Ye Guo

SALL4, a stem cell factor, affects the side population by regulation of the ATP-binding cassette drug transport genes.

Our previous work shows that the stem cell factor SALL4 plays a central role in embryonic and leukemic stem cells. In this study, we report that SALL4 expression was higher in drug resistant primary acute myeloid leukemic patients than those from drug-responsive cases. In addition, while overexpression of SALL4 led to drug resistance in cell lines, cells with decreased SALL4 expression were more sensitive to drug treatments than the parental cells. This led to our investigation of the implication of SALL4 in drug resistance and its role in side population (SP) cancer stem cells. SALL4 expression was higher in SP cells compared to non-SP cells by 2–4 fold in various malignant hematopoietic cell lines. Knocking down of SALL4 in isolated SP cells resulted in a reduction of SP cells, indicating that SALL4 is required for their self-renewal. The SP phenotype is known to be mediated by members of the ATP-binding cassette (ABC) drug transport protein family, such as ABCG2 and ABCA3. Using chromatin-immunoprecipitation (ChIP), quantitative reverse transcription polymerase chain reaction (qRT-PCR) and electrophoretic mobility shift assay(EMSA), we demonstrated that SALL4 was able to bind to the promoter region of ABCA3 and activate its expression while regulating the expression of ABCG2 indirectly. Furthermore, SALL4 expression was positively correlated to those of ABCG2 and ABCA3 in primary leukemic patient samples. Taken together, our results suggest a novel role for SALL4 in drug sensitivity, at least in part through the maintenance of SP cells, and therefore may be responsible for drug-resistance in leukemia. We are the first to demonstrate a direct link between stem cell factor SALL4, SP and drug resistance in leukemia.

Patterns of circulating tumor cells identified by CEP8, CK and CD45 in pancreatic cancer

To improve the identification for CTCs with weak or negative CK and diploid CTCs in pancreatic cancer, we combined immune‐staining of CK, CD45, DAPI and fluorescence in situ hybridization with the centromere of chromosome 8 (CEP8) probe method. CTCs in 3.75 mL of blood were depleted for CD45 positive cells with anti‐CD45 antibodies and identified by combining CK, CD45, DAPI and CEP8 in 61 cases including 22 pancreatic cancers, 3 borderline pancreatic solid pseudopapillary tumors, 6 pancreatic benign tumors, and 30 healthy individuals. We found that enriched cells could be classified into 5 patterns: CK+CD45‐DAPI+CEP8=2 (2 hybridization signals), CK+CD45‐DAPI+CEP8>2 (>2 hybridization signals), CK‐CD45‐DAPI+CEP8>2, CK‐CD45‐DAPI+CEP8=2, and CK+/‐CD45+DAPI+CEP8=2 or >2. Among 22 pancreatic cancers, CK+CD45‐DAPI+CEP8=2 and CK+CD45‐DAPI+CEP8>2 patterns were identified in two cases, and CK‐CD45‐ DAPI+CEP8>2 pattern was identified in 16 cases. CK‐CD45‐DAPI+CEP8=2 and CK+/‐CD45+DAPI+CEP8=2 or >2 patterns were detected in pancreatic cancers, other pancreatic diseases and healthy individuals. Among the five patterns, CK+CD45‐DAPI+CEP8=2, CK+CD45‐DAPI+CEP8>2 and CK‐CD45‐DAPI+CEP8>2 were considered as CTCs, while CK‐CD45‐DAPI+CEP8=2 and CK+/‐CD45+DAPI+CEP8=2 or >2 were considered as indeterminate cells. When the cutoff value was set as 2 cells/3.75 mL based on ROC curve, the sensitivity and specificity in the diagnosis of pancreatic cancer was 68.18 and 94.87%, respectively. Dynamically monitoring CTCs changes prior to and after surgery in pancreatic patients revealed that CTCs count decreased in 3 days after surgery, but increased in 10 days after surgery in most patients. During our one and a half year follow‐up, CTCs positive patients showed metastasis and worse survival rate.

Elevated Homocysteine Level and Folate Deficiency Associated with Increased Overall Risk of Carcinogenesis: Meta-Analysis of 83 Case-Control Studies Involving 35,758 Individuals

Background Results of the association of folate metabolism and carcinogenesis are conflicting. We performed a meta-analysis to examine the effect of the interaction of serum concentration of homocysteine (Hcy), folate, and vitamin B12 and 5,10-methylenetetrahydrofolate reductase (MTHFR) polymorphism on risk of cancer overall. Method Two reviewers independently searched for all published studies of Hcy and cancer in PubMed, EMBASE-MEDLINE and Chinese databases. Pooled results were reported as odds ratios (ORs) and mean differences and presented with 95% confidence intervals (95% CIs) and 2-sided probability values. Results We identified 83 eligible studies of 15,046 cases and 20,712 controls. High level of Hcy but low level of folate was associated with risk of cancer overall, with little effect by type of cancer or ethnicity. Vitamin B12 level was inversely associated with only urinary-system and gastrointestinal carcinomas and for Asian and Middle Eastern patients. As well, MTHFR C677T, A1298C and G1793A polymorphisms were related to elevated serum level of Hcy, and folate and vitamin B12 deficiency. However, only MTHFR C677T homogeneity/wild-type (TT/CC) polymorphism was positively associated with overall risk of cancer. Conclusion Elevated serum Hcy level and folate deficiency are associated with increased overall risk of cancer.

Lung cancer circulating tumor cells isolated by the EpCAM-independent enrichment strategy correlate with Cytokeratin 19-derived CYFRA21-1 and pathological staging

BACKGROUNDnCytokeratin 19-derived CYFRA21-1 is an acceptable lung cancer biomarker. However, whether CYFRA21-1 correlates with lung cancer circulating tumor cells (CTCs) remains unclear.nnnMETHODSnCTCs in 42 lung cancer patients and 10 nonmalignant pulmonary disease patients were isolated by means of an EpCAM-independent enrichment strategy. Correlation of lung cancer CTCs with serum concentration of CYFRA21-1 and pathological staging was investigated.nnnRESULTSnAmong lung cancer patients in this study, 39% (7/18) of those with normal CYFRA21-1 (≤3.3 ng/ml) and 62% (13/21) of high CYFRA21-1 (>3.3 ng/ml) patients were found to have ≥3 CTCs/7.5 ml blood. The CTCs-positive rate of stage I to IV lung cancer patients was 20% (2/10), 45% (5/11), 54% (6/11) and 70% (7/10), respectively. Comparing M0 vs M1 patients, the CTCs-positive rate was 43% (13/30) and 70% (7/10), respectively. All M1 patients (10/10) had one or more CTCs detected, whereas none of the nonmalignant pulmonary disease patients had detectable CTCs.nnnCONCLUSIONnLung cancer CTCs isolated by the EpCAM-independent enrichment approach correlate with CYFRA21-1 and TNM staging. Correlation of CTCs and CYFRA21-1 in lung cancer patients is of potential clinical utility in terms of early diagnosis and predicting prognosis.

Serum miRNAs panel (miR-16-2*, miR-195, miR-2861, miR-497) as novel non-invasive biomarkers for detection of cervical cancer

miRNAs have been established as critical layer of regulation during tumorigenesis; extracellular miRNAs are extraordinarily stable; and, quantitative reverse transcript polymerase chain reaction (qRT-PCR) provides a sensitive platform for quantifying miRNAs with a broad dynamic range. Herein, we aimed to establish a serum miRNA signature for diagnosing cervical cancer (CC). In this study, we recruited a cohort of 184 CC, 186 cervical intraepithelial neoplasia (CIN) patients and 193 healthy control subjects. qRT-PCR was performed with serum samples to screen a pool of 444 miRNAs at the initial phase, 66 miRNAs at the training phase, and 7 miRNAs at the validation phase. The profile of 4 circulating miRNAs (miR-16-2*, miR-195, miR-2861, miR-497) was established for CC diagnosis. By Receiver Operating Characteristic (ROC) curve analysis, this 4-miRNA signature showed high accuracy in discriminating CC (AUCu2009=u20090.849), and CIN individuals (AUCu2009=u20090.734) from healthy controls. Among these 4 miRNAs, only miR-16-2*, but not miR-195, miR-2861 or miR497, shared a similar pattern in sera of breast cancer and ovarian cancer patients. Overall, our studies have identified a novel noninvasive biomarker constituted with a panel of four miRNAs (miR-16-2*, miR-195, miR-2861, miR-497).

Improvement of specific detection of circulating tumor cells using combined CD45 staining and fluorescence in situ hybridization

BACKGROUNDnConventional cytokeratin (CK)-based methods to detect circulating tumor cells (CTCs) often present suboptimal sensitivities for decreased or non-expression of cytokeratin in some CTCs. We clinically investigated a new method that combines immunocytochemistry staining (ICC) of CD45 and fluorescence in situ hybridization (FISH).nnnMETHODSnCirculating epithelial cells from 141 subjects were enriched using an EpCAM-independent strategy and then identified by either a combination of FISH with chromosome 8 centromere probe (CEP8) and ICC staining of CD45 (CD45-FISH) or ICC staining of CK.nnnRESULTSnFor detecting CTCs enriched from lung cancers, CD45-FISH had larger areas under ROC curves of 0.963 (P=0.000) compared to ICC (0.653; P=0.031) using cut-off values of 2 and 1 cell/3.75ml blood with sensitivities of 83.3% and 43.3%, specificities of 98.6% and 89.5%, respectively. Moreover, CD45-FISH showed 76.2% sensitivity in detecting CTCs in ovarian cancers (P<0.001). Four of six ovarian cancers showed dramatical decrease in both CTCs and serum CA125 on the 7th day after surgery.nnnCONCLUSIONnCD45-FISH method had improved sensitivity and specificity in detecting CTCs of lung and ovarian cancers compared to ICC-CK. This combined detection strategy may be useful in detecting or monitoring CTCs after ovarian cancer surgery.

Stem cell factor SALL4, a potential prognostic marker for myelodysplastic syndromes

BackgroundMyelodysplastic syndromes (MDS) are a group of heterogeneous diseases with variable clinical course. Predicting disease progression is difficult due to lack of specific molecular marker(s). SALL4 plays important roles in normal hematopoiesis and leukemogenesis. SALL4 transgenic mice develop MDS prior to acute myeloid leukemia (AML) transformation. However, the role of SALL4 in human MDS has not been extensively investigated. In this study, we evaluate the diagnostic/prognostic value of SALL4 in MDS by examining its expression levels in a cohort of MDS patients.MethodsFifty-five newly diagnosed MDS, twenty MDS-AML, and sixteen post-treatment MDS patients were selected for our study along with ten healthy donors.ResultsWe demonstrated that SALL4 was over-expressed in MDS patients and proportionally increased in MDS patients with high grade/IPSS scores. This expression pattern was similar to that of Bmi-1, an important marker in predicting MDS/AML progression. In addition, the level of SALL4 was positively correlated with increased blast counts, high-risk keryotypes and increased significantly in MDS-AML transformation. Furthermore, higher level of SALL4 expression was associated with worse survival rates and SALL4 level decreased following effective therapy.ConclusionsTo the best of our knowledge, this is the largest series and the first to report the expression pattern of SALL4 in detail in various subtypes of MDS in comparison to that of Bmi-1. We conclude that SALL4 is a potential molecular marker in predicting the prognosis of MDS.

Biological and analytical variations of 16 parameters related to coagulation screening tests and the activity of coagulation factors.

To accurately estimate longitudinal changes in individuals, it is important to take into consideration the biological variability of the measurement. The few studies available on the biological variations of coagulation parameters are mostly outdated. We confirmed the published results using modern, fully automated methods. Furthermore, we added data for additional coagulation parameters. At 8:00 am, 12:00 pm, and 4:00 pm on days 1, 3, and 5, venous blood was collected from 31 healthy volunteers. A total of 16 parameters related to coagulation screening tests as well as the activity of coagulation factors were analyzed; these included prothrombin time, fibrinogen (Fbg), activated partial thromboplastin time, thrombin time, international normalized ratio, prothrombin time activity, activated partial thromboplastin time ratio, fibrin(-ogen) degradation products, as well as the activity of factor II, factor V, factor VII, factor VIII, factor IX, and factor X. All intraindividual coefficients of variation (CVI) values for the parameters of the screening tests (except Fbg) were less than 5%. Conversely, the CVI values for the activity of coagulation factors were all greater than 5%. In addition, we calculated the reference change value to determine whether a significant difference exists between two test results from the same individual.

Establishment and development of the personalized criteria for microscopic review following multiple automated routine urinalysis systems.

BACKGROUNDnThis study intends to develop the microscopic review criteria for automated urine chemistry analyzer and integrated urine chemistry and formed-element analyzer.nnnMETHODSnA total of 1058 samples were analyzed using chemistry analyzer (Siemens Atlas) for proteins (PRO), blood (BLD) and WBC. Cast, RBC and WBC were analyzed using 4 different instruments, IRIS IQ200, AVE-766, US 2026, and Sysmex UF-1000i. A phase-contrast microscopy was used as reference to evaluate false-negative rate (FNR) and review rate (RR).nnnRESULTSnThe optimized review criteria for Atlas were either PRO 2+, or BLD 2+, or WBC 2+, or specimen from nephrology department. FNR was 4.65% and RR was 40.41%. The optimized criteria for integrated Atlas and IQ200, or AVE-766 or US 2026 or UF-1000i were either BLD≥2+ (>2+ for females), or RBC count ≥ 2 times reference range, or different WBC results between chemistry and formed-element analysis, or PRO ≥ 2+ or CAST>the reference range. One additional rule for integrated Atlas and UF-1000i was different results between BLD and RBC counts. FNR was 1.94%, 2.03%, 1.74%, 1.65%, and RR was 41.09%, 40.12%, 44.86%, 50.39%, respectively.nnnCONCLUSIONSnThese specific review criteria ensured a low missed-diagnosis rate and a reasonable workload in practice.

Intra-day and inter-day biological variations of peripheral blood lymphocytes.

BACKGROUNDnThe proportion and absolute numbers of lymphocyte subsets are important indices that reflect the immunological status of the body. Few studies on biological variations of absolute lymphocyte subset counts and no study in Asian population are currently available. Furthermore, there have been few reports on the biological variation of these indices in the short term.nnnMETHODSnAt 8:00AM, 12:00PM, and 4:00PM on days 1, 3, and 5, venous blood was collected from 20 healthy volunteers. All participants maintained their normal lifestyles. The percent and absolute lymphocyte subset counts were measured using single -platform method.nnnRESULTSnIntraday and interday biological variations for the absolute and percent counts of lymphocyte subsets were relatively constant. The intra-individual coefficient of variation (CVI) and inter-individual coefficient of variation (CVG) were similar to those in previous studies. Biological variations in the percent and absolute counts for the CD3(+)CD4(-)CD8(-), CD3(+)CD4(+)CD8(+), and CD3(+)CD16(+)CD56(+) subsets were relatively high.nnnCONCLUSIONSnThese observations are clinically valuable. Investigation on the CVI and CVG may allow us to determine the utility of traditional population based reference ranges. Documentation of the reference change values may be used as objective delta-check values in quality management and decide whether the change that occur in an individuals serial results before the change is significant. The present study also enriched the database regarding the biological variations of lymphocyte subsets in Asian population.