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Supplementary Table 1 from Novel Susceptibility Loci for Second Primary Tumors/Recurrence in Head and Neck Cancer Patients: Large-Scale Evaluation of Genetic Variants

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. Cancer Prev Res; 9(2); 172–9. ©2015 AACR.

Table S1: Detailed information on all CpGs of interest Table S2: Cancer-associated CpGs in TRGs by pathway at FDR < 0.05, with unspecified skin malignancies excluded Table S3: Methylation trajectory over time by cancer status Table S4: Logistic regression results for DNA methylation measured over 8 years before diagnosis/censoring Table S5: Regulatory elements enrichment analysis results Figure S1: Plots of Beta-coefficients by genomic location for all CpGs on six genes of interest Figure S2: DNA methylation by years to cancer diagnosis/censoring and cancers status with 95% confidence intervals Figure S3: DNA methylation by years to cancer diagnosis/censoring and cancer status for CpG additional sites Figure S4: Regulatory enrichment analysis results

Supplementary Table 5 from Novel Susceptibility Loci for Second Primary Tumors/Recurrence in Head and Neck Cancer Patients: Large-Scale Evaluation of Genetic Variants

Supplementary Table 6 from Novel Susceptibility Loci for Second Primary Tumors/Recurrence in Head and Neck Cancer Patients: Large-Scale Evaluation of Genetic Variants

Data description and detailed statistical information regarding model development. Supplementary Table 1. Patient information on samples from the University of Michigan. Supplementary Table 2. Patient information on samples from HALT-C. Supplementary Table 3. Patient information on samples from EDRN. Supplementary Table 4. Patient information on samples from Thomas Jefferson University. Supplementary Table 5. Patient information on samples from the University of Texas Southwestern Medical Center. Supplementary Table 6. Odds ratios for each predictor in univariate and multivariate logistic regressions. Supplementary Table 7. Indices of goodness-of-fit and apparent validation of candidate logistic regression models. Supplementary Table 8. Performance of leave one out cross validation (LOOCV). Supplementary Table 9. Cross Validation of bootstrap method. Supplementary Table 10. AUCs and IDIs from 3-fold Cross-Validation. Supplementary Table 11. Summary statistics of Doylestown model. Supplementary Table 12. Comparison of logistic regression, classification and regression tree (CART) and conditional inference tree (CTREE). Supplementary Figure 1. The distribution of AFP in each data set by cases and controls: Supplementary Figure 2. Quartiles for AFP in the individual patient sets. Supplementary Figure 3. Histogram of predictions and observe occurrence proportion of HCC for the top 4 models.

Supplementary Figure 1 from Novel Susceptibility Loci for Second Primary Tumors/Recurrence in Head and Neck Cancer Patients: Large-Scale Evaluation of Genetic Variants

Supplementary Table 7 from Novel Susceptibility Loci for Second Primary Tumors/Recurrence in Head and Neck Cancer Patients: Large-Scale Evaluation of Genetic Variants

Perspective on this Article from Novel Susceptibility Loci for Second Primary Tumors/Recurrence in Head and Neck Cancer Patients: Large-Scale Evaluation of Genetic Variants

Perspective on This Article from Global Assessment of Genetic Variation Influencing Response to Retinoid Chemoprevention in Head and Neck Cancer Patients

Supplementary Table 4 from Novel Susceptibility Loci for Second Primary Tumors/Recurrence in Head and Neck Cancer Patients: Large-Scale Evaluation of Genetic Variants

Supplementary Table 3 from Novel Susceptibility Loci for Second Primary Tumors/Recurrence in Head and Neck Cancer Patients: Large-Scale Evaluation of Genetic Variants

Head and neck squamous cell carcinoma (HNSCC) patients are at an increased risk of developing a second primary tumor (SPT) or recurrence following curative treatment. 13-cis-retinoic acid (13-cRA) has been tested in chemoprevention clinical trials, but the results have been inconclusive. We genotyped 9,465 single nucleotide polymorphisms (SNP) in 450 patients from the Retinoid Head and Neck Second Primary Trial. SNPs were analyzed for associations with SPT/recurrence in patients receiving placebo to identify prognosis markers and further analyzed for effects of 13-cRA in patients with these prognostic loci. Thirteen loci identified a majority subgroup of patients at a high risk of SPT/recurrence and in whom 13-cRA was protective. Patients carrying the common genotype of rs3118570 in the retinoid X receptor (RXRA) were at a 3.33-fold increased risk (95% CI, 1.67–6.67) and represented more than 70% of the study population. This locus also identified individuals who received benefit from chemoprevention with a 38% reduced risk (95% CI, 0.43–0.90). Analyses of cumulative effect and potential gene–gene interactions also implicated CDC25C:rs6596428 and JAK2:rs1887427 as 2 other genetic loci with major roles in prognosis and 13-cRA response. Patients with all 3 common genotypes had a 76% reduction in SPT/recurrence (95% CI, 0.093–0.64) following 13-cRA chemoprevention. Carriers of these common genotypes constituted a substantial percentage of the study population, indicating that a pharmacogenetic approach could help select patients for 13-cRA chemoprevention. The lack of any alternatives for reducing risk in these patients highlights the need for future clinical trials to prospectively validate our findings. Cancer Prev Res; 4(2); 185–93. ©2011 AACR.

This study was aimed to identify novel susceptibility variants for second primary tumor (SPT) or recurrence in curatively treated early-stage head and neck squamous cell carcinoma (HNSCC) patients.We constructed a custom chip containing a comprehensive panel of 9,645 chromosomal and mitochondrial single nucleotide polymorphisms (SNP) representing 998 cancer-related genes selected by a systematic prioritization schema. Using this chip, we genotyped 150 early-stage HNSCC patients with and 300 matched patients without SPT/recurrence from a prospectively conducted randomized trial and assessed the association of these SNPs with risk of SPT/recurrence.Individually, six chromosomal SNPs and seven mitochondrial SNPs were significantly associated with risk of SPT/recurrence after adjustment for multiple comparisons. A strong gene-dosage effect was observed when these SNPs were combined, as evidenced by a progressively increasing SPT/recurrence risk as the number of unfavorable genotypes increased (P for trend < 1.00 × 10−20). Several polygenic analyses suggest an important role of interconnected functional network and gene-gene interaction in modulating SPT/recurrence. Furthermore, incorporation of these genetic markers into a multivariate model improved significantly the discriminatory ability over the models containing only clinical and epidemiologic variables.This is the first large-scale systematic evaluation of germ-line genetic variants for their roles in HNSCC SPT/recurrence. The study identified several promising susceptibility loci and showed the cumulative effect of multiple risk loci in HNSCC SPT/recurrence. Furthermore, this study underscores the importance of incorporating germ-line genetic variation data with clinical and risk factor data in constructing prediction models for clinical outcomes.

Supplementary Data from MicroRNA Expression Signatures in Barrett's Esophagus and Esophageal Adenocarcinoma

Purpose: Esophageal adenocarcinoma is a highly aggressive malignancy that frequently develops from Barrett's esophagus, a premalignant pathologic change occurring in the lower end of the esophagus. Identifying Barrett's esophagus patients at high risk of malignant transformation is essential to the prevention of esophageal adenocarcinoma. Although microRNA (miRNA) expression signatures have been associated with the etiology and prognosis of several types of cancers, their roles in the development of esophageal adenocarcinoma have not been extensively evaluated.Experimental Design: In this study, we analyzed the expression patterns of 470 human miRNAs using Agilent miRNA microarray in 32 disease/normal-paired tissues from 16 patients diagnosed with Barrett's esophagus of either low- or high-grade dysplasia, or esophageal adenocarcinoma.Results: Using unsupervised hierarchical clustering and class comparison analyses, we found that miRNA expression profiles in tissues of Barrett's esophagus with high-grade dysplasia were significantly different from their corresponding normal tissues. Similar findings were observed for esophageal adenocarcinoma, but not for Barrett's esophagus with low-grade dysplasia. The expression patterns of selected miRNAs were further validated using quantitative reverse transcription real-time PCR in an independent set of 75 pairs of disease/normal tissues. Finally, we identified several miRNAs that were involved in the progressions from low grade-dysplasia Barrett's esophagus to esophageal adenocarcinoma.Conclusions: We showed that miRNAs were involved in the development and progression of esophageal adenocarcinoma. The identified significant miRNAs that may become potential targets for early detection, chemoprevention, and treatment of esophageal cancer. (Clin Cancer Res 2009;15(18):5744–52)

Background: Cumulative data have shown that microRNAs (miRNA) are involved in the etiology and prognosis of colorectal cancer (CRC). Genetic polymorphisms in pre-miRNA genes may influence the biogenesis and functions of their host miRNAs. However, whether these polymorphisms are associated with CRC prognosis remains unknown.Methods: We analyzed the effects of seven single-nucleotide polymorphisms (SNP) in pre-miRNA genes on the prognosis of a Chinese population with 408 CRC patients with surgically-resected adenocarcinoma.Results: Two SNPs were identified to be significantly associated with recurrence-free survival and overall survival of the patients. The most significant SNP was rs6505162 in pre-miR-423. Compared with the homozygous wild-type genotype, the variant-containing genotypes of this SNP were significantly associated with both the overall survival (HR = 2.12, 95% CI = 1.34–3.34, P = 0.001) and the recurrence-free survival (HR = 1.59, 95% CI = 1.08–2.36, P = 0.019). Another SNP, rs4919510 in pre-miR-608, was also associated with altered recurrence-free survival (HR = 0.61, 95% CI = 0.41–0.92, P = 0.017). These effects were evident only in patients receiving chemotherapy but not in those without chemotherapy. In addition, the combined analysis of the two SNPs conferred a 2.84-fold (95% CI = 1.50–5.37, P = 0.001) increased risk of recurrence and/or death. Similarly, this effect was only prominent in those receiving chemotherapy (P < 0.001) but not in those without chemotherapy (P = 0.999).Conclusions: Our data suggest that genetic polymorphisms in pre-miRNA genes may impact CRC prognosis especially in patients receiving chemotherapy, a finding that warrants further independent validation.Impact: This is one of the first studies showing a prognostic role of pre-miRNA gene SNPs in CRC. Cancer Epidemiol Biomarkers Prev; 21(1); 217–27. ©2011 AACR.

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Background: Although different patterns and changes in DNA methylation play an important role in cancer progression and tumor subtype differentiation, methylation remains relatively less understood in the context of tumor heterogeneity. Intra-tumor heterogeneity influences chemotherapy response, resistance development, and metastatic progression, and can be identified with liquid biopsy, a non-invasive approach to monitor cancer progression and provide predictive information. In this study, we sequenced circulating tumor cells (CTCs) to interrogate whole-genome methylation at single-cell level and single-base resolution. Methods: We enumerated CTCs in 7.5ml of whole blood samples from over 130 metastatic breast cancer patients using CellSearch, and selected blood samples with more than 20 CTCs to be further processed by DEPArray. After single-cell library construction using the Swift Accel-NGS Adaptase Module, whole-genome bisulfite sequencing (WGBS) was performed. The sequencing data were then aligned to the reference genome, and the methylation information was extracted by Bismark. We compared methylation profiles between CTC and WBC samples from each patient to identify differentially methylated regions (DMRs) using Methylkit. Multiple-comparison was conducted by SMART2 to identify DMRs within CTCs. Heatmap was plotted based on the regional methylation rates of DMRs. CpG and genic annotations were performed by annotatr. The pathway and function enrichment analyses (KEGG and GO) were conducted using clusterProfiler. Genomic region-based enrichment was conducted using LOLA. t-SNE clustering was used to investigate intra-patient heterogeneity. Results: A total of 376 cells from nine patients that passed our selection criteria were isolated for WGBS. The mean sequencing depth of all single cells was 0.8×, and an average mapping rate of 54% was achieved. We first compared CTCs and WBCs, and observed a global hypo-methylation pattern in CTCs (average methylation rate 68.8% in CTCs vs. 76.7% in WBCs). Moreover, approximately 2,000 highly differentially methylated regions were found in each patient, with hundreds of hypo- or hyper-methylated genes related to these DMRs. Hypo-methylated DMRs showed genomic region-based enrichment in breast-, prostate-, and fibroblast-related region sets. Then, we investigated methylation heterogeneity within CTCs, where t-SNE clustering identified four different subsets in one representative patient. About 1,500 hypo-methylated DMRs were found, differentiating those four subgroups. KEGG pathway analysis indicated enrichment in the Rap1 signaling pathway and focal adhesion. GO enrichment analysis highlighted the regulation of GTPase activity and membrane potential. Conclusions: Our study identified differentially methylated regions in CTCs of metastatic breast cancer patients, and demonstrated intra-patient heterogeneity based on cellular methylation information. Future studies are warranted to validate our findings and explore their biological mechanisms and clinical relevance. Citation Format: Rui Luo, Weelic Chong, Zhenchao Zhang, Maysa Abu-Khalaf, Daniel Silver, Frederick Fellin, Rebecca Jaslow, AnaMaria Lopez, Terrence Cescon, Kevan Ip, Ronald Myers, Qiang Wei, Bingshan Li, Chun Wang, Hushan Yang. Whole-genome bisulfite sequencing of single circulating tumor cells identifies cellular methylation heterogeneity in metastatic breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-06-05.

Background: Structural variation is a hallmark of breast cancer. Previous single-cell genomic analysis of 10 untreated early-stage primary TNBC tumors identified only one or two subclones per tumor, found that copy number variation (CNV) occurs early in the evolution of the tumor, and asserted that CNV changes do not contribute to genomic variation at later time points of a tumor’s growth. This PCNE model (punctuated copy number evolution) is analogous to the big-bang theory of colorectal cancer. However, cancer exists in a dynamic environment, and systemic chemotherapy provides evolutionary pressure, making stasis quite unlikely. An investigation of late-stage cancers could shed light on CNV evolution. Methods: We performed a longitudinal, prospective observational study of over 130 patients with metastatic breast cancer, with regular follow-up and detailed treatment histories. CTCs were enumerated by CellSearch, and samples with >20 CTCs in 7.5ml of whole blood were further processed for single CTC isolation via DEPArray, single-cell library construction, and whole-genome sequencing. CNV counts for each cell were obtained using previously published binning methods (Ginkgo). Three mathematical models were used to evaluate the evolution of CNVs: a punctuated model, a gradual model, and a gradual-on-punctuated hybrid model. Results: In total, 150 patient-years of data were collected. Among samples with sufficient CTC counts for study inclusion, a total of nine patients and 376 cells were isolated for sequencing. A mean sequencing depth of 0.8× was achieved from each single cell. The degree of CNV heterogeneity varies from patient to patient. CNV changes occurred over time in a gradual manner on a background of punctuated changes. The adjusted R2 value for the punctuated model was 0.46, the adj R2 value for the gradual model was 0.76, and the hybrid model achieved an adj R2 of 0.99. Patient samples with large CNV heterogeneity across single cells tend to be from patients with longer treatment histories and from the last blood draw (i.e., the blood collection closest to patient’s point of death). We also identified multiple whole-genome duplication (WGD) events from the same patient, in contrast to previous findings that WGD events are usually early clonal events in most instances. Finally, X loss events are among the most frequent chromosomal aberrations identified in CTCs (in up to 80% of CTCs in some patients), which supports previous literature on the loss of the Barr body in hastening metastatic spread. Discussion and Conclusion: In contrast to the previous study, our findings support a gradual-on-punctuated hybrid model, and this model can explain how copy number changes can be a source from which tumors gain resistance to systemic therapies. The model explains how CNV heterogeneities are seen in patients with longer treatment histories, suggesting that CNV continues to be a source of genetic variation at the metastatic stage of the disease course. Citation Format: Weelic Chong, Rui Luo, Zhenchao Zhang, Maysa Abu-Khalaf, Daniel Silver, Frederick Fellin, Rebecca Jaslow, AnaMaria Lopez, Terrence Cescon, Ronald Myers, Mariel Becker, Qiang Wei, Bingshan Li, Chun Wang, Hushan Yang. Circulating tumor cells in metastatic breast cancer highlight potential role of copy number evolution in late-stage cancer mutational profile [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-06-02.

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.

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

Endometrial cancer (EC) is one of the most common gynecological malignancies. The aim of this study was to evaluate the combined prognostic value of D-dimer and fibrinogen in EC patients. We evaluated the prognostic value of preoperative plasma fibrinogen and D-Dimer concentration in EC patients using Kaplan-Meier curve analysis and Cox proportional hazards regression model. Kaplan-Meier curve analysis demonstrated that EC patients with higher levels of plasma fibrinogen or D-Dimer had a significantly poorer overall survival (OS) and recurrence-free survival (RFS). Furthermore, combination of fibrinogen and D-Dimer levels significantly improved the efficacy for predicting RFS of EC patient. Patients with higher fibrinogen or D-Dimer levels exhibited a significantly poorer RFS in both the univariate and multivariate analysis. In conclusions, the preoperative plasma fibrinogen and D-Dimer concentrations may serve as potential prognostic factors for EC patients, and combination of fibrinogen and D-Dimer concentration may be a potential marker for predicting RFS of EC patients.

Background: Circulating tumor cells (CTCs) and their clusters (CTC-clusters) are promising prognostic markers in breast cancer. Recently studies showed that platelets interact with tumor cells in the circulation and promote metastasis. However, no study has been reported to explore the effects of interactions between CTCs/CTC-clusters and platelets on the prognosis of breast cancer at the population level. Methods: A total of 146 female advanced-stage breast cancer patients were included in this study. CTCs and CTC-clusters were enumerated using the CellSearch system. Platelet count was obtained from laboratory tests. The individual and joint effects of CTCs/CTC-clusters and platelets on progression free survival (PFS) were evaluated using the Cox proportional hazards model. Results: Elevated CTCs (≥ 5 CTCs/7.5 mL), presence of CTC-clusters, or high platelet counts (≥ 400 × 109/L) were individually associated with PFS (hazard ratio [HR] 2.69, 2.71, and 1.79, respectively). We further stratified patients into three risk groups according to the status of CTCs/CTC-clusters in combination with the level of platelet counts, including: 1) low-risk group: for CTC-related analyses, low CTCs (< 5 CTCs/7.5 mL) and platelet counts (< 400 × 109/L); for CTC-cluster-related analyses, absence of CTC-cluster and low platelet counts; 2) medium-risk group: for CTC-related analyses, either elevated CTCs (≥ 5 CTCs/7.5 mL) or high platelet counts (≥ 400 × 109/L), but not both; for CTC-cluster-related analyses, either presence of CTC-clusters or high platelet counts, but not both; 3) high-risk group: for CTC-related analyses, both elevated CTCs and high platelet counts; for CTC-cluster-related analyses, presence of CTC-clusters and high platelet counts. A significantly increased risk for disease progression was observed in high-risk patients, especially in patients with both CTC-clusters and high platelet counts (adjusted HR 16.06, P = 0.0003, log-rank P< 0.0001). Furthermore, we identified a multiplicative interaction between CTC-clusters and platelets on PFS (P for interaction = 0.0483), and the patients with both CTC-clusters and high platelet counts had the shortest time of development of new metastatic sites. Conclusions: Our results indicated the effects of interactions between the presence of CTC-clusters and high platelet counts on tumor progression and dissemination in breast cancer. Future studies are warranted to validate our findings and explore their biological mechanisms and clinical relevance. Citation Format: Chun Wang, Zhong Ye, Zhenchao Zhang, Maysa Abu-Khalaf, Daniel Silver, AnaMaria Lopez, Frederick Fellin, Saveri Bhattacharya, Rebecca Jaslow, Kaelan Yao, Darayus Toorkey, Neha Pancholy, Ronald Myers, Hushan Yang. Presence of circulating tumor cell clusters and elevated platelet counts interactively predict disease progression in advanced-stage breast cancer patients [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-10-23.

Background: Palbociclib is the first-in-class cyclin dependent kinase (CDK) 4/6 inhibitor approved for the treatment of patients (pts) with hormone receptor (HR) positive/human epidermal growth factor receptor 2 (HER2) negative metastatic breast cancer (MBC). Despite an initial response, pts eventually develop resistance to therapy; however, the underlying molecular mechanisms remain elusive. This study aimed to identify genomic aberrations in circulating tumor DNA (ctDNA) associated with treatment resistance and disease progression in MBC pts receiving palbociclib. Methods: Blood samples were collected from pts with HR+/HER2− MBC who were treated with palbociclib plus endocrine therapies (letrozole or fulvestrant). Circulating cell-free DNAs were isolated from plasma and assayed by targeted-capture next-generation sequencing using an in-house developed panel that covers the exons in 92 breast cancer-related genes. Unique molecular index (UMI)-based sequencing was performed using the Illumina HiSeq 4000 system. Progression free survival (PFS) differences between pts with different mutation status were compared using the log-rank test. The Cox proportional hazards model with time-dependent covariates was used to evaluate associations between mutations and PFS. Results: The study included 16 pts with a median age of 54.7 years (range: 32.6-74.8 years). A total of 42 blood samples were collected, including 16 baseline samples before the initiation of palbociclib +/- endocrine therapies (within 1.5 months prior to initiation), 10 samples during treatment (1.0-3.9 months after initiation), and 16 after treatment completion (within 1.2 months after completion). During a median follow-up of 23.7 weeks (range: 6.0-99.6 weeks), 8 pts had disease progression at initial blood collection after treatment initiation, and 11 had disease progression at final blood collection during follow-up. Among the 3 pts without progression, 2 were lost to follow-up and 1 had a change in regimen due to adverse events. Mutations were detected in 77 out of 92 genes. Mutations were identified for PIK3CA, ALK, ERBB2, NOTCH1, IDH1 in the baseline samples of 7, 3, 7, 6, and 12 patients, respectively. A shorter time to progression was observed in the pts with PIK3CA, ALK, ERBB2, or NOTCH1 mutations (log-rank P = 0.011, 0.016, 0.057, and 0.059, respectively), whereas those pts with IDH1 mutations had a prolonged PFS (log-rank P = 0.005). Similar results were obtained when the events and survival time at the final blood collection were used for analyses (log-rank P = 0.007, 0.004, 0.046, 0.023, and 0.001 for PIK3CA, ALK, ERBB2, NOTCH1, and IDH1 mutations, respectively). To analyze the association between a given mutation and disease progression, we separated pts into four risk groups according to the status change of the mutation prior to and after treatment: Group 1, persistent absence of the mutation; Group2, loss of the mutation after treatment; Group 3, acquisition of the mutation after treatment; Group 4, persistent presence of the mutation. Longitudinal analyses showed that PIK3CA E542K, ERBB2 P1035H, and NOTCH1 K1069Q mutations were associated with unfavorable survival. For PIK3CA E542K mutation, pts in Group 2 had an increased risk for progression, as compared with those in Group 1 (hazard ratio [HR] =9.90, P = 0.110). For ERBB2 P1035H and NOTCH1 K1069Q mutations, when compared with pts in Group 1, those in Group 4 had the highest risk of progression (HR = 17.24, P = 0.049; HR = 19.17, P = 0.043, respectively), whereas pts in Group 2 had a non-significant slightly increased risk. Conclusions: Results of this study show that genomic aberrations in ctDNA were associated with progression in MBC pts receiving palbociclib-containing regimens. Further research is needed to validate these findings in larger independent studies. Citation Format: Maysa M Abu-Khalaf, Chun Wang, Zhong Ye, Zhenchao Zhang, Daniel Silver, Frederick Fellin, Saveri Bhattacharya, Rebecca Jaslow, Terrence Cescon, AnaMaria Lopez, Ronald Myers, Qiang Wei, Bingshan Li, Hushan Yang. Genomic aberrations in circulating tumor DNAs associated with progression in palbociclib-treated metastatic breast cancer patients [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-10-27.

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.

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 underling mechanism by Drp1 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 microscope and confocal microscopy. Mitochondrial function was evaluated by mitochondrial membrane potential, ROS and ATP measurement. Xenograft nude mice model was performed in BALB/c nude mice to confirm 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, which is recognized as cytosolic mtDNA stress. Mechanistically, cytosolic mtDNA stress 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 mitochondrial DNA stress, which subsequently activates the cGAS-STING pathway, triggers autophagy and promotes ESCC progression.

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.

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: Since IBC is rare and burdened by a particularly unfavorable prognosis, biomarkers able to enhance diagnosis and risk assessment are of pivotal importance and a current unmet need. The aim of this study is to integrate standard clinico-pathological features with CTCs-based biomarkers for a more objective and detailed characterization of IBC. Methods: This study analyzed retrospectively 251 Advanced Breast Cancer (BC) patients (pts) longitudinally characterized for CTCs and CTCs-based biomarkers at Thomas Jefferson University (Philadephia, PA) and Northwestern University (Chicago. IL). CTCs were enumerated through the CellSearch system (Menarini Silicon Biosystems), and characterized for HER2 expression using the CellSearch CXC Kit. Pts were defined as stage IV aggressive based on the previously reported ≥5 CTCs cut-off (Davis et al. 2018). Associations between clinical features, CTC-derived biomarkers and IBC were tested through uni and multivariate logistic regression. Survival was tested though log-rank test. Results: Within the analyzed cases, 46% were diagnosed with IBC and among them, 38% was stage IV aggressive. CTC clusters (CTC_CL) were detectable in 12.5% of pts and HER2 positive CTCs (HER2_CTC) in 29.5%. Notably, IBC patients (pts) had a significantly lower CTC count with respect to non-IBC (median 2.5 vs 0 respectively for non-IBC and IBC; P=0.019). BC subtype (HER2 positive BC: OR 2.97; Triple negative BC: OR 2.13), liver and bone involvement (liver: OR 0.46; bone involvement: OR 0.31) were the only significant clinico-pathological features associated with IBC at univariate logistic regression. Interestingly, a marginal significance was observed for soft tissue involvement (OR 1.65, 95%CI 0.95 - 2.87, P=0.07). Stage IV aggressive and presence of HER2_CTC at baseline were moreover inversely associated with IBC. The multivariate model confirmed the significant association between IBC and HER2 positive BC subtype (OR 2.64, 95%CI 1.08 - 6.48, P=0.034), absence of bone involvement (OR 0.31, 95%CI 0.14 - 0.68, P=0.003) and absence of HER2_CTC (OR 0.38, 95%CI 0.15 - 0.98, P=0.045). The baseline detection of CTC_CL was a strong predictor of prognosis for OS in IBC pts (median OS (mOS) 7.6 months (mts) vs not reached (NR) respectively for detectable vs non-detectable CTC_CL; P Conclusion: HER2_CTC and in particular CTC_CL are promising prognostic predictors in IBC. Stage IV aggressive IBC pts could benefit from a longitudinal CTCs assessment, being more prone to develop CTC_CL and therefore at higher risk of rapid disease progression. Probably due to the tropism for soft tissue, IBC is characterized by a lower number of HER2_CTC. Citation Format: Gerratana L, Zhang Q, Wang C, Shah A, Davis AA, Ye Z, Zhang Y, Abu-Khalaf M, Flaum L, Strickland K, Rossi G, Behdad A, Gradishar W, Platanias L, Yang H, Cristofanilli M. How is inflammatory breast cancer (IBC) different? Integration of clinico-pathological features and circulating tumor cells (CTCs)-based biomarkers for disease and prognostic assessment [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-17-03.

Background: The biological characteristics conferring Inflammatory BC's (IBC) distinctive and aggressive clinical features are currently not fully clarified. The aim of this study is to dissect IBC's biology through the integration of DNA and CTC-based circulating biomarkers. Methods: This study retrospectively analyzed 251 Advanced BC (ABC) patients (pts) treated and longitudinally characterized for CTCs and circulating tumor DNA (ctDNA) at Thomas Jefferson University (Philadephia, PA) and Northwestern University (Chicago, IL). CTCs were enumerated through CellSearch (Menarini Silicon Biosystems), and characterized for HER2 expression using the CellSearch CXC Kit, while ctDNA was analyzed using the Guardant360 NGS assay (Guardant Health) and its percentage (%ctDNA) was classified based on the previously reported cut-off of 5.7% (Gerratana et al 2018). A subset of 117 pts was further characterized for circulating cell-free DNA (ccfDNA) through Qubit® dsDNA HS quantitation Assay (Thermo Fisher Scientific) and quantitative real-time PCR assay for ALU DNA repeats on chromosome 1.Associations between clinical characteristics, CTCs-derived biomarkers and IBC were explored through Fisher's exact test; survival was tested though Cox regression and log-rank test. Results: Of the total 251 pts, 115 were diagnosed with IBC. Among the 117 patients characterized for ccfDNA, 70 had IBC. Median ccfDNA was 1.59 for IBC (IQR 1.02-3.19) and 2.37 for non-IBC (nIBC) (IQR 1.13-3.52), P=0.27. Consistent results were observed for %ctDNA levels (median value: 2 vs 1.6). The impact on OS of ccfDNA after log transformation was significant for the total population (HR 1.73 95%CI: 1.11-2.69) but not in IBC pts (HR 1.40 95%CI: 0.84-2.34). On the other hand, ctDNA high pts had a significantly worse OS (nIBC: HR 5.34 95%CI: 1.70-18.81 P=0.004; IBC: HR 4.05 95%CI: 1.91-8.58 P< 0.001). In the ctDNA high subgroup no differences in total number of CTCs were observed between IBC and nIBC, while significantly lower CTCs were observed in ctDNA low IBC pts (P=0.0097). The ctDNA low IBC subgroup had a higher incidence of HER2 positive BC (P=0.003) and a significantly lower incidence of CTCs clusters (P=0.006), HER2 positive CTCs (P=0.041). Notably, no associations were observed with stage at baseline, number of metastatic sites, liver, lung and visceral involvement. On the other hand, the ctDNA_high IBC subgroup was characterized by a lower incidence in liver, bone and visceral involvement (P=0.017, P=0.014 and P=0.03 respectively) and a marginally high incidence in soft tissue involvement (0.084). Moreover, IBC diagnosis conferred a significantly worse prognosis only in the ctDNA low subgroup (OS at 12 months nIBC: 100% vs IBC: 70%; P=0.049), while no differences were observed in the ctDNA_high subgroup (OS at 12 months nIBC: 29% vs IBC: 26%; P=0.767). Conclusion: ctDNA is able to stratify BC according to aggressiveness independently from the sites and type of metastases, both in the IBC and nIBC subgroups. IBC has a distinctive CTCs/ctDNA-based signature, in particular ctDNAlow pts have a lower incidence of HER2 positive CTCs and CTC clusters. This signature is probably due to predominant lymphatic metastatic spread and aggressive phenotype. Citation Format: Gerratana L, Zhang Q, Wang C, Shah A, Davis AA, Ye Z, Zhang Y, Abu-Khalaf M, Flaum L, Strickland K, Rossi G, Behdad A, Gradishar W, Platanias L, Yang H, Cristofanilli M. Dissecting the biology of inflammatory breast cancer (BC) through cell free DNA and a circulating tumor cells (CTC)-derived signature [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-17-02.

BACKGROUND: Both circulating tumor 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 PCR and Qubit fluorometer. The individual and joint effects of CTC and ccfDNA levels on patient progression-free survival (PFS) and overall survival (OS) were analyzed 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.

PURPOSE 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.

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 (&ge, 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 &lt, 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.

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.

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(+)

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.

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.