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Background: Identification of emerging molecular biomarkers on circulating tumor cells (CTCs) represents an attractive feature of liquid biopsy that facilitates precision and tailored medicine in the management of metastatic castration-resistant prostate cancer (mCRPC). Prostein is an androgen-regulated transmembrane protein with high prostate specificity. Prostein-positive circulating tumor cell (CTC) was recently suggested to have diagnostic potential; however, no study has been conducted to evaluate its prognostic value in mCRPC., Methods: CTCs from mCRPC patients were enumerated using the CellSearch System. Prostein-positive CTCs were identified by immunostaining results. The relationships between prostein expression on CTCs and PSA response rate, PSA progression-free survival (PSA-PFS), radiographic progression-free survival (PFS), and overall survival (OS) were tested by Fisher's exact test or evaluated using Kaplan-Meier and multivariate Cox analyses., Results: Prostein-positive CTCs were identified in 31 of 87 baseline samples from mCRPC patients and 16 of 51 samples collected at the first follow-up visit. PSA response rates were significantly lower in baseline prostein-positive patients (0%, 0/31) than in prostein-negative patients (19.6%, 11/56) (p = 0.007). The 31 prostein-positive patients had significantly shorter PSA-PFS (p < 0.001), radiographic PFS (p < 0.001), and OS (p = 0.018), compared to the 56 prostein-negative patients at baseline. The association with PSA-PFS maintained its significance (p = 0.028) in multivariate analyses. Analyzing prostein expression at the first follow-up as well as the conversion of prostein expression from baseline to follow-up samples not only confirmed the association with PSA-PFS, but also demonstrated prognostic significance with OS., Conclusion: Our study provides the first evidence to support the potential of prostein expression on CTCs to serve as a novel prognostic marker in mCRPC patients. Future large-scale prospective studies are needed to validate our findings., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Genomic tests are being developed for use in cancer screening. As most screening is offered in primary care settings, primary care provider and patient perceptions of such tests are likely to affect uptake. We conducted a scoping review to synthesize information on factors likely to affect patient and provider use of biospecimen collection and analysis for cancer screening, methods referred to as liquid biopsy or multi-cancer early detection (MCED) testing when used to detect multiple cancers. We ultimately identified 7 articles for review and analyzed them for major themes. None reported on primary care provider perspectives. Six articles focused on patient perceptions about testing for a single cancer (colorectal), and 1 reported on patient views related to testing for multiple cancers. Factors favoring this type of testing included its non-invasiveness, and the perceived safety, convenience, and effectiveness of testing. There is a dearth of information in the literature on primary care provider perceptions about liquid biopsy and MCED testing. The limited information on patient perceptions suggests that they are receptive to such tests. Research on primary care provider and patient test-related knowledge, attitudes, and behavior is needed to guide future implementation in primary care settings.

The dynamics of mitochondrial biogenesis regulation is critical in maintaining cellular homeostasis for immune regulation and tumor prevention. Here, we report that mitochondrial biogenesis disruption through TFAM reduction significantly impairs mitochondrial function, induces autophagy, and promotes esophageal squamous cell carcinoma (ESCC) growth. We found that TFAM protein reduction promotes mitochondrial DNA (mtDNA) release into the cytosol, induces cytosolic mtDNA stress, subsequently activates the cGAS-STING signaling pathway, thereby stimulating autophagy and ESCC growth. STING depletion or mtDNA degradation by DNase I abrogates mtDNA stress response, attenuates autophagy, and decreases the growth of TFAM depleted cells. In addition, autophagy inhibitor also ameliorates mitochondrial dysfunction-induced activation of the cGAS-STING signaling pathway and ESCC growth. In conclusion, our results indicate that mtDNA stress induced by mitochondria biogenesis perturbation activates the cGAS-STING pathway and autophagy to promote ESCC growth, revealing an underappreciated therapeutic strategy for ESCC., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Previously undescribed molecular mechanisms of resistance will emerge with the increased use of cyclin-dependent kinase 4/6 inhibitors in clinical settings. To identify genomic aberrations in circulating tumor DNA associated with treatment resistance in palbociclib-treated metastatic breast cancer (MBC) patients, we collected 35 pre- and post-treatment blood samples from 16 patients with estrogen receptor-positive (ER + ) MBC, including 9 with inflammatory breast cancer (IBC). Circulating cell-free DNAs (cfDNAs) were isolated for sequencing using a targeted panel of 91 genes. Our data showed that FBXW7 and CDK6 were more frequently altered in IBC than in non-IBC, whereas conversely, PIK3CA was more frequently altered in non-IBC than in IBC. The cfDNA samples collected at follow-up harbored more mutations than baseline samples. By analyzing paired samples, we observed a higher percentage of patients with mutations in RB1 , CCNE1 , FBXW7 , EZH2 , and ARID1A , but a lower proportion of patients with mutated TSC2 at the post-treatment stage when they developed progression. Moreover, acquisition of CCNE1 mutations or loss of TSC2 mutations after treatment initiation conferred an unfavorable prognosis. These data provide insights into the relevance of novel genomic alterations in cfDNA to palbociclib resistance in MBC patients. Future large-scale prospective studies are warranted to confirm our findings.

Hexavalent chromium [Cr(VI)] is a well-known carcinogen that can cause several types of cancer including lung cancer. NF-E2-related factor 2 (Nrf2), the redox sensitive transcription factor, can protect normal cells from a variety of toxicants and carcinogens by inducing the expression of cellular protective genes and maintaining redox balance. However, Nrf2 also protects cancer cells from radio- and chemo-therapies and facilitates cancer progression. Although Cr(VI) treatment has been demonstrated to upregulate Nrf2 expression, the mechanisms for Nrf2 regulation upon chronic Cr(VI) exposure remain to be elucidated. We found that Nrf2 was upregulated in BEAS-2B cells exposed to Cr(VI) from 1 to 5 months, and also in Cr(VI)-induced transformed (Cr-T) cells with Cr(VI) treatment for 6 months. We showed that KEAP1, the classic negative regulator of Nrf2, was downregulated after Cr(VI) exposure for 4 months, suggesting that Nrf2 induction by Cr(VI) treatment is through KEAP1 decrease at late stage. To further decipher the mechanisms of Nrf2 upregulation at early stage of Cr(VI) exposure, we demonstrated that miR-27a and miR-27b were redox sensitive miRNAs, since reactive oxygen species (ROS) scavengers induced miR-27a/b expression. After Cr(VI) exposure for 1 month, the expression levels of miR-27a/b was dramatically decreased. The changes of miR-27a/b and their target Nrf2 were confirmed in vivo by mouse model intranasally exposed to Cr(VI) for 12 weeks. Nrf2 was a direct target of miR-27a/b, which acted as tumor suppressors in vitro and in vivo to inhibit tumorigenesis and cancer development of Cr-T cells. The results suggested that the inhibition of miR-27a/b was responsible for Nrf2 upregulation at both early stage and late stage of Cr(VI) exposure. This novel regulation of Nrf2 upon chronic Cr(VI) exposure through redox-regulated miR-27a/b will provide potential targets for preventing and treating Cr(VI)-induced carcinogenesis in the future., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Mitochondrial dynamics homeostasis is important for cell metabolism, growth, proliferation, and immune responses. The critical GTPase for mitochondrial fission, Drp1 is frequently upregulated in many cancers and is closely implicated in tumorigenesis. However, the mechanism underling Drp1 to influence tumor progression is largely unknown, especially in esophageal squamous cell carcinoma (ESCC)., Methods: Immunohistochemistry was used to examine Drp1 and LC3B expression in tissues of ESCC patients. Autophagic vesicles were investigated by transmission electron microscopy. Fluorescent LC3B puncta and mitochondrial nucleoid were observed by fluorescent and confocal microscopy. Mitochondrial function was evaluated by mitochondrial membrane potential, ROS and ATP levels. Xenograft tumor model was performed in BALB/c nude mice to analyze the role of Drp1 on ESCC progression., Results: We found that Drp1 high expression is correlated with poor overall survival of ESCC patients. Drp1 overexpression promotes cell proliferation and xenograft ESCC tumor growth by triggering autophagy. Furthermore, we demonstrated that Drp1 overexpression disturbs mitochondrial function and subsequent induces mitochondrial DNA (mtDNA) released into the cytosol thereby inducing cytosolic mtDNA stress. Mechanistically, cytosolic mtDNA activates the cGAS-STING pathway and facilitates autophagy, which promotes ESCC cancer growth. Moreover, mtDNA digestion with DNase I and autophagy inhibition with chloroquine attenuates the cGAS-STING pathway activation and ESCC cancer growth., Conclusions: Our finding reveals that Drp1 overexpression induces mitochondrial dysfunction and cytosolic mtDNA stress, which subsequently activates the cGAS-STING pathway, triggers autophagy and promotes ESCC progression., (© 2022. The Author(s).)

Background: Esophageal squamous cell carcinoma (ESCC) is the most predominant primary malignant tumor among worldwide, especially in China. To date, the successful treatment remains a mainly clinical challenge, it is imperative to develop successful therapeutic agents., Methods: The anti-proliferative effect of ivermectin on ESCC is investigated in cell model and in nude mice model. Cell apoptosis was assessed using flow cytometry, TUNEL assay and western blotting. Mitochondrial dysfunction was determined by reactive oxygen species accumulation, mitochondrial membrane potential and ATP levels., Results: Our results determined that ivermectin significantly inhibited the proliferation of ESCC cells in vitro and in vivo. Furthermore, we found that ivermectin markedly mediated mitochondrial dysfunction and induced apoptosis of ESCC cells, which indicated the anti-proliferative effect of ivermectin on ESCC cells was implicated in mitochondrial apoptotic pathway. Mechanistically, ivermectin significantly triggered ROS accumulation and inhibited the activation of NF-κB signaling pathway and increased the ratio of Bax/Bcl-2., Conclusions: These finding indicated that ivermectin has significant anti-tumour potential for ESSC and may be a potential therapeutic candidate against ESCC., (© 2021. The Author(s).)

Liquid biopsy-based biomarkers, including circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA), are increasingly important for the characterization of metastatic breast cancer (MBC). The aim of the study was to explore CTCs and ctDNA dynamics to better understand their potentially complementary role in describing MBC., Methods: The study retrospectively analyzed 107 patients with MBC characterized with paired CTCs and ctDNA assessments and a second prospective cohort, which enrolled 48 patients with MBC. CTCs were immunomagnetically isolated and ctDNA was quantified and then characterized through next-generation sequencing in the retrospective cohort and droplet digital polymerase chain reaction in the prospective cohort. Matched pairs variations at baseline, at evaluation one (EV1), and at progression were tested through the Wilcoxon test. The prognostic role of ctDNA parameters was also investigated., Results: Mutant allele frequency (MAF) had a significant decrease between baseline and EV1 and a significant increase between EV1 and progression. Number of detected alterations steadily increased across timepoints, CTCs enumeration (nCTCs) significantly increased only between EV1 and progression. MAF dynamics across the main altered genes was then investigated. Plasma DNA yield did not vary across timepoints both in the retrospective cohort and in the prospective cohort, while the short fragments fraction showed a potential role as a prognostic biomarker., Conclusion: nCTCs and ctDNA provide complementary information about prognosis and treatment benefit. Although nCTCs appeared to assess tumor biology rather than tumor burden, MAF may be a promising biomarker for the dynamic assessment of treatment response and resistance., Competing Interests: Massimo Cristofanilli Honoraria: Pfizer, Foundation Medicine Consulting or Advisory Role: Novartis, CytoDyn, Lilly, Foundation Medicine, Menarini Research Funding: Lilly, Angle, Merck No other potential conflicts of interest were reported.Massimo Cristofanilli Honoraria: Pfizer, Foundation Medicine Consulting or Advisory Role: Novartis, CytoDyn, Lilly, Foundation Medicine, Menarini Research Funding: Lilly, Angle, Merck No other potential conflicts of interest were reported., (© 2021 by American Society of Clinical Oncology.)

Background: The neutrophil-lymphocyte ratio (NLR), platelet-lymphocyte ratio (PLR), and circulating tumor cells (CTCs) have been associated with survival in castration-resistant prostate cancer (CRPC). However, no study has examined the prognostic value of NLR and PLR in the context of CTCs., Methods: Baseline CTCs from mCRPC patients were enumerated using the CellSearch System. Baseline NLR and PLR values were calculated using the data from routine complete blood counts. The associations of CTC, NLR, and PLR values, individually and jointly, with progression-free survival (PFS) and overall survival (OS), were evaluated using Kaplan-Meier analysis, as well as univariate and multivariate Cox models., Results: CTCs were detected in 37 (58.7%) of 63 mCRPC patients, and among them, 16 (25.4%) had ≥5 CTCs. The presence of CTCs was significantly associated with a 4.02-fold increased risk for progression and a 3.72-fold increased risk of death during a median follow-up of 17.6 months. OS was shorter among patients with high levels of NLR or PLR than those with low levels (log-rank P = 0.023 and 0.077). Neither NLR nor PLR was individually associated with PFS. Among the 37 patients with detectable CTCs, those with a high NLR had significantly shorter OS (log-rank P = 0.024); however, among the 26 patients without CTCs, the OS difference between high- and low-NLR groups was not statistically significant. Compared to the patients with CTCs and low NLR, those with CTCs and high levels of NLR had a 3.79-fold risk of death (P = 0.036). This association remained significant after adjusting for covariates (P = 0.031). Combination analyses of CTC and PLR did not yield significant results., Conclusion: Among patients with detectable CTCs, the use of NLR could further classify patients into different risk groups, suggesting a complementary role for NLR in CTC-based prognostic stratification in mCRPC.

Inflammatory breast cancer (IBC) is the most aggressive form of breast cancer. Although it is a rare subtype, IBC is responsible for roughly 10% of breast cancer deaths. In order to obtain a better understanding of the genomic landscape and intratumor heterogeneity (ITH) in IBC, we conducted whole-exome sequencing of 16 tissue samples (12 tumor and four normal samples) from six hormone-receptor-positive IBC patients, analyzed somatic mutations and copy number aberrations, and inferred subclonal structures to demonstrate ITH. Our results showed that KMT2C was the most frequently mutated gene (42%, 5/12 samples), followed by HECTD1, LAMA3, FLG2, UGT2B4, STK33, BRCA2, ACP4, PIK3CA, and DNAH8 (all nine genes tied at 33% frequency, 4/12 samples). Our data indicated that PTEN and FBXW7 mutations may be considered driver gene mutations for IBC. We identified various subclonal structures and different levels of ITH between IBC patients, and mutations in the genes EIF4G3, IL12RB2, and PDE4B may potentially generate ITH in IBC.

Liquid biopsy-based biomarkers have advantages in monitoring the dynamics of metastatic castration-resistant prostate cancer (mCRPC), a bone-predominant metastatic disease. Previous studies have demonstrated associations between circulating tumor cells (CTCs) and clinical outcomes of mCRPC patients, but little is known about the prognostic value of CTC-clusters. In 227 longitudinally collected blood samples from 64 mCRPC patients, CTCs and CTC-clusters were enumerated using the CellSearch platform. The associations of CTC and CTC-cluster counts with progression-free survival (PFS) and overall survival (OS), individually and jointly, were evaluated by Cox models. CTCs and CTC-clusters were detected in 24 (37.5%) and 8 (12.5%) of 64 baseline samples, and in 119 (52.4%) and 27 (11.9%) of 227 longitudinal samples, respectively. CTC counts were associated with both PFS and OS, but CTC-clusters were only independently associated with an increased risk of death. Among patients with unfavorable CTCs (≥5), the presence of CTC-clusters signified a worse survival (log-rank p = 0.0185). mCRPC patients with both unfavorable CTCs and CTC-clusters had the highest risk for death (adjusted hazard ratio 19.84, p = 0.0072), as compared to those with <5 CTCs. Analyses using longitudinal data yielded similar results. In conclusion, CTC-clusters provided additional prognostic information for further stratifying death risk among patients with unfavorable CTCs.

Purpose: Discordance between HER2 expression in tumor tissue (tHER2) and HER2 status on circulating tumor cells (cHER2) has been reported. It remains largely underexplored whether patients with tHER2 - /cHER2 + can benefit from anti-HER2 targeted therapies., Methods: cHER2 status was determined in 105 advanced-stage patients with tHER2 - breast tumors. Association between cHER2 status and progression-free survival (PFS) was analyzed by univariate and multivariate Cox models and survival differences were compared by Kaplan-Meier method., Results: Compared to the patients with low-risk cHER2 (cHER2 +  < 2), those with high-risk cHER2 (cHER2 +  ≥ 2) had shorter survival time and an increased risk for disease progression (hazard ratio [HR] 2.16, 95% confidence interval [CI] 1.20-3.88, P = 0.010). Among the patients with high-risk cHER2, those who received anti-HER2 targeted therapies had improved PFS compared with those who did not (HR 0.30, 95% CI 0.10-0.92, P = 0.035). In comparison, anti-HER2 targeted therapy did not affect PFS among those with low-risk cHER2 (HR 0.70, 95% CI 0.36-1.38, P = 0.306). Similar results were obtained after adjusting covariates. A longitudinal analysis of 67 patients with cHER2 detected during follow-ups found that those whose cHER2 status changed from high-risk at baseline to low-risk at first follow-up exhibited a significantly improved survival compared to those whose cHER2 remained high-risk (median PFS: 11.7 weeks vs. 2.0 weeks, log-rank P = 0.001)., Conclusion: In advanced-stage breast cancer patients with tHER2 - tumors, cHER2 status has the potential to guide the use of anti-HER2 targeted therapy in patients with high-risk cHER2.

Heteroplasmic mutations in mitochondrial DNA (mtDNA) play critical roles in mitochondrial disease, aging, and cancer. Recently, next-generation sequencing (NGS) has been widely used to detect mtDNA mutations for diagnosis and monitoring of the above-mentioned diseases. However, little attention is paid on inherent cross-contamination generated during mtDNA capture and sequencing of mixed samples, which may seriously reduce the detection accuracy of mtDNA heteroplasmic mutations. In this study, a novel sequencing strategy based on a unique double-barcode design was established. The results showed that when single barcode-based analysis strategy was used, cross-contamination level of 20 DNA samples ranged from 0.27% to 11.90% on HiSeq 2500 and from 0.93% to 17.70% on HiSeq X ten, whereas double barcode-based strategy could effectively eliminate cross-contamination. Moreover, the data indicated that cross-contamination was mainly derived from capture process and was significantly affected by different NGS platforms. In addition, contamination level was negatively related to sequencing depth. Moreover, cross-contamination significantly increased the false-positive calling of mtDNA heteroplasmic mutations and remarkably affected the heteroplasmy level of mtDNA mutations. In contrast, cross-contamination had no notable effect on classification of mtDNA haplogroup. Taken together, our novel double barcode-based sequencing strategy is effective in eliminating cross-contamination, enhancing the detection accuracy of mtDNA NGS, and improving its application in diagnosis or monitoring of diseases associated with mtDNA mutations., (Copyright © 2019 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.)

Purpose: To update the American Society of Clinical Oncology (ASCO)/American Society of Hematology (ASH) recommendations for use of erythropoiesis-stimulating agents (ESAs) in patients with cancer., Methods: PubMed and the Cochrane Library were searched for randomized controlled trials (RCTs) and meta-analyses of RCTs in patients with cancer published from January 31, 2010, through May 14, 2018. For biosimilar ESAs, the literature search was expanded to include meta-analyses and RCTs in patients with cancer or chronic kidney disease and cohort studies in patients with cancer due to limited RCT evidence in the cancer setting. ASCO and ASH convened an Expert Panel to review the evidence and revise previous recommendations as needed., Results: The primary literature review included 15 meta-analyses of RCTs and two RCTs. A growing body of evidence suggests that adding iron to treatment with an ESA may improve hematopoietic response and reduce the likelihood of RBC transfusion. The biosimilar literature review suggested that biosimilars of epoetin alfa have similar efficacy and safety to reference products, although evidence in cancer remains limited., Recommendations: ESAs (including biosimilars) may be offered to patients with chemotherapy-associated anemia whose cancer treatment is not curative in intent and whose hemoglobin has declined to < 10 g/dL. RBC transfusion is also an option. With the exception of selected patients with myelodysplastic syndromes, ESAs should not be offered to most patients with nonchemotherapy-associated anemia. During ESA treatment, hemoglobin may be increased to the lowest concentration needed to avoid transfusions. Iron replacement may be used to improve hemoglobin response and reduce RBC transfusions for patients receiving ESA with or without iron deficiency. Additional information is available at www.asco.org/supportive-care-guidelines and www.hematology.org/guidelines .

Purpose: To update the American Society of Clinical Oncology (ASCO)/American Society of Hematology (ASH) recommendations for use of erythropoiesis-stimulating agents (ESAs) in patients with cancer., Methods: PubMed and the Cochrane Library were searched for randomized controlled trials (RCTs) and meta-analyses of RCTs in patients with cancer published from January 31, 2010, through May 14, 2018. For biosimilar ESAs, the literature search was expanded to include meta-analyses and RCTs in patients with cancer or chronic kidney disease and cohort studies in patients with cancer due to limited RCT evidence in the cancer setting. ASCO and ASH convened an Expert Panel to review the evidence and revise previous recommendations as needed., Results: The primary literature review included 15 meta-analyses of RCTs and two RCTs. A growing body of evidence suggests that adding iron to treatment with an ESA may improve hematopoietic response and reduce the likelihood of RBC transfusion. The biosimilar literature review suggested that biosimilars of epoetin alfa have similar efficacy and safety to reference products, although evidence in cancer remains limited., Recommendations: ESAs (including biosimilars) may be offered to patients with chemotherapy-associated anemia whose cancer treatment is not curative in intent and whose hemoglobin has declined to < 10 g/dL. RBC transfusion is also an option. With the exception of selected patients with myelodysplastic syndromes, ESAs should not be offered to most patients with nonchemotherapy-associated anemia. During ESA treatment, hemoglobin may be increased to the lowest concentration needed to avoid transfusions. Iron replacement may be used to improve hemoglobin response and reduce RBC transfusions for patients receiving ESA with or without iron deficiency. Additional information is available at www.asco.org/supportive-care-guidelines and www.hematology.org/guidelines.

Background: Both circulating tumour cell (CTC) and total circulating cell-free DNA (ccfDNA) predict cancer patient prognosis. However, no study has explored the prognostic value of the combined use of CTC and ccfDNA. We aimed to investigate individual and joint effects of CTC and ccfDNA on clinical outcomes of metastatic breast cancer (MBC) patients., Methods: We collected 227 blood samples from 117 MBC patients. CTCs were enumerated using the CellSearch System. ccfDNAs were quantified by quantitative real-time polymerase chain reaction and Qubit fluorometer. The individual and joint effects of CTC and ccfDNA levels on patient progression-free survival (PFS) and overall survival (OS) were analysed using Cox proportional hazards models., Results: Compared to patients with <5 CTCs, patients with ≥5 CTCs had a 2.58-fold increased risk of progression and 3.63-fold increased risk of death. High level of ccfDNA was associated with a 2.05-fold increased risk of progression and 3.56-fold increased risk of death. These associations remained significant after adjusting for other important clinical covariates and CTC/ccfDNA levels. CTC and ccfDNA levels had a joint effect on patient outcomes. Compared to patients with low levels of both CTC and ccfDNA, those with high levels of both markers exhibited a >17-fold increased death risk (P < 0.001). Moreover, longitudinal analysis of 132 samples from 22 patients suggested that the inconsistency between CTC level and outcome in some patients could possibly be explained by ccfDNA level., Conclusions: CTC and total ccfDNA levels were individually and jointly associated with PFS and OS in MBC patients., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Background: Use of chemotherapy in stage II colorectal cancer (CRC) is controversial because it improves survival only in some patients. We aimed to develop a statistical model using routine and readily available blood tests to predict the prognosis of patients with stage II CRC and to identify which patients are likely to benefit from chemotherapy. Methods: We divided 422 patients with stage II CRC into a training and a testing set. The association of routine laboratory variables and disease-free survival (DFS) was analyzed. A prognostic model was developed incorporating clinically relevant laboratory variables with demographic and tumor characteristics. A prognostic score was derived by calculating the sum of each variable weighted by its regression coefficient in the model. Model performance was evaluated by constructing receiver operating characteristic curves and calculating the area under the curve (AUC). Results: Significant associations were seen between 5 laboratory variables and patient DFS in univariate analyses. After stepwise selection, 3 variables (carcinoembryonic antigen, hemoglobin, creatinine) were retained in the multivariate model with an AUC of 0.75. Compared with patients with a low prognostic score, those with a medium and high prognostic score had a 1.99- and 4.78-fold increased risk of recurrence, respectively. The results from the training set were validated in the testing set. Moreover, chemotherapy significantly improved DFS in high-risk patients, but not in low- and medium-risk patients. Conclusions: A routine laboratory variable-based model may help predict DFS of patients with stage II CRC and identify high-risk patients more likely to benefit from chemotherapy., (Copyright © 2018 by the National Comprehensive Cancer Network.)

Recent studies have revealed significant intratumor heterogeneity (ITH) of nuclear genome mutations and highlighted its function in tumor progression and treatment resistance. However, the ITH of somatic mitochondrial DNA (mtDNA) mutations detected in cancers remains unknown. In this study, we performed multiregional mtDNA sequencing of tumor and paratumor tissue samples from 12 hepatocellular carcinoma (HCC) and 13 colorectal cancer (CRC) patients. A substantial level of mtDNA mutations was found in paired non-HCC inflammatory tissues, suggesting that these tissues might not be mtDNA-genetically "normal." Moreover, our data indicated that the ITH of somatic mtDNA mutations was a common feature in HCC and CRC patients. In addition, we found that shared mutations which were observed in at least 2 samples in each patient exhibited a significantly higher heteroplasmic level than mutations that were private to a specific tumor region from both HCC (p = 0.039) and CRC patients (p = 0.001). The heteroplasmic level of shared mutations was positively correlated with intratumoral recurrence of mtDNA mutations. We also found that shared mutations in tumor tissues with a higher degree of pathogenicity risk exhibited a higher heteroplasmic level and intratumoral recurrence in both HCC and CRC patients. These findings suggest that some mtDNA mutations may undergo positive selection during the clonal expansion. Taken together, our analyses identified various levels of ITH of somatic mtDNA mutations in HCC and CRC patients and provided evidence supporting the positive selection working on some somatic mtDNA mutations in tumor tissues., (© 2018 UICC.)

Researchers hypothesized that telomere shortening facilitates carcinogenesis. Previous studies found inconsistent associations between blood leukocyte telomere length (LTL) and cancer. Epigenetic reprogramming of telomere maintenance mechanisms may help explain this inconsistency. We examined associations between DNA methylation in telomere-related genes (TRG) and cancer. We analyzed 475 participants providing 889 samples 1 to 3 times (median follow-up, 10.1 years) from 1999 to 2013 in the Normative Aging Study. All participants were cancer-free at each visit and blood leukocytes profiled using the Illumina 450K array. Of 121 participants who developed cancer, 34 had prostate cancer, 10 melanoma, 34 unknown skin malignancies, and 43 another cancer. We examined 2,651 CpGs from 80 TRGs and applied a combination of Cox and mixed models to identify CpGs prospectively associated with cancer (at FDR < 0.05). We also explored trajectories of DNA methylation, logistic regression stratified by time to diagnosis/censoring, and cross-sectional models of LTL at first blood draw. We identified 30 CpGs on 23 TRGs whose methylation was positively associated with cancer incidence (β = 1.0-6.93) and one protective CpG in MAD1L1 (β = -0.65) , of which 87% were located in TRG promoters. Methylation trajectories of 21 CpGs increased in cancer cases relative to controls; at 4 to 8 years prediagnosis/censoring, 17 CpGs were positively associated with cancer. Three CpGs were cross-sectionally associated with LTL. TRG methylation may be a mechanism through which LTL dynamics reflect cancer risk. Future research should confirm these findings and explore potential mechanisms underlying these findings, including telomere maintenance and DNA repair dysfunction. Cancer Prev Res; 11(8); 511-22. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Aim: Previous studies suggest telomere shortening represses PGC1A and PGC1B expression leading to mitochondrial dysfunction. Methylation of CpG sites within these genes may interact with these factors to affect cancer risk., Materials & Methods: Among 385 men, we identified 84 incidents of cancers (predominantly prostate and nonmelanoma skin). We examined associations between leukocyte DNA methylation of 41 CpGs from PGC1A and PGC1B with telomere length, mitochondrial 8-OHdG lesions, mitochondrial abundance and cancer incidence., Results: Methylation of five and eight CpG sites were significantly associated with telomere length and mitochondrial abundance at p < 0.05. Two CpG sites were independently associated with cancer risk: cg27514608 (PGC1A, TSS1500; HR: 1.55, 95% CI: 1.19-2.03, FDR = 0.02), and cg15219393 (PGC1B, first exon/5'UTR; HR: 3.71, 95% CI: 1.82-7.58, FDR < 0.01). Associations with cg15219393 were observed primarily among men with shorter leukocyte telomeres., Conclusion: PGC1A and PGC1B methylation may serve as early biomarkers of cancer risk.

Aims: Levels of the mitochondrial calcium uniporter regulator 1 (MCUR1) increases during development of hepatocellular carcinoma (HCC). However, mechanisms of how mitochondrial Ca 2+ homeostasis is modulated and its function remain limited in cancers., Results: MCUR1 was frequently upregulated in HCC cells to enhance the Ca 2+ uptake into mitochondria in an MCU-dependent manner, which significantly facilitated cell survival by inhibiting mitochondria-dependent intrinsic apoptosis and promoting proliferation of HCC cells, and thus led to poor prognosis. In vivo assay confirmed these results, indicating that overexpressed MCUR1 notably decreased the fraction of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells and increased the positive Ki67 staining in xenograft tumors, while reduced MCUR1 expression was associated with impaired growth capacity of HCC cells in nude mice. The survival advantage conferred by MCUR1-mediated mitochondrial Ca 2+ uptake was majorly caused by elevated production of mitochondrial reactive oxygen species and subsequent AKT/MDM2- induced P53 degradation, which regulated the expression level of apoptosis-related molecules and cell cycle-related molecules. Treatment of mitochondrial Ca 2+ -buffering protein parvalbumin remarkably inhibited the growth of HCC cells. Conclusions and Innovation: Our study provides evidence supporting a possible tumor-promoting role for MCUR1-mediated mitochondrial Ca 2+ uptake and uncovers a mechanistic understanding that links change of mitochondrial Ca 2+ homeostasis to cancer cell survival, which suggests a potential novel therapeutic target for HCC. Antioxid. Redox Signal. 28, 1120-1136.

Background: Tumor necrosis factor-α has been proven an effective anticancer agent in preclinical studies. However, the translation of TNFα from research to clinic has been blocked by significant systemic toxicity and limited efficacy at maximal tolerated dose, which need urgently to be solved., Methods: The level of cytosolic Ca 2+ was assessed by Fura-2 in HCC cells. After changing cytosolic Ca 2+ level by using agonists or inhibitors, cell apoptosis was detected by flow cytometry. We also detected the effect of ionomycin or parvalbumin on the anti-tumor activity of TNFα in a mice model. Lastly, we studied the roles of cytosolic Ca 2+ in the mitochondrial-dependent intrinsic apoptosis pathway., Results: Here, we demonstrated that TNFα induced extracellular Ca 2+ influx into cytoplasm through transient receptor potential channel in HCC cells. Both cytosolic Ca 2+ scavenger and Ca 2+ -binding protein PV effectively desensitized hepatocellular carcinoma cells to TNFα, whereas combination ionomycin or 1,4,5-inositol triphosphate significantly sensitized HCC cells to TNFα, indicating that the increased level of cytosolic Ca 2+ was positively correlated with the TNFα-induced cell apoptosis in vitro. In a nude mice xenograft model, our data revealed that TNFα combined with ionomycin remarkably synergized the anti-tumor effect of TNFα. Furthermore, we found that TNFα-mediated extracellular Ca 2+ influx accelerated TNFα-induced extrinsic apoptosis through activating calpain/IAP/caspase3 pathway., Conclusions: Our study provides the evidence supporting a novel mechanism by which TNFα induces extracellular Ca 2+ influx to enhance cell apoptosis and suggests that increasing the level of cytosolic Ca 2+ might be an alternative strategy to improve the pro-apoptotic activity of TNFα in HCC cells, although suitable chemical or biological reagents need to be further tested.

Background: Previous studies have indicated that tumor mitochondrial DNA (mtDNA) mutations are primarily shaped by relaxed negative selection, which is contradictory to the critical roles of mtDNA mutations in tumorigenesis. Therefore, we hypothesized that site-specific selection may influence tumor mtDNA mutations., Methods: To test our hypothesis, we developed the largest collection of tumor mtDNA mutations to date and evaluated how natural selection shaped mtDNA mutation patterns., Results: Our data demonstrated that both positive and negative selections acted on specific positions or functional units of tumor mtDNAs, although the landscape of these mutations was consistent with the relaxation of negative selection. In particular, mutation rate (mutation number in a region/region bp length) in complex V and tRNA coding regions, especially in ATP8 within complex V and in loop and variable regions within tRNA, were significantly lower than those in other regions. While the mutation rate of most codons and amino acids were consistent with the expectation under neutrality, several codons and amino acids had significantly different rates. Moreover, the mutations under selection were enriched for changes that are predicted to be deleterious, further supporting the evolutionary constraints on these regions., Conclusion: These results indicate the existence of site-specific selection and imply the important role of the mtDNA mutations at some specific sites in tumor development.

The epithelial-mesenchymal transition (EMT) process is a crucial step for tumor invasion and metastasis. Previous research investigating EMT has mostly focused on its role in cancer progression. Recent studies showed that EMT and EMT-driving transcription factor (EMT-TF) expression are early events in lung cancer pathogenesis, implying a potential association between EMT and lung cancer risk. In this study, we examined whether genetic variants in EMT-related genes are associated with risk of non-small cell lung cancer (NSCLC). We used data from a genome-wide association study of 1482 NSCLC cases and 1544 healthy controls as the discovery phase, in which we analyzed 1602 single-nucleotide polymorphisms (SNPs) within 159 EMT-related genes. We then validated the significant SNPs in another 5699 cases and 5815 controls from the National Cancer Institute lung cancer genome-wide association study. Cumulative effects were evaluated for validated SNPs, and a gene-based test was performed to explore gene-level association with disease risk. In the discovery phase, 174 SNPs demonstrated significant associations with NSCLC risk. In the validation phase, seven SNPs mapped to EGFR, NOTCH3, ADGRF1 and SMAD3 were confirmed. Cumulative effect analysis of the significant SNPs demonstrated increasing risk with the number of unfavorable genotypes in the discovery and validation datasets. Gene-based analysis implicated ADGRF1, NOTCH3 and CDH1 as significant for NSCLC risk. Functional prediction revealed several potential mechanisms underlying these associations. Our results suggest that EMT-related gene variants may be involved in susceptibility to NSCLC; if confirmed, they might help identify higher-risk individuals., (© The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Purpose: Circulating cancer associated macrophage-like cells (CAMLs) have been detected in the peripheral blood of patients with solid tumors including breast cancer. However, the prognostic relevance of CAMLs in metastatic breast cancer (MBC) has not been evaluated. In the present study, we aimed to measure CAMLs and circulating tumor cells (CTCs) at baseline and examine their prognostic value in patients with MBC., Methods: Peripheral blood samples from 127 MBC patients were collected at baseline before starting a new treatment. The detection and enumeration of CAMLs and CTCs in 7.5 ml whole blood were performed using the CellSearch™ system. The associations of CAMLs and CTCs with the progression-free survival (PFS) and overall survival (OS) in the patients were evaluated using Kaplan-Meier curves and Cox proportional hazards modeling., Results: Among 127 MBC patients, 21 (16.5%) were detected with CAMLs and 38 (29.9%) had elevated CTCs (≥5 CTCs/7.5 ml). Patients with CAMLs at baseline had worse PFS and OS with an adjusted hazard ratio (HR) of 1.75 (95% CI 1.03-2.98, P = 0.0374) and 3.75 (95% CI 1.52-9.26, P = 0.0042), compared to patients without CAMLs. Compared with patients with <5 CTCs and without CAMLs, patients with <5 CTCs and with CAMLs, with ≥5 CTCs but without CAMLs, or with ≥5 CTCs and with CAMLs, had an increasing trend of risk of disease progression (HR = 0.84, 3.42 and 4.04 respectively, P for trend <0.0001) and death (HR = 2.66, 6.14, and 9.13, respectively, P for trend <0.0001)., Conclusion: Baseline enumeration of individual CAMLs is an independent indicator for MBC patients' survival. Evaluation of CAMLs in peripheral blood might provide a potential biomarker with additional prognostic values over CTC enumeration alone in MBC patients.

Background: Previous studies have indicated that mitochondrial genetic variations were associated with the risk of many cancers. However, there are few reports on the association between single nucleotide polymorphisms (SNPs) or haplogroups of mitochondrial DNA (mtDNA) and the risk or prognosis of hepatocellular carcinoma (HCC)., Methods: In order to investigate the predictive and prognostic role of mtDNA SNPs and haplogroups in HCC, the mitochondrial genome of 188 HCC patients and 344 healthy controls were sequenced by next generation sequencing technology. Then, logistic regression analysis was used to determine the effect of mtDNA SNP or haplogroup on risk and prognosis of HCC patients., Results: The haplogroup M7 had an odds ratio (OR) of 0.47 (95% CI=0.24-0.91; P=0.026) to develop HCC. The frequency of 152T/C, 199T/C, 4048G/A, 9824T/C, 15784T/C, 16185C/T and 16399A/G was significantly different between HCC patients and the controls. In addition, multivariate analysis with COX hazards model showed that the patients with haplogroup M8 had lower survival rate than the patients with haplogroup D4 (HR=2.62, 95% CI=1.03-6.68; P=0.044). Three SNPs 15784T/C, 16185C/T and 16399A/G were also identified to have a statistically significant association with postoperative survival in HCC., Conclusions: To date, these results provide the first evidence that mtDNA SNPs and haplogroups may be potential risk factors for susceptibility and survival of HCC patients., (Copyright © 2017. Published by Elsevier Masson SAS.)

Prospective and longitudinal epidemiological evidence is needed to assess the association between telomere length and risk of hepatocellular carcinoma (HCC). In 323 cancer-free Korean-American HBV patients with 1-year exclusion window (followed for >1 year and did not develop HCC within 1 year), we measured the relative telomere length (RTL) in baseline serum DNAs and conducted extensive prospective and longitudinal analyses to assess RTL-HCC relationship. We found that long baseline RTL conferred an increased HCC risk compared to short RTL [hazard ratio (HR) = 4.93, P = 0.0005). The association remained prominent when the analysis was restricted to patients with a more stringent 5-year exclusion window (HR = 7.51, P = 0.012), indicating that the association was unlikely due to including undetected HCC patients in the cohort, thus minimizing the reverse-causation limitation in most retrospective studies. Adding baseline RTL to demographic variables increased the discrimination accuracy of the time-dependent receiver operating characteristic analysis from 0.769 to 0.868 (P = 1.0 × 10-5). In a nested longitudinal subcohort of 16 matched cases-control pairs, using a mixed effects model, we observed a trend of increased RTL in cases and decreased RTL in controls along 5 years of follow-up, with a significant interaction of case/control status with time (P for interaction=0.002) and confirmed the association between long RTL and HCC risk [odds ratio [OR] = 3.63, P = 0.016]. In summary, serum DNA RTL may be a novel non-invasive prospective marker of HBV-related HCC. Independent studies are necessary to validate and generalize this finding in diverse populations and assess the clinical applicability of RTL in HCC prediction., (© The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Motivation: Comprehensive catalogue of genes that drive tumor initiation and progression in cancer is key to advancing diagnostics, therapeutics and treatment. Given the complexity of cancer, the catalogue is far from complete yet. Increasing evidence shows that driver genes exhibit consistent aberration patterns across multiple-omics in tumors. In this study, we aim to leverage complementary information encoded in each of the omics data to identify novel driver genes through an integrative framework. Specifically, we integrated mutations, gene expression, DNA copy numbers, DNA methylation and protein abundance, all available in The Cancer Genome Atlas (TCGA) and developed iDriver, a non-parametric Bayesian framework based on multivariate statistical modeling to identify driver genes in an unsupervised fashion. iDriver captures the inherent clusters of gene aberrations and constructs the background distribution that is used to assess and calibrate the confidence of driver genes identified through multi-dimensional genomic data., Results: We applied the method to 4 cancer types in TCGA and identified candidate driver genes that are highly enriched with known drivers. (e.g.: P < 3.40 × 10 -36 for breast cancer). We are particularly interested in novel genes and observed multiple lines of supporting evidence. Using systematic evaluation from multiple independent aspects, we identified 45 candidate driver genes that were not previously known across these 4 cancer types. The finding has important implications that integrating additional genomic data with multivariate statistics can help identify cancer drivers and guide the next stage of cancer genomics research., Availability and Implementation: The C ++ source code is freely available at https://medschool.vanderbilt.edu/cgg/ ., Contacts: hai.yang@vanderbilt.edu or bingshan.li@Vanderbilt.Edu., Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com)

Cardiomyocytes can be differentiated from human pluripotent stem cells (hPSCs) in defined conditions, but efficient and consistent cardiomyocyte differentiation often requires expensive reagents such as B27 supplement or recombinant albumin. Using a chemically defined albumin-free (E8 basal) medium, we identified heparin as a novel factor that significantly promotes cardiomyocyte differentiation efficiency, and developed an efficient method to differentiate hPSCs into cardiomyocytes. The treatment with heparin helped cardiomyocyte differentiation consistently reach at least 80% purity (up to 95%) from more than 10 different hPSC lines in chemically defined Dulbecco's modified Eagle's medium/F-12-based medium on either Matrigel or defined matrices like vitronectin and Synthemax. One of heparin's main functions was to act as a Wnt modulator that helped promote robust and consistent cardiomyocyte production. Our study provides an efficient, reliable, and cost-effective method for cardiomyocyte derivation from hPSCs that can be used for potential large-scale drug screening, disease modeling, and future cellular therapies. Stem Cells Translational Medicine 2017;6:527-538., (© 2016 The Authors Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.)

Purpose: Circulating tumor cell (CTC) is a well-established prognosis predictor for metastatic breast cancer (MBC), and CTC-cluster exhibits significantly higher metastasis-promoting capability than individual CTCs. Because measurement of CTCs and CTC-clusters at a single time point may underestimate their prognostic values, we aimed to analyze longitudinally collected CTCs and CTC-clusters in MBC prognostication., Methods: CTCs and CTC-clusters were enumerated in 370 longitudinally collected blood samples from 128 MBC patients. The associations between baseline, first follow-up, and longitudinal enumerations of CTCs and CTC-clusters with patient progression-free survival (PFS) and overall survival (OS) were analyzed using Cox proportional hazards models., Results: CTC and CTC-cluster counts at both baseline and first follow-up were significantly associated with patient PFS and OS. Time-dependent analysis of longitudinally collected samples confirmed the significantly unfavorable PFS and OS in patients with ≥5 CTCs, and further demonstrated the independent prognostic values by CTC-clusters compared to CTC-enumeration alone. Longitudinal analyses also identified a link between the size of CTC-clusters and patient OS: compared to the patients without any CTC, those with 2-cell CTC-clusters and ≥3-cell CTC-clusters had a hazard ratio (HR) of 7.96 [95 % confidence level (CI) 2.00-31.61, P = 0.003] and 14.50 (3.98-52.80, P < 0.001), respectively., Conclusions: In this novel time-dependent analysis of longitudinally collected CTCs and CTC-clusters, we showed that CTC-clusters added additional prognostic values to CTC enumeration alone, and a larger-size CTC-cluster conferred a higher risk of death in MBC patients.

Lessons Learned: Patients with metastatic castration-resistant prostate cancer did not tolerate the combination of alisertib with abiraterone and prednisone.There was no clear signal indicating that adding alisertib might be beneficial for those patients progressing on abiraterone., Background: We hypothesized that Aurora A kinase (AK) contributes to castrate resistance in prostate cancer (PCa) and that inhibiting AK with alisertib can resensitize PCa cells to androgen receptor (AR) inhibitor abiraterone., Methods: This was a phase I/II trial to determine the safety and efficacy of alisertib when given in combination with abiraterone plus prednisone (AP). Metastatic castration-resistant prostate cancer (mCRPC) patients were treated with dose escalation (alisertib at 30, 40, and 50 mg orally b.i.d., days 1-7 every 21 days) per standard 3+3 design., Results: Nine of 43 planned subjects were enrolled. The maximum tolerated dose (MTD) was not reached, and the dose-limiting toxicities (DLTs) included neutropenic fever (1 of 9), neutropenia (1 of 9), fatigue with memory impairment (1 of 9), and diarrhea/mucositis (1 of 9). No prostate-specific antigen (PSA) decrease or circulating tumor cell (CTC) changes were observed during the study. Pharmacodynamically, adding alisertib did not affect total testosterone or dehydroepiandrosterone (DHEA) levels. There was some change in neuroendocrine markers after therapy. Mean duration on study was 2.5 months. The trial was terminated early., Conclusion: A tolerable dose of alisertib in combination with AP in mCRPC was not established in this study. There was no clear signal indicating that alisertib might be beneficial for patients with mCRPC progressing on abiraterone., (©AlphaMed Press; the data published online to support this summary is the property of the authors.)

Purpose: Increasing evidence suggests that alterations in mitochondrial DNA (mtDNA) copy number (mtDNAcn) and relative telomere length (RTL) may be implicated in the tumorigenesis of several malignancies. Alterations of both RTL and mtDNAcn are generally accepted as independent biomarkers for predicting risk and prognosis in various cancers. The aim of this study was to evaluate the prognostic value of combining leukocyte RTL with mtDNAcn (RTL-mtDNAcn) in hepatocellular carcinoma (HCC)., Methods: RTL and mtDNAcn in peripheral blood leukocytes (PBLs) were measured using a real-time PCR-based method in a total of 250 HCC patients treated with transcatheter arterial chemoembolization (TACE). We evaluated the associations between RTL and/or mtDNAcn and HCC overall survival using Kaplan-Meier curve analysis and Cox proportional hazards regression model., Results: We found that patients with longer leukocyte RTL or lower mtDNAcn had shorter overall survival time. The univariate analysis (HR 1.63, 95 % CI 1.23-2.17, P = 7.7 × 10(-4)) and multivariate analysis (HR 1.78, 95 % CI 1.31-2.42, P = 2.4 × 10(-4)) indicated that longer leukocyte RTL was significantly associated with poorer OS in HCC patients. Kaplan-Meier curve analysis showed that patients with longer RTL had shorter overall survival time than those with shorter RTL (log-rank P = 0.001). Patients with lower mtDNA copy number was significantly associated with poorer OS by Cox proportional hazards model using both univariate (HR 1.60, 95 % CI 1.21-2.13, P = 0.001) and multivariate analyses (HR 1.77, 95 % CI 1.30-2.41, P = 2.8 × 10(-4)). Kaplan-Meier curve analysis showed that patients with lower mtDNA content had significantly shorter overall survival time than those with higher mtDNA content (log-rank P = 0.001). Furthermore, combination of leukocyte RTL and mtDNAcn significantly improved the efficacy of predicting HCC prognosis. Patients with longer RTL and lower mtDNAcn exhibited a significantly poorer overall survival in both the univariate analysis (HR 2.21, 95 % CI 1.52-3.22, P = 3.5 × 10(-5)) and multivariate analysis (HR 2.60, 95 % CI 1.73-3.90, P = 4.3 × 10(-6)). The effect on patient prognosis was more evident in patients with longer RTL and lower mtDNAcn than in those with shorter RTL and lower mtDNA (HR 2.11, 95 % CI 1.34-3.32, P = 0.001) or in those with longer RTL and higher mtDNA (HR 2.10, 95 % CI 1.34-3.27, P = 0.001)., Conclusions: Our data suggest that combination of leukocyte RTL-mtDNAcn may be a potential efficient prognostic marker for HCC patients receiving the TACE treatment.

The availability of blood-based diagnostic testing using a non-invasive technique holds promise for real-time monitoring of disease progression and treatment selection. Circulating tumor cells (CTCs) have been used as a prognostic biomarker for the metastatic breast cancer (MBC). The molecular characterization of CTCs is fundamental to the phenotypic identification of malignant cells and description of the relevant genetic alterations that may change according to disease progression and therapy resistance. However, the molecular characterization of CTCs remains a challenge because of the rarity and heterogeneity of CTCs and technological difficulties in the enrichment, isolation and molecular characterization of CTCs. In this pilot study, we evaluated circulating tumor associated cells in one blood draw by size exclusion technology and cytological analysis. Among 30 prospectively enrolled MBC patients, CTCs, circulating tumor cell clusters (CTC clusters), CTCs of epithelial-mesenchymal transition (EMT) and cancer associated macrophage-like cells (CAMLs) were detected and analyzed. For molecular characterization of CTCs, size-exclusion method for CTC enrichment was tested in combination with DEPArray™ technology, which allows the recovery of single CTCs or pools of CTCs as a pure CTC sample for mutation analysis. Genomic mutations of TP53 and ESR1 were analyzed by targeted sequencing on isolated 7 CTCs from a patient with MBC. The results of genomic analysis showed heterozygous TP53 R248W mutation from one single CTC and pools of three CTCs, and homozygous TP53 R248W mutation from one single CTC and pools of two CTCs. Wild-type ESR1 was detected in the same isolated CTCs. The results of this study reveal that size-exclusion method can be used to enrich and identify circulating tumor associated cells, and enriched CTCs were characterized for genetic alterations in MBC patients, respectively., Competing Interests: The authors declare no conflict of interest.

Background: Methylation of repetitive elements Alu and LINE-1 in humans is considered a surrogate for global DNA methylation. Previous studies of blood-measured Alu/LINE-1 and cancer risk are inconsistent., Methods: We studied 1259 prospective methylation measurements from blood drawn 1-4 times from 583 participants from 1999 to 2012. We used Cox regression to evaluate time-dependent methylation as a biomarker for cancer risk and mortality, and linear regression to compare mean differences in methylation over time by cancer status and analyse associations between rate of methylation change and cancer., Results: Time-dependent LINE-1 methylation was associated with prostate cancer incidence (HR: 1.38, 95% CI: 1.01-1.88) and all-cancer mortality (HR: 1.41, 95% CI: 1.03-1.92). The first measurement of Alu methylation (HR: 1.39, 95% CI: 1.08-1.79) was associated with all-cancer mortality. Participants who ultimately developed cancer had lower mean LINE-1 methylation than cancer-free participants 10+ years pre-diagnosis (P<0.01). Rate of Alu methylation change was associated with all-cancer incidence (HR: 3.62, 95% CI: 1.09-12.10)., Conclusions: Our results add longitudinal data on blood Alu and LINE-1 methylation and cancer, and potentially contribute to their use as early-detection biomarkers. Future larger studies are needed and should account for the interval between blood sample collection and cancer diagnosis.

Background & Aims: We evaluated differences in treatment of black vs white patients with colon cancer and assessed their effects on survival, based on cancer stage., Methods: We collected data from the Surveillance, Epidemiology, and End Results-Medicare database and identified 6190 black and 61,951 white patients with colon cancer diagnosed from 1998 through 2009 and followed up through 2011. Three sets of 6190 white patients were matched sequentially, using a minimum distance strategy, to the same set of 6190 black patients based on demographic (age; sex; diagnosis year; and Surveillance, Epidemiology, and End Results registry), tumor presentation (demographic plus comorbidities, tumor stage, grade, and size), and treatment (presentation plus therapies) variables. We conducted sensitivity analyses to explore the effects of socioeconomic status in a subcohort that included 2000 randomly selected black patients. Racial differences in treatment were assessed using a logistic regression model; their effects on racial survival disparity were evaluated using the Kaplan-Meier method and the Cox proportional hazards model., Results: After patients were matched for demographic variables, the absolute 5-year difference in survival between black and white patients was 8.3% (white, 59.2% 5-y survival; blacks, 50.9% 5-y survival) (P < .0001); this value decreased significantly, to 5.0% (P < .0001), after patients were matched for tumor presentation, and decreased to 4.9% (P < .0001) when patients were matched for treatment. Differences in treatment therefore accounted for 0.1% of the 8.3% difference in survival between black and white patients. After patients were matched for tumor presentation, racial disparities were observed in almost all types of treatment; the disparities were most prominent for patients with advanced-stage cancer (stages III or IV, up to an 11.1% difference) vs early stage cancer (stages I or II, up to a 4.3% difference). After patients were matched for treatment, there was a greater reduction in disparity for black vs white patients with advanced-stage compared with early-stage cancer. In sensitivity analyses, the 5-year racial survival disparity was 7.7% after demographic match, which was less than the 8.3% observed in the complete cohort. This reduction likely was owing to the differences between the subcohort and the complete cohort in those variables that were not included in the demographic match. This value was reduced to 6.5% (P = .0001) after socioeconomic status was included in the demographic match. The difference decreased significantly to 2.8% (P = .090) after tumor presentation match, but was not reduced further after treatment match., Conclusions: We observed significant disparities in treatment and survival of black vs white patients with colon cancer. The disparity in survival appears to have been affected more strongly by tumor presentation at diagnosis than treatment. The effects of treatment differences on disparities in survival were greater for patients with advanced-stage vs early-stage cancer., (Copyright © 2016 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Recent studies have demonstrated a potential link between circulating cell-free mitochondrial DNA (mtDNA) content and cancers. However, there is no study evaluating the association between circulating mtDNA as a non-invasive marker of hepatocellular carcinoma (HCC) risk. We conducted a nested case-control study to determine circulating mtDNA content in serum samples from 116 HBV-related HCC cases and 232 frequency-matched cancer-free HBV controls, and evaluate the retrospective association between mtDNA content and HCC risk using logistic regression and their temporal relationship using a mixed effects model. HCC cases had significantly lower circulating mtDNA content than controls (1.06 versus 2.47, P = 1.7 × 10(-5)). Compared to HBV patients with higher mtDNA content, those with lower mtDNA content had a significantly increased risk of HCC with an odds ratio (OR) of 2.19 (95% confidence interval [CI] 1.28-3.72, P = 0.004). Quartile analyses revealed a significant dose-dependent effect (Ptrend = 0.001) for this association. In a pilot longitudinal sub-cohort of 14 matched cases-control pairs, we observed a trend of dramatically decreased mtDNA content in cases and slightly decreased mtDNA content in controls, with a significant interaction of case-control status with time (Pinteraction = 0.049). Our findings suggest that circulating mtDNA is a potential novel non-invasive biomarker of HCC risk in HBV patients.

High levels of arsenic in drinking water, soil, and air are associated with the higher incidences of several kinds of cancers worldwide, but the mechanism is yet to be fully discovered. Recently, a number of evidences show that dysregulation of microRNAs (miRNAs) induces carcinogenesis. In this study, we found miR-222 was upregulated in arsenic-transformed human lung epithelial BEAS-2B cells (As-T cells). Anti-miR-222 inhibitor treatment decreased cell proliferation, migration, tube formation, and induced apoptosis. In addition, anti-miR-222 inhibitor expression decreased tumor growth in vivo. We also found that inhibition of miR-222 induced the expression of its direct targets ARID1A and phosphatase and tensin homolog deleted on chromosome 10 (PTEN), and activated apoptosis of As-T cells in part through ARID1A downregulation. These results indicate that miR-222 plays an important role in arsenic-induced tumor growth.

Biomarkers for the early diagnosis of hepatocellular carcinoma (HCC) are needed to decrease mortality from this cancer. However, as new biomarkers have been slow to be brought to clinical practice, we have developed a diagnostic algorithm that utilizes commonly used clinical measurements in those at risk of developing HCC. Briefly, as α-fetoprotein (AFP) is routinely used, an algorithm that incorporated AFP values along with four other clinical factors was developed. Discovery analysis was performed on electronic data from patients who had liver disease (cirrhosis) alone or HCC in the background of cirrhosis. The discovery set consisted of 360 patients from two independent locations. A logistic regression algorithm was developed that incorporated log-transformed AFP values with age, gender, alkaline phosphatase, and alanine aminotransferase levels. We define this as the Doylestown algorithm. In the discovery set, the Doylestown algorithm improved the overall performance of AFP by 10%. In subsequent external validation in over 2,700 patients from three independent sites, the Doylestown algorithm improved detection of HCC as compared with AFP alone by 4% to 20%. In addition, at a fixed specificity of 95%, the Doylestown algorithm improved the detection of HCC as compared with AFP alone by 2% to 20%. In conclusion, the Doylestown algorithm consolidates clinical laboratory values, with age and gender, which are each individually associated with HCC risk, into a single value that can be used for HCC risk assessment. As such, it should be applicable and useful to the medical community that manages those at risk for developing HCC., (©2015 American Association for Cancer Research.)

Background & Aims: CD147 is a transmembrane glycoprotein which is highly expressed in various human cancers including hepatocellular carcinoma (HCC). A drug Licartin developed with (131)Iodine-labeled antibody against CD147 has been approved by the Chinese Food and Drug Administration (FDA) and enters into clinical use for HCC treatment. Increasing lines of evidence indicate that CD147 is implicated in the metabolism of cancer cells, especially glycolysis. However, the molecular mechanism underlying the relationship between CD147 and aberrant tumor lipid metabolism remains elusive., Methods: We systematically investigated the role of CD147 in the regulation of lipid metabolism, including de novo lipogenesis and fatty acid β-oxidation, in HCC cells and explored the underlying molecular mechanisms., Results: Bioinformatic analysis and experimental evidence demonstrated that CD147 significantly contributed to the reprogramming of fatty acid metabolism in HCC cells mainly through two mechanisms. On one hand, CD147 upregulated the expression of sterol regulatory element binding protein 1c (SREBP1c) by activating the Akt/mTOR signaling pathway, which in turn directly activated the transcription of major lipogenic genes FASN and ACC1 to promote de novo lipogenesis. On the other hand, CD147 downregulated peroxisome proliferator-activated receptor alpha (PPARα) and its transcriptional target genes CPT1A and ACOX1 by activating the p38 MAPK signaling pathway to inhibit fatty acid β-oxidation. Moreover, in vitro and in vivo assays indicated that the CD147-mediated reprogramming of fatty acid metabolism played a critical role in the proliferation and metastasis of HCC cells., Conclusion: Our findings demonstrate that CD147 is a critical regulator of fatty acid metabolism, which provides a strong line of evidence for this molecule to be used as a drug target in cancer treatment., (Copyright © 2015 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

The enumeration of circulating tumor cells (CTCs) provides important prognostic values in patients with metastatic breast cancer. Recent studies indicate that individual CTCs form clusters and these CTC-clusters play an important role in tumor metastasis. We aimed to assess whether quantification of CTC-clusters provides additional prognostic value over quantification of individual CTCs alone. In 115 prospectively enrolled advanced-stage (III and IV) breast cancer patients, CTCs and CTC-clusters were counted in 7.5 ml whole blood using the CellSearch system at baseline before first-line therapy. The individual and joint effects of CTC and CTC cluster counts on patients' progression-free survival (PFS) were analyzed using Cox proportional hazards modeling. Of the 115 patients, 36 (31.3 %) had elevated baseline CTCs (≥5 CTCs/7.5 ml) and 20 (17.4 %) had CTC-clusters (≥2 CTCs/7.5 ml). Patients with elevated CTCs and CTC-clusters both had worse PFS with a hazard ratio (HR) of 2.76 [95 % confidence interval (CI) 1.57-4.86, P log-rank = 0.0005] and 2.83 (1.48-5.39, P log-rank = 0.001), respectively. In joint analysis, compared with patients with <5 CTCs and without CTC-clusters, patients with elevated CTCs but without clusters, and patients with elevated CTCs and with clusters, had an increasing trend of progression risk, with an HR of 2.21 (1.02-4.78) and 3.32 (1.68-6.55), respectively (P log-rank = 0.0006, P trend = 0.0002). The additional prognostic value of CTC-clusters appeared to be more pronounced in patients with inflammatory breast cancer (IBC), the most aggressive form of breast cancer with the poorest survival. Baseline counts of both individual CTCs and CTC-clusters were associated with PFS in advanced-stage breast cancer patients. CTC-clusters might provide additional prognostic value compared with CTC enumeration alone, in patients with elevated CTCs.

Current clinical guidelines state that the use of erythropoiesis-stimulating agents (ESAs) may be considered to treat chemotherapy-induced anemia in the non-curative setting to alleviate anemia-related symptoms. However, no convincing survival benefit has been demonstrated to support the use of ESAs in these patients. Using the comprehensive data collected in the National Cancer Institute (NCI)-surveillance epidemiology and end results (SEER) and Medicare-linked database, we analyzed the effect of ESA use on the short-term (18-month) and long-term (60-month) survival rates of chemotherapy-treated metastatic breast cancer patients. Confounding variables were adjusted using a propensity score approach. We also analyzed the effects of ESA on the survival of patients receiving trastuzumab, a commonly prescribed targeted therapy agent in treating HER2-positive tumors. Metastatic breast cancer patients who received ESA treatment exhibited similar 60-month survival rate to those without ESA treatment (22.8 vs. 24.9%, p = 0.8). ESA-treated patients had a trend toward better 18-month survival [crude hazard ratio (HR) 0.86, 95% confidence intervals (CI) 0.68-1.09, p = 0.21]. This protective effect during the first 18 months of chemotherapy became marginally significant after adjusting for the propensity of receiving ESAs (HR 0.80, 95% CI 0.63-1.01, p = 0.070). An interaction effect between ESA and trastuzumab on patient survival was noticeable but not statistically significant. ESAs did not negatively affect the long-term survival of metastatic breast cancer patients. Moreover, ESAs improved patients' survival during the first 18 months of chemotherapy treatment. These findings endorse the current clinical guideline. Given the short survival of these patients, the potential short-term beneficial effects of ESAs are clinically meaningful.

Background and Aims: Accumulating evidence has indicated that variations of mitochondrial DNA (mtDNA) content may affect the susceptibility to hepatocellular carcinoma (HCC). However, no study has been conducted to evaluate the association of circulating mtDNA content and the risk of liver cirrhosis, a leading cause of HCC., Methods: We conducted a nested case-control study including 136 cirrhotic hepatitis B virus (HBV) cases and 136 frequency-matched non-cirrhotic HBV controls. We determined mtDNA content in serum DNA using quantitative real-time PCR and analyzed its association with cirrhosis risk., Results: We found that cirrhotic HBV patients had significantly lower levels of mtDNA content than non-cirrhotic HBV controls (P = 0.0184). Compared to patients with high mtDNA content, those with low mtDNA content had a 2.25-fold increased risk of cirrhosis [odds ratio (OR) 2.25, 95 % confidence interval (CI) 1.26-4.02]. This association exhibited a significant dose relationship as evidenced in both tertile and quartile analyses (P for trend = 0.0018 and 0.0008, respectively). Stratified analyses showed that the association was prominent in younger patients (P = 0.0122), males (P = 0.0069), never smokers (P = 0.0063), never drinkers (P = 0.0078), patients with a family history of HBV infection (P = 0.0062), and patients with low values of aspartate aminotransferase to platelet ratio index (APRI), a commonly used noninvasive marker for cirrhosis (P = 0.0109). Moreover, a joint effect was observed between low mtDNA content and high APRI values on cirrhosis risk (OR 24.07, 95 % CI 6.72-86.24)., Conclusions: Low circulating mtDNA content may confer an increased cirrhosis risk in HBV patients. Further prospective studies are warranted to validate these findings and explore the clinical significance.

Non-small cell lung cancer (NSCLC) is characterized by poor prognosis and only a few molecular markers may be potentially used to predict the risk of progression. This study aims to assess the effects of single nucleotide polymorphisms (SNPs) in the CD98 gene on prognosis of NSCLC patients. We genotyped three potential functional SNPs in CD98 gene using Sequenom iPLEX genotyping system in a cohort of 482 NSCLC patients. Multivariate cox proportional hazards model and Kaplan-Meier curve were used for the survival analysis. The variant-containing genotypes of rs1059292 in 5'-flanking region of CD98 gene were significantly associated with an increased risk of death in the multivariate analysis (Hazard ratio [HR], 1.49; 95% confidence interval [95% CI]: 1.04-2.14 in a dominant model). In stratified analysis, the association remained significant in patients with poor differentiation (HR=1.81, 95% CI=1.01-3.25). In addition, rs1059292 also showed a borderline significant association with T stage (OR=1.49; 95% CI: 0.96-2.35) and N stage (OR=1.53; 95% CI: 0.98-2.39). Functional analysis demonstrated that variant genotype of SNP rs1059292 significantly enhanced the transcription activity of CD98 gene promoter. Our data suggest that genetic variation of rs1059292 in CD98 gene may affect clinical outcome of NSCLC in Chinese population., (© 2014 Wiley Periodicals, Inc.)

Non-small cell lung cancer (NSCLC) is characterized by poor prognosis and only a few molecular markers may be potentially used to predict clinical outcomes. Previous studies have demonstrated that monocarboxylate transporters (MCTs) play important roles in the development and progression of many cancers. The purpose of this study was to assess the effects of single nucleotide polymorphisms (SNPs) of MCT genes on prognosis of NSCLC patients in Chinese Han population. Nine functional SNPs in MCT1, MCT2, and MCT4 genes were selected and genotyped using Sequenom iPLEX genotyping system in 500 Chinese NSCLC patients receiving surgery. Multivariate Cox proportional hazards model and Kaplan-Meier curve were used for the prognostic analysis. TT genotype of SNP rs1049434 (MCT1) was significantly associated with better overall survival (OS) (HR = 0.56, P = 0.026) and recurrence-free survival (RFS) (HR = 0.57, P = 0.016) of NSCLC patients. TT genotype of another SNP rs995343 (MCT2) exhibited an association with worse RFS of NSCLC patients (HR = 1.46, P =  .039). Unfavorable genotypes of SNP rs1049434 and rs995343 showed a significant cumulative effect on OS and RFS of NSCLC patients. Moreover, we found that patients carrying AA+AT genotypes of rs1049434 showed significant OS and RFS benefits from adjuvant chemotherapy, but those with TT genotype did not. Our findings suggest that SNPs in MCT1 and MCT2 genes may affect clinical outcomes and can be used to predict the response to adjuvant chemotherapy in NSCLC patients who received surgical treatment once validated in future study.

Alterations of activity and expression in tricarboxylic acid (TCA) cycle key enzymes have been indicated in several malignancies, including hepatocellular carcinoma (HCC). They play an important role in the progression of cancer. However, the impact of single nucleotide polymorphisms (SNPs) in genes encoding these key enzymes on the recurrence of HCC has not been investigated. In this study, we genotyped 17 SNPs in genes encoding TCA cycle key enzymes and analyzed their association with recurrence-free survival (RFS) in a cohort of 492 Chinese HCC patients by Cox proportional hazard model and survival tree analysis. We identified 7 SNPs in SDHC, SDHD, FH, and IDH2 genes to be significantly associated with the RFS of HCC patients. Moreover, all these SNPs were associated with the early recurrence (within 2 years after surgery) risk of diseases. Cumulative effect analysis showed that these SNPs exhibited a dose-dependent effect on the overall and early recurrence. Further stratified analysis suggested that number of risk genotypes modified the protective effect on HCC recurrence conferred by transcatheter arterial chemoembolization treatment. Finally, the survival tree analysis revealed that SNP rs10789859 in SDHD gene was the primary factor contributing to HCC recurrence in our population. To the best of our knowledge, we for the first time observed the association between SNPs in genes encoding TCA cycle key enzymes and HCC recurrence risk. Further observational and functional studies are needed to validate our findings and generalize its clinical usage.

AT-rich interactive domain 2 (ARID2), catenin (cadherin-associated protein), beta 1, 88kDa (β-catenin), tumor protein 53 (p53), and phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (p110α) mutations are implicated in hepatocellular carcinoma (HCC); and previous work has contributed to thorough molecular characterization of these events. However, studies that assess the impact of these mutations on downstream protein expression, especially those that evaluate all 4 cancer markers simultaneously, are relatively lacking. Hence, the present study uses immunohistochemistry to assess protein expression patterns of ARID2, β-catenin, p53, and p110α in HCCs and adjacent nonneoplastic cirrhotic tissues from 58 explanted livers. Notably, this study is the first to our knowledge to investigate ARID2 protein expression in the liver. The frequency of ARID2 mutations detected using our immunohistochemistry method was similar to that reported in previous molecular studies. Furthermore, we found that loss of ARID2 protein expression may be associated with recurrence, although further studies must be done to validate these findings in a larger population. We found that expression patterns of the 4 cancer markers were independent of each other, suggesting separate pathways of hepatocarcinogenesis. We also did not observe an association between viral etiology and protein expression. Consistent with previous studies, overexpression of p53 correlated with poor differentiation. Lastly, 17.5% of HCCs paradoxically had diffuse loss of the oncoprotein p110α compared with strong expression in background cirrhotic liver. The exact mechanism is unclear, but enigmatic loss of oncoprotein function has been described in other carcinomas and could potentially have significant implications for the use of targeted mechanistic target of rapamycin (serine/threonine kinase) drug therapies., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Introduction: Non-small cell lung cancer (NSCLC) is characterized by poor prognosis and only a few molecular markers may be potentially used to predict the outcome. Metabolic reprogramming is a hallmark of cancer, including the alterations of tricarboxylic acid (TCA) cycle key enzymes. However, the significance of single nucleotide polymorphisms (SNPs) in genes encoding these key enzymes has not been investigated in NSCLC., Patients and Methods: In this study, we genotyped 18 potentially functional SNPs in 7 genes belonging to 3 TCA cycle enzyme families (SDH, FH and IDH) using Sequenom iPLEX genotyping system in a cohort of 500 NSCLC patients. Multivariate Cox proportional hazards model and Kaplan-Meier curve were used for the survival analysis., Results: Our results showed that SDHC gene: SNP rs12064957, IDH2 gene: SNP rs11540478 and FH gene: SNP rs1414493 were associated with overall survival (OS) and SDHA gene: SNP rs13173911, IDH2 gene: SNP rs4932158 were associated with recurrence-free survival (RFS) of NSCLC patients. Unfavorable genotypes of these SNPs showed a significant cumulative effect on OS and RFS of NSCLC patients (both P<0.001). Furthermore, survival tree analysis indicated that FH: rs1414493 was the primary risk factor contributing to OS of NSCLC patients and the IDH2: rs4932158 was the primary risk factor contributing to RFS of NSCLC patients., Conclusion: Our data suggest that SNPs in TCA cycle key enzyme genes may serve as potential biomarkers to predict the outcomes of NSCLC. Further studies with different ethnicities are needed to validate our findings and generalize their clinical utility., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Cancer patients undergo routine clinical monitoring with an array of blood tests that may carry long-term prognostic information. We aimed to develop a new prognostic model predicting survival for patients with advanced non-small cell lung cancer (NSCLC), based on laboratory tests commonly performed in clinical practice. A cohort of 1,161 stage IIIB or IV NSCLC patients was divided into training (n = 773) and testing (n = 388) cohorts. We analyzed the associations of 32 commonly tested laboratory variables with patient survival in the training cohort. We developed a model based on those significant laboratory variables, together with important clinical variables. The model was then evaluated in the testing cohort. Five variables, including albumin, total protein, alkaline phosphatase, blood urea nitrogen and international normalized ratio, were significantly associated with patient survival after stepwise selection. A model incorporating these variables classified patients into low-, medium- and high-risk groups with median survival of 16.9, 7.2 and 2.1 months, respectively (p < 0.0001). Compared with low-risk group, patients in the medium- and high-risk groups had a significantly higher risk of death at 1 year, with hazard ratio (HR) of 1.95 (95% CI 1.62-2.36) and 5.22 (4.30-6.34), respectively. These results were validated in the testing cohort. Overall, we developed a prognostic model relying entirely on readily available variables, with similar predictive power to those which depend on more specialized and expensive molecular assays. Further study is necessary to validate and further refine this model, and compare its performance to models based on more specialized and expensive testing., (© 2014 UICC.)

Purpose: A survival disparity of black versus white breast cancer patients has been extensively documented but not adequately explained. Blacks and whites also have significant differences in hematologic traits including hemoglobin (HGB). However, a link between survival disparity and hematologic differences has not been reported. We aimed to explore the effect of pre-treatment hematologic variables on this survival disparity., Methods: We sequentially matched 443 black patients, using a minimum distance approach, to four different sets of 443 whites on demographics (age, year of diagnosis, smoking, and drinking status), tumor presentation (all demographic variables plus tumor stage, grade, and hormone receptor status), treatment (all presentation variables plus surgery, chemotherapy, radiation therapy, and hormone therapy), and presentation plus pre-treatment hematologic variables. Racial survival for each matched dataset was analyzed by Cox proportional hazards model., Results: We found that white patients matched on demographic characteristics had more favorable survival than blacks [hazard ratio (HR) 0.57, 95 % confidence interval (CI) 0.42-0.77, p log-rank = 0.0002]. Presentation match diminished this disparity [HR 0.72 (0.54-0.95), p log-rank = 0.0199], which was not further reduced in treatment match [HR 0.73 (0.55-0.96), p log-rank = 0.0249]. However, the survival disparity was largely reduced when pre-treatment level of HGB or red blood cell distribution width was further matched in addition to presentation match [HR 0.83 (0.64-1.09), p log-rank = 0.1819 and HR 0.83 (0.64-1.09), p log-rank = 0.1760, respectively]., Conclusions: We found that in our patient population, differences in tumor presentation and certain pre-treatment hematologic traits, but not treatment, were associated with the survival disparity between black and white breast cancer patients.