD4+/CD8+ among the cervical cancer group, the CIN group and the healthy control group. However, compared with the healthy control group, the percentages of CD4+ CD25+ Treg, CD4+CD25+CD127- Treg, CD4+IL17+ Th17, CD4+CD25+Foxp3+, CD4+CD25- Foxp3+, CD8+CD25+CD127-Treg and CD8+CD25+Foxp3 were significantly higher in the cervical cancer group and the CIN group. Similar results were also found in the Th17/Treg ratio and the related cytokines. There was no significant difference between the cervical cancer group and the CIN group. Additionally, Th17 cell levels were positively correlated with IL-6, IL-23 and IL-17. Also, Treg cell levels were positively correlated with TGF-β, IL-10 and IL-6. Contrarily, Treg cell levels and IFN-γ were negatively correlated.ConclusionsOur data indicated that the Th17/Treg balance was broken in peripheral blood of cervical cancer patients. Analysis of Th17/Treg balance may have a significant implication in diagnosing cervical cancer.Virtual slidesThe virtual slide for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1813823795931511

Exosomal-miRNA profiles as diagnostic biomarkers in cervical cancer.

10557 Background: MicroRNA (miRNA) expression is altered in cancer cells and associated with the development and progression of various types of cancer. miRNA could serve as diagnostic or prognostic biomarker for cancer patients. Our study was designed to analyze circulating exosomal-miRNA in patients with cervical cancer. Methods: Total RNA was extracted from serum in healthy women and patients with cervical intraepithelial neoplasia (CIN) and cervical cancer. We first explored miRNA expression profiles using miRCURY LNA microRNA Array (Exiqon) in each six malignant and healthy serum samples. miRNAs with significant differences in expression were validated in larger sample sets by a quantitative real-time RT-PCR using TaqMan gene expression assays (Applied Biosystems). Results: Six of 1223 miRNAs compared in serum samples from cervical cancer and normal control were found to have a &gt;3.0-fold change with a P value &lt;0.01 using miRCURY LNA microRNA Array. In a validation set (n=101), expression of 3 of the 6 miRNAs, miR-483-5p, miR-1246 and miR-1275, was significantly different between the cervical cancer (n=40) and control (n=20) samples. We also found that the median levels of these miRNAs were significantly higher in patients with cervical cancer (n=40) than those in CIN (n=41). Circulating miRNAs were not correlated with clinical-pathological parameters except for miR-1246. Compared to patients with squamous cell carcinoma, the miR-1246 level was significantly higher in those with adenocarcinoma. Whereas, receiver-operator characteristic (ROC) curve analyses suggest that these serum miRNAs may be useful markers for discriminating patients with cervical cancer from healthy controls. Conclusions: Expression of circulating miR-483-5p, miR-1246 and miR-1275 is significantly higher in the blood of cervical cancer patients compared to controls and may potentially serve as a useful biomarker for cervical cancer diagnosis. Especially, miR-1246 may be a candidate for early detection of cervical adenocarcinoma. Larger-scaled studies are warranted to fully explore the role of circulating exosomal-miRNAs in cervical cancer.

Lack of HPV 16 and 18 detection in serum of colposcopy clinic patients

Background Persistent infection with high-risk human papillomavirus (HPV) types is necessary for the development of high-grade cervical dysplasia and cervical carcinoma. The presence of HPV DNA in the blood of cervical cancer patients has been reported; however, whether HPV DNA is detectable in the blood of patients with pre-invasive cervical disease is unclear. Objectives The objectives of this study were to determine if HPV 16 and HPV 18 DNA could be detected in the serum of colposcopy clinic patients, and if serum HPV detection was associated with grade of cervical disease and HPV cofactors. Study design Samples were selected from a biorepository collected from non-pregnant, HIV-negative women ages 18–69 attending colposcopy clinics at two urban public hospitals. Cervical disease status was based on review of colposcopy, biopsy and cytology findings. Serum HPV DNA detection was conducted using a novel PCR and mass spectroscopy-based assay. Results Of the 116 adequate serum samples, all (100%) were negative for HPV 16 and HPV 18. Over half (51.7%) of participants had cervical HPV 16 and/or HPV 18 infection. Nearly one-third (31.1%) had high grade, 10.3% had low grade, and 50.9% had no cervical disease. Nearly one-third (28.5%) had ever regularly smoked cigarettes, 70.7% had early onset of sexual intercourse, and 75% had ever used oral contraceptives. Conclusions In this colposcopy clinic population with a range of clinical characteristics and established HPV cofactors, HPV DNA was undetectable in their serum. Our findings suggest that serum HPV DNA detection is not a cervical cancer screening tool.

Increase of human papillomavirus‐16 E7‐specific T helper type 1 response in peripheral blood of cervical cancer patients after radiotherapy

It has been suggested that tumour cell lysis by gamma-radiation induces a tumoral antigen release eliciting an immune response. It is not clear how a specific immune response in cervical cancer patients is developed after radiotherapy. This study is an attempt to investigate the role of the human papillomavirus type 16 (HPV-16) E7-specific T helper response before and after radiotherapy. Lymphocytes were isolated from 32 cervical cancer patients before and after radiotherapy and from 16 healthy women. They were stimulated for 12 hr with autologous HPV-16 E7-pulsed monocyte-derived dendritic cells or directly with HPV-16 E7 synthetic peptides: E7(51-70), E7(65-84) and E7(79-98). The cells were stained for CD4, CD69, intracellular interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) cytokines and analysed by flow cytometry. A specific CD4(+) CD69(+) IFN-gamma(+) immune response against HPV-16 E7(79-98) peptide was observed in 10 of 14 patients (71.4%) after treatment, compared with 4 of 14 (28.5%) before radiotherapy (P = 0.039); however, this response was not associated with a successful clinical response. Before treatment, 5 of 31 patients showed a HPV-16 E7(79-98)-specific T helper type 2 (Th2) response. Interestingly, this response was significantly associated with a decrease in disease-free survival (P = 0.027). These results suggest that a Th2-type cellular response could be useful as a predictor of recurrence and poor prognosis. An increase of the HPV-specific immune response was observed after radiotherapy; however, it is not enough to control completely the disease after treatment. Our results support that the E7-specific T-cell IFN-gamma response in cervical cancer patients, rather than reflecting the host's capability of controlling tumour growth, might be an indicator for disease severity.

Detection and quantitation of human papillomavirus type 16, 18 and 52 DNA in the peripheral blood of cervical cancer patients

Detection and quantitation of human papillomavirus type 16, 18 and 52 DNA in the peripheral blood of cervical cancer patients

Detection and Quantitation of Human Papillomavirus Type 16, 18 and 52 DNA in the Peripheral Blood of Cervical Cancer Patients

Detection and Quantitation of Human Papillomavirus Type 16, 18 and 52 DNA in the Peripheral Blood of Cervical Cancer Patients

Detection and quantitation of human papillomavirus type 16, 18 and 52 DNA in the peripheral blood of cervical cancer patients

Objective. To prospectively evaluate the feasibility of detecting human papillomavirus (HPV) type 16, 18 and 52 DNA in the peripheral blood of patients with cervical cancer using real-time polymerase chain reaction (PCR) and to determine its prognostic importance. Methods. Blood and cervical swab specimens from 135 consecutive patients with 60 invasive cervical cancers, 10 microinvasions, 20 cervical intraepithelial neoplasias (CIN) III, 10 CIN II, 10 CIN I and 25 controls were collected and examined for HPV type 16, 18 and 52 DNA using real-time PCR to investigate the prevalence and viral load of HPV DNA at the time of diagnosis and during follow-up in patients with positive blood samples. Results. Of the 60 patients with invasive cervical cancer, 27% had positive test results for HPV DNA in blood samples in contrast to 0% of patients with microinvasions, CIN III, CIN II, CIN I and normal controls. The DNA detection rates of viral subtypes in blood samples of cervical cancer patients were 5% for HPV-16, 16.7% for HPV-18, 8.3% for HPV-52, 1.7% for both HPV-16 and HPV-18 and 1.7% for both HPV-18 and HPV-52, while the detection rates in cervical swab specimens were 36.2% for HPV-16, 15.5% for HPV-18 and 17.2% for HPV-52. During follow-up, 8 of 10 cervical cancer patients with viral DNA detected in blood within 3 months after treatment had recurrence, and a high percentage (87.5%, 7/8) of this recurrence involved distant metastases. Conclusions. In this study, real-time PCR detected HPV-16, -18 or -52 DNA in the peripheral blood of more than one-fourth of invasive cervical cancer patients. The association between risk of cancer recurrence and the amount of viral DNA detected in blood among cervical cancer patients after treatment is intriguing and deserves further investigation.

Detection of epidermal growth factor receptor mRNA in peripheral blood of cervical cancer patients

Objective Epidermal growth factor receptor (EGFR) has been reported to be expressed by immunohistochemistry in invasive cervical cancers. We evaluated the feasibility of detecting EGFR mRNA by EGFR-based reverse transcription polymerase chain reaction (RT-PCR) in peripheral blood of patients with cervical cancer. Methods Expression of EGFR mRNA, cytokeratin (CK)-19 mRNA, and CK-20 mRNA was examined by RT-PCR in 12 human cervical cancer cell lines. All 12 cell lines expressed both EGFR mRNA and CK-19 mRNA, but only 4 of 12 (33.3%) cell lines expressed CK-20 mRNA. Peripheral blood samples from 20 healthy donors and 45 cervical cancer patients were also examined. Results In peripheral blood from 20 healthy donors, neither EGFR mRNA nor CK-20 mRNA was expressed, but CK-19 mRNA was expressed in 13 of 20 (65%). In contrast, EGFR mRNA was expressed in 12 of 45 (26.7%) patients with cervical cancer ( P = 0.0071, χ 2 test, patient vs control). On the other hand, expression of EGFR was observed in 98% of tumor tissues by immunohistochemistry. CK-19 mRNA and CK-20 mRNA were found in 35 of 45 (77.8%) and 0 of 45 (0%) patients, respectively (NS, χ 2 test, patient vs control). The rate of detection of EGFR mRNA in peripheral blood correlated with FIGO stage ( P = 0.049). Conclusion Both CK-19 mRNA and CK-20 mRNA showed no diagnostic value as markers of circulating tumor cells in cervical cancers. However, EGFR mRNA in blood might be a useful marker of circulating tumor cells in cervical cancers.

Infiltration of cervical cancer tissue with human papilloma virus-specific cytotoxic T-lymphocytes

CTLs specific for high-risk human papillomaviruses (HPVs) have recently been found in the peripheral blood of cervical cancer patients. Although cell-mediated immunity is thought to be important in the control of HPV infection, the functional relevance and site of activation of HPV-specific CTLs are unclear. We identified HLA-A*0201-restricted HPV-16 E7 peptide-specific CTLs in the peripheral blood (four of five patients), draining lymph nodes (three of four patients) and tumors (one of three patients) of cervical cancer patients. In four of four cancer patients, the frequency of CTLs specific for a recombinant vaccinia virus expressing HPV-16 and -18 E6/E7 gene products was found to be higher in tumors and lymph nodes compared with that of peripheral blood. HPV-specific CTLs were not identified in any of seven healthy controls, but primary responses could be generated by peptide-pulsed dendritic cells (four of four controls). In a non-HLA-A*0201 subject with invasive carcinoma, other HLA alleles also presented HPV antigens. This is the first demonstration that virus-specific CTLs infiltrate the virus-associated tumor, where they may play an important role in restricting disease progression.

Detection of human papillomavirus mRNA and cervical cancer cells in peripheral blood of cervical cancer patients with metastasis.

To determine the presence of cervical cancer cells in circulating peripheral blood of stage IVb cervical cancer patients with metastasis to distant organs. Cervical cancer tissue from 15 stage IVb cervical cancer patients with metastasis were analyzed for the presence of human papillomavirus (HPV) type 16 DNA by nested polymerase chain reaction (PCR). The presence of transcriptional products of the HPV type 16 E6-transforming gene in the peripheral blood of the same 15 cancer patients was analyzed by reverse transcription and PCR. Cervical tissues and peripheral-blood specimens from 12 normal healthy individuals served as controls. Thirteen of 15 (86.7%) cervical cancer tissues from same number of patients were found to contain HPV type 16 DNA. Peripheral-blood specimens from 12 of 13 (92.3%) cervical HPV DNA-positive patients were found to contain HPV-specific mRNA detectable by reverse transcription (RT) and PCR. Cervical tissues from all 12 normal controls were HPV-free. None of the peripheral-blood specimens from two cervical HPV-negative cancer patients and 12 normal controls contained detectable amounts of mRNA of HPV type 16 E6-transforming gene. The most likely source of the HPV-specific mRNA detected in the peripheral blood of cervical cancer patients with metastasis is the cervical cancer cells derived from or shed from the cervix. The presence of HPV E6 mRNAs in peripheral blood may be a sensitive indicator of circulating cervical cancer cells. If PCR positivity is proven to be able to predict disease progression reliably, these findings may have clinical applications in the treatment of cervical and many other cancers.