Construction of Novel Prognostic Nomogram for Mucinous and Signet Ring Cell Colorectal Cancer Patients with a Survival Longer Than 5 Years.

Purpose: Mucinous adenocarcinoma (MA) and signet ring cell carcinoma (SRCC) are aggressive colorectal cancer histological subtypes with dismal prognosis. This study investigated prognostic factors and constructed novel nomograms for MA and SRCC patients who survived for over 5 years to optimize the follow-up regime, especially for early-onset patients. Patients and Methods: Data from the Surveillance, Epidemiology, and End Results (SEER) database registered between 2004 and 2018 were extracted. MA and SRCC patients were divided into two groups with survival time of 5 years as a cut-off point. Prognostic factors for overall survival (OS) and cancer-specific survival (CSS) were determined by Cox regression models, and survival curves were plotted by the Kaplan-Meier method. Results: We identified 8286 MA patients (45.73%) and 551 SRCC patients (20.32%) who survived for over 5 years. Multivariable Cox analyses identified age, tumor location, N stage, metastasis, CEA level, surgery, and lymph nodes dissection as independent risk factors for MACSS. SRCC was more aggressive and only N2 stage (P = 0.011) and metastasis (P = 0.043) were inversely associated with SRCCSS. Furthermore, we observed that small tumor size, well differentiation, and chemotherapy no longer provided survival benefit to ≥5-year survivors. Therefore, we constructed novel nomograms appropriate for MA patients who survived for over 5 years. The consistency indexes for predicting 10-year OS and CSS were respectively 0.717, 0.712 in the training cohort and 0.727, 0.735 in the validation cohort. Conclusion: Our well-calibrated nomograms represent the first clinical prognostic models developed especially for MA patients with a survival longer than 5 years. For both MA and SRCC patients, TNM stage was a stable prognostic factor, while the prognostic values of tumor size, differentiation grade, and chemotherapy changed over time. We are hopeful that our prognostic models will help define personalized follow-up managements to further prolong patient survival.

Serum miR-27a is a biomarker for the prognosis of non-small cell lung cancer patients receiving chemotherapy.

BACKGROUND: Lung cancer has a high incidence and a 5-year survival rate of less than 15%. Non-small cell lung cancer (NSCLC) accounts for approximately 85% of lung cancer cases. Chemotherapy and immunotherapy are the most frequently used alternative treatments for patients with advanced-stage NSCLC in whom surgery failed. Previous studies have suggested that miR-27a is involved in cancer development and progression. The purpose of this study was to investigate the clinical value of miR-27a in the prognosis of NSCLC patients after chemotherapy. METHODS: Flow cytometry was used to detect the apoptosis rate of SPC-A1 cells treated with optical cisplatin at different times. Simultaneously, the expression of miR-27a in supernatants and cells was detected. Fifty-two newly diagnosed NSCLC patients were recruited. All patients received gemcitabine and cisplatin as first-line chemotherapy and docetaxel as second-line chemotherapy. At the end of every chemotherapy cycle, a therapeutic evaluation was performed according to the RECIST criteria. The expression of serum miR-27a was detected in each cycle. RESULTS: After treatment with 2.5 µg/mL cisplatin, the apoptosis rates of SPC-A1 cells were significantly greater than those of the paired untreated control groups at 12, 24, 48 and 72 h. The expression of miR-27a in supernatants and cells was also consistent with the apoptosis rate and changed a time-dependent manner. The chi-square test showed that an increase in miR-27a after chemotherapy was more common in patients who achieved partial response (PR) than in those who achieved no response (NR) (61.5% vs. 30.8%, P=0.026). Kaplan-Meier survival analysis indicated that patients with decreased miR-27a levels had poorer outcomes than those with increased miR-27a levels (P<0.05). Furthermore, dynamic changes in serum miR-27a with a gradual increasing trend during chemotherapy predicted a good prognosis. CONCLUSIONS: Collectively, our results suggest that miR-27a is involved in the apoptosis of lung cancer cells and that serum miR-27a levels are related to the prognosis of NSCLC patients. The expression levels of miR-27a in the serum may be an independent predictor for the prognosis of NSCLC.

Reaction of HIV screening test for a renal cancer patient confirmed to be uninfected with HIV virus: a case report.

Diagnosis of HIV infection and AIDS need to rely on HIV antibody or HIV antigen test internationally, and the test process is divided into preliminary screening test and confirmatory test. The HIV/AIDS screening laboratory of the First Affiliated Hospital of Nanjing Medical University has been using electrochemiluminescence (ECL) to screen the patients. A 50-year-old man with his enhanced computed tomography (CT) scan revealing two soft tissue density images were seen in the left kidney came to the hospital for medical treatment. The patient's postoperative pathological examination revealed (left kidney) renal clear cell carcinoma. The HIV screening tests of the patient several times in perioperative period showed reactivity, but the result of the confirmatory tests of Nanjing Municipal Center for Disease Control and Prevention were negative all the time. False positive HIV results are quite rare in the setting of renal clear cell carcinoma. There must be some substances that can react with the HIV Combi PT designed to detect anti-HIV antibodies of the IgG- and IgM-class as well as HIV p24 antigen in this patient. In conclusion, false positive results of HIV screening test may occur in serum of patients with renal cell carcinoma, and necessary confirmatory tests are needed. When clinicians encounter such problems, confirmatory tests should be conducted according to the guidelines to avoid misdiagnosis.

MAb NJ001 inhibits lung adenocarcinoma invasiveness by directly regulating TIMP-3 promoter activity via FOXP1 binding sites.

BACKGROUND: Previously, we developed a monoclonal antibody (mAb) NJ001 that binds to the antigen SP70 in human non-small cell lung cancer (NSCLC) cells and showed it could inhibit lung adenocarcinoma (AD) growth. Here, we investigated the effect and mechanisms of NJ001 in lung AD metastasis. METHODS: Human lung AD cells (SPC-A1 and A549) were treated with different concentrations of mAb NJ001, and the effects of NJ001 on cell migration and invasive activity were investigated using wound-healing and Matrigel assays, respectively. The molecular mechanism of this inhibition was explored by microarrays, qRT-PCR, western blot, luciferase assays and electrophoretic mobility shift assays (EMSA). RESULTS: MAb NJ001 markedly suppressed lung AD cell migration; and the invasiveness of SPC-A1 and A549 cells treated with mAb NJ001 was diminished by 65%. Tissue inhibitor of matrix metalloproteinase-3 (TIMP-3) was highly expressed in SPC-A1 cells treated with mAb NJ001, whereas knockdown of TIMP-3 by shRNA significantly increased SPC-A1 and A549 invasiveness. MAb NJ001 affects lung AD by inhibiting TIMP-3 through direct transcriptional regulation of FOXP1 binding sites in the TIMP-3 promoter region, as shown in luciferase assays and EMSA. CONCLUSIONS: MAb NJ001 inhibits invasiveness and metastasis in lung AD through the FOXP1 binding sites in the TIMP-3 promoter region. It may have clinical applications in preventing and treating metastatic lung AD.

SP70-Targeted Imaging for the Early Detection of Lung Adenocarcinoma.

NJ001 is a monoclonal antibody that can specifically recognize the SP70 antigen on lung adenocarcinoma cells. The goal of this study was to explore its utility in targeted imaging. Subcutaneous xenograft and orthotopic lung tumor implantation BALB/c mouse models were established. Near-infrared fluorescent CF750-labeled NJ001 was injected into two tumor mouse models. Mice that received orthotopic lung tumor implantation were also injected with NJ001-conjugated nanomagnetic beads intravenously, and then underwent micro-CT scanning. Meanwhile, mice with lung tumor were intravenously injected with normal saline and bare nanomagnetic beads as a control. Fluorescence could be monitored in the mice detected by anti-SP70 fluorescence imaging, which was consistent with tumor burden. Signal intensities detected with SP70-targeted micro-CT scans were greater than those in control mice. More importantly, orthotopic tumor lesions could be found on the fourth week with SP70-targeted imaging, which was 2 weeks earlier than detection in the control. Our results suggest that SP70 is a promising target for molecular imaging, and molecularly targeted imaging with an NJ001-labeled probe could be applied for the early detection of lung adenocarcinoma.

Correction of serum NSE reference intervals includes the unidentified hemolysis sample: 1-year data analysis from healthcare individuals.

BACKGROUND: Reference intervals (RIs) are important for interpretation of laboratory results. Neuron-specific enolase (NSE) can be utilized to aid the diagnosis of various tumors. However, while red blood cells contain NSE αγ-isozymes, unrecognized slight hemolysis will result in increasing of NSE levels in serum. The aim of this study was to correct the NSE RIs from healthcare individuals results which may have unidentified microhemolysis. METHODS: A total of 15 047 healthy individuals undergoing regular health care were recruited to redefine the NSE reference interval according to the CA28-A3 document. Volunteers with NSE level between 16.3 ng/mL and the upper limit of new RIs were performed venipuncture for NSE retest. Simultaneously, serum free hemoglobin (fHb) was performed with o-tolidine test. RESULTS: Reestablishment of NSE RIs is 0-18.9 ng/mL, which is wider than 0-16.3 ng/mL provided by the manufacturer. Seventy-four volunteers with the NSE level between 16.3 and 18.9 ng/mL were performed venipuncture for NSE retest. The ratio of NSE level drop to normal is 85.1% (63/74) in the subsequent results; there are significant differences between the median NSE of two groups (18.15 vs 14.15 ng/mL). Subsequently, the fHb concentration of 22 healthy individuals from 74 individuals was measured; there are significant differences between the median fHb of two groups (58 vs 30 mg/L). CONCLUSIONS: Some specimens with slightly elevated NSE may be attributed to the unrecognized slight hemolysis. The correction RIs may be expected to decrease the abnormal NSE results.

Failure of internal quality control in detecting significant reagent lot shift in serum creatinine measurement.

BACKGROUND: Internal quality control (IQC) in clinical laboratories is carried out to monitor analytical stability. Usually, the satisfactory results of the IQC ensure the acceptability of the examination results. Here, we reported that patients' creatinine results are unreliable, although the internal quality control is satisfactory. METHODS: Creatinine levels were analyzed from two quality control materials and twenty patients' specimens using two different lots of reagents. Lot-to-lot comparison was performed. The daily median values of serum creatinine levels of patients were calculated from the test results recorded in our laboratory information system. RESULTS: Although IQC was consistent, serum creatinine concentrations were higher using lot B (median: 153 µmol/L; interquartile range: 122-522 µmol/L) than using lot A (median: 133 µmol/L; interquartile range: 76-508 µmol/L) for 20 patients (P = .001). The Deming linear regression showed a best fit of y = 0.9394 × x + 45.66. R2  = .8919, and mean percentage difference between two lots was 34%. The new lot was considered unacceptable. Likewise, the median serum creatinine level from the 360 patients using lot B was 102 µmol/L, which was significantly higher than the daily medians of patients using lot A (median: 66 µmol/L; range: 61-70 µmol/L) in the previous month. CONCLUSION: The variations in creatinine concentrations proved to be due to different lots of reagents. However, IQC materials tested using both lots of reagent exhibited minimal variation. Therefore, IQC alone is insufficient for assessing laboratory analytical results. This finding prompts us to be vigilant in potential pitfall of interpreting test results based on satisfactory IQC alone.

Liquid biopsy for non-invasive assessment of liver injury in hepatitis B patients.

BACKGROUND: Hepatitis B is a major public health problem in China. Accurate liver injury assessment is essential for clinical evidence-based treatment. Liver biopsy is considered the gold standard method to stage liver disease, but it is not widely used in resource-limited settings. Therefore, non-invasive liquid biopsy tests are needed. AIM: To assess liver injury in hepatitis B patients using quantified cell free DNA combined with other serum biomarker as a liquid biopsy-based method. METHODS: A cohort of 663 subjects including 313 hepatitis B patients and 350 healthy controls were enrolled. Ultrasound-guided liver biopsies followed by histopathological assessments were performed for the 263 chronic hepatitis B patients to determine the degree of liver injury. Cell-free DNA was quantified using a novel duplex real-time polymerase chain reaction assay. RESULTS: Compared with healthy controls, patients with hepatitis B virus (HBV) infection had significantly higher plasma DNA, serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), bilirubin, and HBV DNA levels (P < 0.01). Serum ALT, AST, bilirubin, and plasma DNA levels of patients with marked-severe inflammation were significantly higher than those with mild-moderate inflammation (P < 0.01). There was a statistically significant correlation between hepatocyte inflammation severity and serum bilirubin (R 2 = 0.673, P < 0.01) or plasma DNA (R 2 = 0.597, P < 0.01) levels. The areas under the curves of serum ALT, bilirubin, plasma DNA, and their combination to distinguish between patients with mild-moderate and marked-severe inflammation were 0.8059, 0.7910, 0.7921, and 0.9564, respectively. CONCLUSION: The combination of plasma DNA, serum ALT, and bilirubin could be a candidate liquid biopsy for non-invasive assessment of liver injury in hepatitis B patients.

Development and Evaluation of a Duplex Real-Time PCR Assay With a Novel Internal Standard for Precise Quantification of Plasma DNA.

BACKGROUND: Circulating levels of cell-free DNA increase in many pathologic conditions. However, notable discrepancies in the quantitative analysis of cell-free DNA from a large number of laboratories have become a considerable pitfall, hampering its clinical application. METHODS: We designed a novel recombinant DNA fragment that could be applied as an internal standard in a newly developed and validated duplex real-time PCR assay for the quantitative analysis of total cell-free plasma DNA, which was tested in 5,442 healthy adults and 200 trauma patients. RESULTS: Compared with two traditional methods, this novel assay showed a lower detection limit of 0.1 ng/mL, lower intra- and inter-assay CVs, and higher accuracy in the recovery test. The median plasma DNA concentration of healthy males (20.3 ng/mL, n=3,092) was significantly higher than that of healthy females (16.1 ng/mL, n=2,350) (Mann-Whitney two-sample rank sum test, P<0.0001). The reference intervals of plasma DNA concentration were 0-45.8 ng/mL and 0-52.5 ng/mL for healthy females and males, respectively. The plasma DNA concentrations of the majority of trauma patients (96%) were higher than the upper normal cutoff values and were closely related to the corresponding injury severity scores (R²=0.916, P<0.0001). CONCLUSIONS: This duplex real-time PCR assay with a new internal standard could eliminate variation and allow for more sensitive, repeatable, accurate, and stable quantitative measurements of plasma DNA, showing promising application in clinical diagnosis.

Non-small-cell lung cancer-induced immunosuppression by increased human regulatory T cells via Foxp3 promoter demethylation.

Patients with non-small-cell lung cancer (NSCLC) have immune defects that are poorly understood. Forkhead box protein P3 (Foxp3) is crucial for immunosuppression by CD4(+) regulatory T cells (Tregs). It is not well known how NSCLC induces Foxp3 expression and causes immunosuppression in tumor-bearing patients. Our study found a higher percentage of CD4(+) Tregs in the peripheral blood of NSCLC compared with healthy donors. NSCLC patients showed demethylation of eight CpG sites within the Foxp3 promoter with methylation ratios negatively correlated with CD4(+)CD25(+)Foxp3(+) T levels. Foxp3 expression in CD4(+) Tregs was directly regulated by Foxp3 promoter demethylation and was involved in immunosuppression by NSCLC. To verify the effect of tumor cells on the phenotype and function of CD4(+) Tregs, we established a coculture system using NSCLC cell line and healthy CD4(+) T cells and showed that SPC-A1 induced IL-10 and TGF-ß1 secretion by affecting the function of CD4(+) Tregs. The activity of DNA methyltransferases from CD4(+) T was decreased during this process. Furthermore, eight CpG sites within the Foxp3 promoter also appeared to have undergone demethylation. Foxp3 is highly expressed in CD4(+) T cells, and this may be caused by gene promoter demethylation. These induced Tregs are highly immunosuppressive and dramatically inhibit the proliferative activity of naïve CD4(+) T cells. Our study provides one possible mechanism describing Foxp3 promoter demethylation changes by which NSCLC down-regulates immune responses and contributes to tumor progression. Foxp3 represents an important target for NSCLC anti-tumor immunotherapy.

High Uric Acid (UA) Negatively Affects Serum Tartrate-Resistant Acid Phosphatase 5b (TRACP 5b) Immunoassay.

BACKGROUND: Bone metastases often occur in the majority of patients with advanced cancer, such as prostate cancer, lung cancer and breast cancer. Serum tartrate-resistant acid phosphatase 5b (TRACP 5b), a novel bone resorption marker, has been used gradually in the clinics as a specific and sensitive marker of bone resorption for the early diagnosis of cancer patients with bone metastasis. Here, we reported that high concentrations of uric acid (UA) lead to decrease of TRACP 5b levels and determined whether TRACP 5b level was associated with UA in interference experiment. METHODS: A total of 77 patients with high concentrations of UA and 77 healthy subjects were tested to evaluate the differences in their TRACP 5b levels. Serial dilutions of UA were respectively spiked with a known concentration of TRACP 5b standard sample, then Serum TRACP 5b was detected by using bone TRAP® Assay. A correction equation was set to eliminate UA-derived TRACP 5b false-decrease. The effect of this correction was evaluated in high-UA individuals. RESULTS: The average TRACP level of the high-UA individuals (1.47 ± 0.62 U/L) was significantly lower than that of the healthy subjects (2.62 ± 0.63 U/L) (t-test, p < 0.0001). The UA correction equation derived: ΔTRACP 5b = -1.9751lgΔUA + 3.7365 with an R2 = 0.98899. Application of the UA correction equation resulted in a statistically non-significant difference in TRACP 5b values between the healthy subjects and high-UA individuals (p = 0.24). CONCLUSIONS: High UA concentrations can falsely decrease TRACP 5b levels due to a method-related systematic error. To avoid misdiagnoses or inappropriate therapeutic decisions, increased attention should be paid to UA interference, when TRACP 5b is used for early diagnosis of cancer patients with bone metastasis, evaluation of the aggressiveness of osteosarcoma or prediction of survival in prostate cancer and breast cancer with bone metastases.

Activation of Toll-like receptors signaling in non-small cell lung cancer cell line induced by tumor-associated macrophages.

BACKGROUND: Inflammation is often linked with the progress and poor outcome of lung cancer. The understanding of the relationship between tumor-associated macrophages (TAMs) and lung cancer cells involves in the underlying mechanism of inflammatory cytokine production. Toll-like receptors (TLRs) are engaged in promoting the production of pro-inflammatory cytokines and play an important role in tumor immunology. METHODS: To investigate the mechanisms by which TAMs influence the production of pro-inflammatory cytokines in lung cancer cells, we established an in vitro coculture system using TAMs and human non-small cell lung cancer (NSCLC) cell line SPC-A1. Levels of interleukin (IL)-1ß, IL-6 and IL-8 in SPC-A1 were evaluated by RT-PCR and cytometric bead array assay after being cocultured with TAMs. Expression changes of TLRs and TLRs signaling pathway proteins in SPC-A1 were further confirmed by RT-PCR and western blot. The level changes of IL-1ß, IL-6 and IL-8 in SPC-A1 were also detected after the stimulation of TLRs agonists. RESULTS: We found that the phenotype markers of TAMs were highly expressed after stimulating human monocyte cell line THP-1 by phorbol-12-myristate-13-acetate (PMA). Higher mRNA and supernate secretion levels of IL-1ß, IL-6 and IL-8 were detected in SPC-A1 after being cocultured with TAMs. We also found that TLR1, TLR6 and TLR7 were up-regulated in SPC-A1 in the coculture system with TAMs. Meanwhile, TLRs signaling pathway proteins were also significantly activated. Moreover, pre-treatment with agonist ligands for TLR1, TLR6 and TLR7 could dramatically promote inductions of IL-1ß, IL-6 and IL-8. CONCLUSIONS: These findings demonstrated that TAMs may enhance IL-1ß, IL-6 and IL-8 expressions via TLRs signaling pathway. We conclude that TAMs contribute to maintain the inflammation microenvironment and ultimately promote the development and progression of lung cancer.

miR-638 is a new biomarker for outcome prediction of non-small cell lung cancer patients receiving chemotherapy.

MicroRNAs (miRNAs), a class of small non-coding RNAs, mediate gene expression by either cleaving target mRNAs or inhibiting their translation. They have key roles in the tumorigenesis of several cancers, including non-small cell lung cancer (NSCLC). The aim of this study was to investigate the clinical significance of miR-638 in the evaluation of NSCLC patient prognosis in response to chemotherapy. First, we detected miR-638 expression levels in vitro in the culture supernatants of the NSCLC cell line SPC-A1 treated with cisplatin, as well as the apoptosis rates of SPC-A1. Second, serum miR-638 expression levels were detected in vivo by using nude mice xenograft models bearing SPC-A1 with and without cisplatin treatment. In the clinic, the serum miR-638 levels of 200 cases of NSCLC patients before and after chemotherapy were determined by quantitative real-time PCR, and the associations of clinicopathological features with miR-638 expression patterns after chemotherapy were analyzed. Our data helped in demonstrating that cisplatin induced apoptosis of the SPC-A1 cells in a dose- and time-dependent manner accompanied by increased miR-638 expression levels in the culture supernatants. In vivo data further revealed that cisplatin induced miR-638 upregulation in the serum derived from mice xenograft models, and in NSCLC patient sera, miR-638 expression patterns after chemotherapy significantly correlated with lymph node metastasis. Moreover, survival analyses revealed that patients who had increased miR-638 levels after chemotherapy showed significantly longer survival time than those who had decreased miR-638 levels. Our findings suggest that serum miR-638 levels are associated with the survival of NSCLC patients and may be considered a potential independent predictor for NSCLC prognosis.

Methylated APC and RASSF1A in multiple specimens contribute to the differential diagnosis of patients with undetermined solitary pulmonary nodules.

BACKGROUND: Inactivation of tumor-suppressor gene (TSG) by promoter hypermethylation has been reported in many tumor types, including lung cancer. This study was designed to determine the methylated APC and RASSF1A genes in tumor tissue, serum and plasma of patients with early stage lung cancer. METHODS: Eighty-nine patients with undetermined solitary pulmonary nodules detected upon CT-scan were recruited in this study. DNA samples were extracted from biopsy tissues, serum and plasma and QMSP of APC and RASSF1A was carried out after bisulfite conversion. The 89 patients consist of 58 stage I lung cancer patients and 31 benign lung disease according to pathological report. Twenty-six cancer patients had matched biopsy tumor tissue, serum and plasma samples. RESULTS: The methylation rates of APC and RASSF1A were 59.0% and 66.1% in biopsy tissues, 42.5% and 52.5% in serum, and 24.1% and 43.1% in plasma of cancer patients. For RASSF1A, different samples all showed a significant difference between cancer group and benign group (P<0.05). However, APC gene only explored the P value less than 0.05 in plasma result. Towards the 26 lung cancer patients with three matched samples, methylation rate in each sample type was more than 50.0% and displayed no difference. CONCLUSIONS: Evaluation of APC and RASSF1A promoter methylation by using QMSP appears to be very useful for the differential diagnosis of patients with undetermined solitary pulmonary nodules. Our results also suggested that plasma might be the best sample for clinical detection of early stage lung.

Relationship between serum CA19-9 and CEA levels and prognosis of pancreatic cancer.

BACKGROUND: To explore the relationship between preoperative serum CA19-9 and CEA levels and prognosis of pancreatic cancer (PC). METHODS: The clinicopathological data of 128 patients with pancreatic adenocarcinoma who were treated in our center between January 2012 and December 2013 were retrospectively analyzed. The relationships between serum CA19-9 and CEA levels and survival were analyzed using Kaplan-Meier method, log-rank test, and Cox regression analysis. The cut-off values for serum CA19-9 and CEA levels were 39 U/mL and 4.7 ng/mL, respectively. RESULTS: Among these 128 patients, the mean age was 62 years, and median survival was 12.2 days. The positive rate of CA19-9 and CEA was 78.1% and 37.5%, respectively. Patients with increased CA19-9 or CEA level suffered a poorer prognosis than those with normal CA19-9 or CEA level (CA19-9: P=0.027; CEA: P=0.036). Cox logistic analysis revealed that lymphatic metastasis, CA19-9 >39 U/mL, and CEA >4.7 ng/mL were independent prognostic factors in patients with pancreatic carcinoma. CONCLUSIONS: Preoperative serum CA19-9 and CEA level are closely related with survival time in PC patients and therefore may be used for evaluating the prognosis for PC.

Downregulation of IFNG in CD4(+) T cells in lung cancer through hypermethylation: a possible mechanism of tumor-induced immunosuppression.

Tumor survival is significantly correlated with the immune response of patients. IFNG plays an important role in the tumor host response and decreased IFNG expression is often observed in lung cancer. Studies have shown that CpG island hypermethylation plays a critical role in transcriptional silencing of IFNG gene expression. However, there is limited understanding regarding the molecular mechanisms of altered methylation, and whether the tumor microenvironment has any effect on DNA methylation and IFNG production. In the current study, we demonstrate that plasma and intra-cellular IFNG levels are significantly lower in lung cancer patients. Hypermethylation of the IFNG promoter in CD4(+) T cells and plasma IFNG was negatively correlated. CD4(+) T cells from healthy individuals co-cultured with SPC-A1 cells generated lower levels of IFNG after activation, elevated expression of DNA methyltransferases (DNMTs), and exhibited hypermethylation of the IFNG promoter. In conclusion, decreased IFNG expression of CD4(+) T cells co-cultured with lung cancer cell is associated with IFNG promoter hypermethylation. Our study suggests that interaction between lung cancer cells and CD4(+) T cells induces DNMT expression and IFNG promoter hypermethylation in CD4(+) T cell, which may serve as an important mechanism of tumor-induced immunosuppression.

[Quantitative detection of APC/RASSF1A promoter methylation in the plasma of patients with cervical diseases].

OBJECTIVE: To quantitatively detect adenomatous polyposis coli(APC) and Ras association domain family 1A( RASSF1A) promoter methylation levels in the plasma of patients with cervical disease and to determine the diagnostic value of the indicators of cervical disease. METHODS: Preoperative blood samples were collected from 25 cases of healthy women and 118 cases of cervical disease, and tissue samples were also collected from 31 cases of them. The APC/RASSF1A promoter methylation levels of plasma and tissue were determined by duplex real-time quantitative methylation specific PCR (qMSP). RESULTS: Among 31 paired plasma and tissue samples, true negative rate of APC and RASSF1A genes were all 100%, and true positive rate of APC and RASSF1A genes were 3/5 and 7/9, respectively. In 143 cases of plasma samples, total positive rate of APC and (or) RASSF1A methylation was 3% (2/59) for control/low-grade lesions groups and 48% (40/84) for high-grade lesions/tumor groups (P < 0.01) . RASSF1A methylation rate was related to lymph node metastasis and International Federation of Gynecology and Obstetrics (FIGO) stage (P < 0.05). CONCLUSION: The plasma APC/RASSF1A methylation detection may be with some application prospect in the diagnosis of cervical diseases.

EDTA-temperature-Induced pseudohematocytopenia in a patient with multiple myeloma.

BACKGROUND: Platelet clumping caused by ethylenediamine tetraacetic acid (EDTA) and erythrocyte agglutination caused by cold agglutinins are often found in clinical findings. However, erythrocyte agglutination induced by EDTA has not been reported as yet. CASE REPORT: Spurious low red blood cell (RBC), white blood cell (WBC), and platelet counts were observed in a patient blood sample collected in EDTA in vitro at room temperature and 37 degrees C. However, the phenomena were only observed in the sodium citrate and heparin anticoagulated blood at room temperature, but not at 37 degrees C. Both erythrocyte agglutination and platelet clumping were observed in the peripheral blood smear. These data suggest an EDTA-temperature-induced pseudohematocytopenia. CONCLUSIONS: It is a very rare phenomenon to observe erythrocyte agglutination induced by EDTA and temperature.