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This study aims to identify pathway involvement in the development of cisplatin (cis-diamminedichloroplatinum (II); CDDP) resistance in A549 lung cancer (LC) cells by utilizing advanced bioinformatics software. We developed CDDP-resistant A549 (A549/DDP) cells through prolonged incubation with the drug and performed RNA-seq on RNA extracts to determine differential mRNA and miRNA expression between A549/DDP and A549 cells. We analyzed the gene dysregulation with Ingenuity Pathway Analysis (IPA; QIAGEN) software. In contrast to prior research, which relied on the clustering of dysregulated genes to pathways as an indication of pathway activity, we utilized the IPA software for the dynamic evaluation of pathway activity depending on the gene dysregulation levels. We predicted 15 pathways significantly contributing to the chemoresistance, with several of them to have not been previously reported or analyzed in detail. Among them, the PKR signaling, cholesterol biosynthesis, and TEC signaling pathways are included, as well as genes, such as PIK3R3, miR-34c-5p, and MDM2, among others. We also provide a preliminary analysis of SNPs and indels, present exclusively in A549/DDP cells. This study's results provide novel potential mechanisms and molecular targets that can be explored in future studies and assist in improving the understanding of the chemoresistance phenotype.

COVID-19 ranges from asymptomatic in 35% of cases to severe in 20% of patients. Differences in the type and degree of inflammation appear to determine the severity of the disease. Recent reports show an increase in circulating monocytic-myeloid-derived suppressor cells (M-MDSC) in severe COVID 19 that deplete arginine but are not associated with respiratory complications. Our data shows that differences in the type, function and transcriptome of granulocytic-MDSC (G-MDSC) may in part explain the severity COVID-19, in particular the association with pulmonary complications. Large infiltrates by Arginase 1+ G-MDSC (Arg+G-MDSC), expressing NOX-1 and NOX-2 (important for production of reactive oxygen species) were found in the lungs of patients who died from COVID-19 complications. Increased circulating Arg+G-MDSC depleted arginine, which impaired T cell receptor and endothelial cell function. Transcriptomic signatures of G-MDSC from patients with different stages of COVID-19, revealed that asymptomatic patients had increased expression of pathways and genes associated with type I interferon (IFN), while patients with severe COVID-19 had increased expression of genes associated with arginase production, and granulocyte degranulation and function. These results suggest that asymptomatic patients develop a protective type I IFN response, while patients with severe COVID-19 have an increased inflammatory response that depletes arginine, impairs T cell and endothelial cell function, and causes extensive pulmonary damage. Therefore, inhibition of arginase-1 and/or replenishment of arginine may be important in preventing/treating severe COVID-19.

COVID-19 ranges from asymptomatic in 35% of cases to severe in 20% of patients. Differences in the type and degree of inflammation appear to determine the severity of the disease. Recent reports show an increase in circulating monocytic-myeloid-derived suppressor cells (M-MDSC) in severe COVID 19, that deplete arginine but are not associated with respiratory complications. Our data shows that differences in the type, function and transcriptome of Granulocytic-MDSC (G-MDSC) may in part explain the severity COVID-19, in particular the association with pulmonary complications. Large infiltrates by Arginase 1+ G-MDSC (Arg+G-MDSC), expressing NOX-1 and NOX-2 (important for production of reactive oxygen species) were found in the lungs of patients who died from COVID-19 complications. Increased circulating Arg+G-MDSC depleted arginine, which impaired T cell receptor and endothelial cell function. Transcriptomic signatures of G-MDSC from patients with different stages of COVID-19, revealed that asymptomatic patients had increased expression of pathways and genes associated with type I interferon (IFN), while patients with severe COVID-19 had increased expression of genes associated with arginase production, and granulocyte degranulation and function. These results suggest that asymptomatic patients develop a protective type I IFN response, while patients with severe COVID-19 have an increased inflammatory response that depletes arginine, impairs T cell and endothelial cell function, and causes extensive pulmonary damage. Therefore, inhibition of arginase-1 and/or replenishment of arginine may be important in preventing/treating severe COVID-19.

Gastric cancer is one of the most common and deadliest cancers worldwide. There is a significant diversity in risk and aggressiveness associated with the disease in terms of factors such as ethnicity, age, and diet. However, one thing that is common is the fact that infection with Helicobacter pylori (H. pylori) is often associated with the development of gastric adenocarcinoma. Infection with H. pylori induces a series of events that lead to non-atrophic gastritis (NAG), multifocal atrophic gastritis (MAG), intestinal metaplasia (IM), dysplasia, and cancer. All the premalignant, proinflammatory stages are treatable, but it has been shown that the rate of progression to more advanced lesions is higher than the rate of regression. Most of the research efforts have focused on understanding the cancer stage, but there is still a large gap in the knowledge of biomarkers of the inflammatory stages and those of the progression/regression. Using samples from African American and Caucasian individuals with gastritis, we have been able to identify specific sets of single-nucleotide polymorphisms (SNPs) and haplotypes in cytokine genes associated with ethnicity. Interestingly, those same SNPs are also associated with advanced gastric lesions. On the other hand, using a cohort of Hispanic/Latino individuals with gastritis, we identified CD44 as a marker of disease progression and DMBT1 as a marker of disease aggressiveness. Both markers were validated in in vitro and in vivo systems.

Breast cancer (BC) is the most common malignancy in women worldwide. Different risk factors are associated with the disease which is classified into several intrinsic subtypes according to expression of hormone receptors (estrogen and progesterone) and human epidermal growth factor receptor 2 (HER2). The advent of high throughput techniques has allowed the molecular classification of BC and development of diagnostic/prognostic tools to predict disease outcome and progression. However, most molecular classifications and diagnostic/prognostic tools were developed using samples from European/Caucasian women. Hispanic/Latino describes an admixed group of individuals with different fractions of European, African, and Indigenous American ancestries. We determined the frequency of the different intrinsic subtypes of BC in Colombia and their association with genetic ancestry and found that luminal B subtype was the most prevalent in Colombian women with BC. We found that African ancestry was associated with more aggressive forms of BC. We then used next-generation sequencing techniques to determine the underlying markers of luminal B subtype of BC in Colombia. We found 67 genes differentially expressed between luminal A and luminal B subtypes, and of those, six were common between patients with high European/low Indigenous American ancestries. Interestingly, three genes (ERBB2, MIEN1, and GRB7) form a genomic “block” in chromosome 17 from which ERBB2 and GRB7 are co-expressed. The expression of ERBB2 was independent of HER-2 labeling. Our results suggest a major role of ethnicity in the modulation of these genes in BC in Hispanic/Latina women and may be the basis for future larger studies to investigate the role of ethnicity in BC susceptibility in minority groups.

Background: Colorectal cancer is the third most deadly cancer among African Americans (AA). When compared to Caucasian Americans (CA), AA present with more advanced disease and lower survival rates. Here, we investigated if differences in tumor immunology could be contributive to disparities observed between these populations.Methods: We examined gene expression of tumor and non-tumor adjacent tissues from AA and CA by whole transcriptome sequencing, and generated scores for immune cell populations by NanoString. In addition, we utilized “The Cancer Genome Atlas” (TCGA) database from AA and CA as a validation cohort. Finally, we measured the secretion of cytokines characteristic of effector T helper cell (Th) subsets by ELISA using plasma from each AA and CA participant.Results: Colon tumors from AA patients showed significant fold-change increase in gene expression when compared to CA for FOXP3 (6.22 vs. 3.22), IL1B (103 vs. 11.4) and IL8 (220 vs. 28.9) (p < 0.05). In contrast, among CA we observed statistically higher gene expression of markers associated with antitumor activity such as GZMB (Granzyme B), IFNG and the immunotherapy targets PDL1 (CD274) and CTLA4 (p < 0.05). TCGA data validated our observed higher gene expression of GZMB and PDL1 in CA patients when compared to AA. Notably, our observations on immune cell populations show that AA tumors have significantly higher number of exhausted CD8+ cells (p < 0.01), mast cells (p < 0.02) and increased T regulatory cells when compared to CA. AA colon cancer patients differed from CA in cytokine production patterns in plasma (i.e., reduced IL-12).Conclusions: Our study demonstrates significant differences of the immunological profiles of colon tumors from AA compared to CA that suggest a deficiency of appropriate immune defense mechanisms in terms of gene expression, recruitment of immune cells and systemic secretion of cytokines. As such, these immune differences could be mitigated through population-specific therapeutic approaches.

Background: Hereditary hemorrhagic telangiectasia is an autosomal dominant vascular disorder caused by heterozygous, loss-of-function mutations in 4 transforming growth factor beta (TGFβ) pathway members, including the central transcriptional mediator of the TGFβ pathway, Smad4. Loss of Smad4 causes the formation of inappropriate, fragile connections between arteries and veins called arteriovenous malformations (AVMs), which can hemorrhage leading to stroke, aneurysm, or death. Unfortunately, the molecular mechanisms underlying AVM pathogenesis remain poorly understood, and the TGFβ downstream effectors responsible for hereditary hemorrhagic telangiectasia–associated AVM formation are currently unknown. Methods: To identify potential biological targets of the TGFβ pathway involved in AVM formation, we performed RNA- and chromatin immunoprecipitation–sequencing experiments on BMP9 (bone morphogenetic protein 9)-stimulated endothelial cells (ECs) and isolated ECs from a Smad4-inducible, EC-specific knockout ( Smad4 -iECKO) mouse model that develops retinal AVMs. These sequencing studies identified the angiopoietin-Tek signaling pathway as a downstream target of SMAD4. We used monoclonal blocking antibodies to target a specific component in this pathway and assess its effects on AVM development. Results: Sequencing studies uncovered 212 potential biological targets involved in AVM formation, including the EC surface receptor, TEK (TEK receptor tyrosine kinase) and its antagonistic ligand, ANGPT2 (angiopoietin-2). In Smad4 -iECKO mice, Angpt2 expression is robustly increased, whereas Tek levels are decreased, resulting in an overall reduction in angiopoietin-Tek signaling. We provide evidence that SMAD4 directly represses Angpt2 transcription in ECs. Inhibition of ANGPT2 function in Smad4 -deficient mice, either before or after AVMs form, prevents and alleviates AVM formation and normalizes vessel diameters. These rescue effects are attributed to a reversion in EC morphological changes, such as cell size and shape that are altered in the absence of Smad4. Conclusions: Our studies provide a novel mechanism whereby the loss of Smad4 causes increased Angpt2 transcription in ECs leading to AVM formation, increased blood vessel calibers, and changes in EC morphology in the retina. Blockade of ANGPT2 function in an in vivo Smad4 model of hereditary hemorrhagic telangiectasia alleviated these vascular phenotypes, further implicating ANGPT2 as an important TGFβ downstream mediator of AVM formation. Therefore, alternative approaches that target ANGPT2 function may have therapeutic value for the alleviation of hereditary hemorrhagic telangiectasia symptoms, such as AVMs.

Background: Breast cancer (BC) is the most common and deadliest cancer in women worldwide. Hormone receptor (HR) positive tumors (luminal) are the most frequent while HER-2 overexpressing (HR-, HER2+) and triple negative (TNBC, HR-/HER2-) are most deadly. HR+ subtypes are more common in non-Hispanic White women while HER-2 and TNBC present either higher prevalence or risk of mortality in minority groups. Hispanic/Latinos (H/L) is the largest and fastest growing minority in the U.S.A. However, H/L women have been underrepresented in BC studies. We reported that luminal B is the most common BC subtype in a group of H/L and that ERBB2, GRB7 and MIEN1 genes have increased expression in women with higher Indigenous American ancestry. Immune infiltration of tumors has become a hallmark for the prediction of outcome. However, contrary to TNBC, luminal tumors have been considered low immunogenic. Immune infiltration of luminal B tumors in H/L has not yet been investigated. Our goal was to predict immune infiltration of luminal B tumors based on RNA-seq data and according to the levels of body mass index (BMI) and ancestral fractions (West African, European, Indigenous American). Methods: We performed RNA-seq in 70 luminal B tumors of H/L from Puerto Rico. Ancestry was estimated by genotyping on the Affymetrix U.K. Biobank array and global ancestry proportions determined with Admixture Software v1.3, using 1000 Genomes reference population for anchoring. BMI, age at diagnosis and tumor size were extracted from the Electronic Medical Records. We used Partek Flow for the RNA-seq analysis based on ancestry fraction and BMI. Prediction of cell populations was done in xCell. Correlation analysis was done in GraphPad Prism v7.05. Results: We found an inverse correlation between Indigenous American and γδ-T cell infiltration (R2= -0.3; p=0.03) while BMI was associated with increased infiltration of basophils (R2=0.3; p=0.026). Among women with high BMI, those with low European ancestry had increased levels of basophils infiltration R2=0.66; p=0.17) and those with high European ancestry had reduced infiltration of naïve CD8+ T cells (R2= -0.53; p=0.008) and reduced plasmacytoid dendritic cells (pDC, R2=-0.51; p=0.01). Conclusions: We found that luminal B tumors have infiltration of immune cells that may contribute to differences in the response to treatment and outcome. To the best of our knowledge, the role of infiltrating basophils into breast cancer tissues has not been described. However, circulating basophils and predicted infiltration of basophils into ovarian and colorectal cancer tissues have been correlated with survival. Our findings have to be confirmed in a larger set of samples and by additional methods, including immunohistochemistry. Citation Format: Jone Garai, Julie Dutil, Douglas W. Cress, Victoria Seewaldt, Jamie K. Teer, Laura Fejerman, Lucio Miele, Jovanny Zabaleta. Cell infiltration prediction of breast cancer tissues identifies basophils as potentially important immune cells in the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2729.

Background and goal: MicroRNAs (miRNA) are small non-coding RNA that regulate post-transcriptional gene expression, influencing cell proliferation, differentiation and death. Extensive research has demonstrated that deregulation of miRNA-associated pathways may participate in the development of cancers. Precursor-B cell acute lymphoblastic leukemia (pre-B ALL) is the most common pediatric malignancy. Over decades, medical advances have helped the risk stratification and treatment modalities for these patients resulting in better favorable prognosis. Unfortunately, approximately 5-10% of standard risk patients and 15-20% of high or very-high risk patients will experience disease relapse. One of the factors involved in risk stratification is the minimal residual disease (MRD) status from bone marrow testing at the end of the initial induction phase of chemotherapy. MRD positivity escalates the risk and may signify some degree of therapy resistance. The aim of this study is to evaluate expression levels of miRNAs in MRD positive samples as compared to MRD negative. Methods: Bone marrow samples of 14 pre-B cell ALL pediatric patients with MRD positivity (n=5) and MRD negative (n=9) at the end of induction chemotherapy were analyzed. Next-generation sequencing technology was utilized to study the miRNAs profiles of these samples (smallRNA-seq) and to identify potential mRNA targets of selected miRNAs overexpressed in NALM6 B-ALL cell lines for 72 hours (RNA-seq). Analysis was done in Partek Flow and with the web-based tool InteractiVenn. Results: Twelve miRNAs were upregulated in patients with MRD positivity compared to those who were MRD negative (p values < 0.05). To investigate the potential role of these mRNAs in pre-B ALL cells, we selected 3 of those miRNAs (hsa-miR-34a-5p, -33b-5p, and -210-3p) to transfect the pre-B-ALL cell line NALM6. We identified 145 potential direct target genes for hsa-miR-33b-5p, 79 potential targets for hsa-miR-34a5-p and 142 potential targets for hsa-miR-210-3p. Thirty-five genes were common among all treatments including IL31A, PDZK1 and INSL3. Individual (per miRNA) pathways associated with these genes included immune response, metabolism and cell signaling. One common pathway associated with the differential expression profile of these overexpressed miRNAs was alcohol intake, known to have deleterious consequences in the fetus and inducing, among other things, transgenerational epigenetic alterations that may be associated with malignancy. Conclusions: Our work identifies potential biomarkers of MRD positivity after induction chemotherapy and identifies pathways associated with this phenomenon. Future plans include the manipulations of several of these pathways to determine their role in the proliferation and aggressiveness of pre-B ALL leukemia cell with the idea of devising new treatment options for those patients with post-induction MRD positivity. Citation Format: Andrea Bauchat, Lauren Raney, Jone Garai, Li Li, Randall Craver, Jovanny Zabaleta. Identification of microRNA upregulated in pediatric precursor-B cell acute lymphoblastic leukemia patients with post-induction MRD positivity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1409.

Background: Colorectal cancer is the third most deadly cancer among African Americans (AA). When compared to Caucasian Americans (CA), AA present with more advanced disease and lower survival rates. We have previously demonstrated that colon tumors from AA and CA differ in their immune cell recruitment and their systemic cytokines secretion profiles. Therefore, in this study we investigated if differences in gene expression and mutational profiles could relate to the disparities observed between these populations. Methods: We examined gene expression of colon tumor and non-tumor adjacent tissues from AA (n=20) and CA (n=20) by whole transcriptome sequencing (Illumina); and determined the differential expression of the 170 genes of the Illumina's TruSight Tumor 170 panel (TST170) in AA (n=12) and CA (n=17) colon tumor and non-tumor adjacent tissues. Results: From our gene expression studies, we found that colon tumors from AA patients showed significant fold-change increases in gene expression when compared to CA for FOXP3 (6.97 vs. 3.15), IL1B (122 vs. 14) and IL8 (262 vs. 28) (p < 0.05). In contrast, among CA we observed statistically higher gene expression of markers associated with antitumor activity such as GZMB (Granzyme B), IFNG and immunotherapy targets PDL1 (CD274) and CTLA4 (p < 0.05). Through our mutational profiling we identified 38 genes in AA and 6 in CA differentially expressed between tumor and non-tumor tissues (p < 0.01). Of those, 5 genes associated with several roles in cancer ETV4 (poor prognosis), CCND1 (tumorigenesis), FGFR2 (activation of the RAS-MAPK and the PI3K-AKT pathways) BRCA2 (genome stability), BCL2 (apoptosis regulator) were commonly altered in both cohorts. Conclusions: Our study demonstrates significant differences in the genetic characteristics of colon tumors from AA compared to CA that suggest a deficiency of appropriate immune defense features in terms of gene expression. We also observed divergence in the mutational profiling between the groups, including numerous genes that are indicative of prognosis, treatment and outcomes in colon cancer. As such, these deficits could be mitigated through population-specific therapeutic approaches. Citation Format: Jenny Paredes, Jone Garai, Li Li, Melody Baddoo, Ping Ji, Sayed Imtiaz, Marzia Spagnardi, Mubarak Akadri, Raavi Gupta, Mohamed Alshal, Maksim Agaronov, Henry Talus, Jennie L. Williams, Laura Martello-Rooney, Jovanny Zabaleta. Mutational analysis of colon tumors from African American patients and potential association with cancer disparities [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3618.

Cervical cancer ranks first in cancer mortality among women of low-middle income countries where 80% of the 570,000 cases and 311,000 worldwide deaths estimated for 2018 occurred. Persistent infections with high-risk HPV (hrHPV) genotypes can lead to cervical high-grade lesions (Cervical Intraepithelial Neoplasia grade 2 or more severe disease, CIN-II+), that if left untreated progress to cancer. hrHPV test has high sensitivity but because many women infected with hrHPV genotypes will clear the infection spontaneously, it has low specificity to detect CIN-II+. MicroRNAs (miRNAs) are small RNAs that regulate gene expression and show differential profiles in cervical intraepithelial lesions. Our aim was to identify miRNAs differentially expressed in CIN-II+ and to evaluate their potential use as biomarkers to distinguish low from high-grade lesions in hrHPV positive women. Methods We compared the miRNAs expression pattern between Formalin-Fixed Paraffin-Embedded (FFPE) tissues from hrHPV positive women with low-grade lesions (n=10) and women with high-grade lesions (n=10) of Medellin, Colombia, using the QIAseq miRNA Library Kit (Qiagen) and sequencing reagents and protocols from Illumina. The miRNAs with low coefficient of variation and high fold change were further validated by RT-PCR (miR-133a-3p, 143-3p, 143-5p, 29a-3p and 30b-5p) using miRCURY LNA miRNA PCR kit (Qiagen) and SNORD44 as housekeeping gene. Receiver Operating Characteristic (ROC) analyses with 95% confidence interval of the area under the curve were used to evaluate the diagnostic accuracy of the 5 miRNA and their combinations and the maxim Youden index to determine the optimal specificity and sensitivity. Results The differential miRNAs expression analysis identified 25 miRNAs overexpressed in high- versus low-grade lesions. The relative expression by RT-PCR of 5 of those miRNAs was higher in high-grade when compared to low-grade, and the levels of miR-143-3p, 143-5p and 30b-5p increased with disease progression (from healthy, CIN-I, CIN-II, CIN-III+). ROC analyses showed that miR-143-5p and the combination with miR-133a-3p exhibits the highest AUC 79% (95% CI: 65-93). However, the highest sensitivity is given by miR-29a-3p, 87.50% (95% CI: 67.64 - 97.34), and the highest specificity is given by miR-30b-5p, 95.45% (95% CI: 77.16-99-88), with cut-offs of >1,157 and >3,345 respectively. In conclusion, we identified 25 candidate miRNAs biomarkers and validated 5 of them with good diagnostic performance to distinguish high-grade from low-grade cervical lesions. Further validation on a larger cohort of samples is needed to confirm the potential role of these microRNAs to triage HPV positive women. Citation Format: Martha I. Gonzalez, Jone Garai, Jovanny Zabaleta, Gloria I. Sanchez. microRNA expression analysis in hrHPV positive FFPE tissues for the detection of cervical disease [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 267.

Colorectal cancer (CRC) is the third most common cancer among African Americans (AAs) in the US. When compared to Caucasian Americans (CAs), AAs present with higher incidence and death rates as well as worse prognosis after treatment with 5-Fluorouacil (5-FU). Previous studies have shown a correlation between microsatellite instability (MSI) and response to 5-FU and our recent findings suggest that differences in the tumor immunology and MSI status of AA and CA CRC patients are associated with the observed disparities between these populations. Therefore, we examined if cytokines secretion, protein production and cellular response to 5-FU treatment, differs between colon cancer cell lines from AAs generated in Dr. Williams’ laboratory (SB521, SB501 and CHTN06) and colon cancer cell lines from CA patients (HT29 and HCT116). Methods: We performed whole transcriptome sequencing of colon cancer cell lines (RNAseq), utilizing the NextSeq 500/550 High Output Kit v2.5 (Illumina) and Partek Flow data analysis to correlate gene expression to immune-oncology pathways. ELISA assays (RayBiotech) were used to examine the secretion of cytokines in supernatants from cells treated with interleukin 1β and tumor necrosis factor alpha. We used western blotting for protein detection of the phosphorylated form of c-Jun N-terminal kinases (JNK) as well as cell viability assays for establishing the IC50 of the cell lines to 5-FU. Results: The gene expression results indicated that the immune profiles of AA cell lines differ from CA in genes and cytokines related to cellular anti-tumor activity, including CD8B, IL-1R, IL-1R2, Granzyme B and NFKB. ELISAs of supernatants from CA and AA CRC cell lines revealed a differential cytokines secretion between the two races, namely IL-8. Lastly, the MSI AA cell line showed sensitivity to 5-FU (tenfold less) when compared to the CA cell lines and a distinct protein production pattern. Conclusions: Our gene expression findings demonstrated the differential expression of immunological pathways involved in immune-surveillance and cancer progression in the CRC cell lines between the two races. These results were in accordance with the cytokines’ and protein’s expression patterns observed in the two cohorts of cell lines. Importantly, our data indicates that 5-FU is more efficient in reducing cell viability in the MSI AA cell line than in the two CA cell lines regardless of their MSI status. Altogether, our results illustrate the value of these in vitro models to study 5-FU treatment in AA patients with MSI and MMR mutations and to elucidate the differences in chemotherapy treatment responses between AAs and CAs. In conclusion, we demonstrated distinct immunological profiles and 5-FU sensitivity of AA and CA CRC cell lines. As such, these differences observed could be used to guide new therapeutic strategies. Citation Format: Marzia Spagnardi, Jenny Paredes, Jone Garai, Ping Ji, Ellen Li, Jovanny Zabaleta, Laura Martello-Rooney, Jennie Williams. Tumor biology and cancer health disparity: Gene expression, cytokine secretion, and protein production in African American colon cancer cell lines [abstract]. In: Proceedings of the Twelfth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2019 Sep 20-23; San Francisco, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl_2):Abstract nr B134.

Introduction: Colorectal cancer (CRC) is the third most common cancer among African Americans (AA) and when compared to Caucasian Americans (CA), they present more advanced CRC disease and lower survival rates. Our previous findings suggest that this may be related to the differential expression in genes linked to cell recruitment and immune response. Therefore, we aimed to investigate the cellular antitumor activity and mutational profile of colon tumors from AAs. We also examined the secretion of cytokines characteristic of immune responses by different effector T helper cells (Th) subsets in AA and CA patients, as well as cell lines, to see if these differences play a role in the health disparities observed between these populations. Lastly, we observed the expression of the Program Death Ligand 1 (PD-L1) in response to the cytokines IL-17A and TNF-α in a microsatellite-unstable (MSI) AA and a microsatellite-stable (MSS) CA colon cancer cell line. Methods: Using IHC, we evaluated the cell recruitment and activation of T and natural killer cells in AA tumors. For mutational analysis, we utilized the TruSight Tumor 170 RUO kit (Illumina). ELISA assays (RayBiotech) were used to examine the secretion of cytokines linked to Th subsets (Th1, Th2, Th17) and inflammation in plasma from the AA and CA CRC patients, as well as in supernatants from the AA and CA colon cancer cell lines. Western blots were used to observe the expression of PD-L1 in the in vitro models. Results: ELISAs of plasma of CA and AA patients revealed a differential Th cytokines production patterns between early-stages (I, II) and late-stage (III) disease. The MSI AA cell line showed an increase on PD-L1 protein expression in response to IL-17A and TNF-α with an additive effect when combined in equal concentrations. Lastly, the mutational sequencing allowed us to further investigate the potential alterations that are responsible for the differences that we observed in gene expression between AAs and CAs in our RNA and cytokine expression profiling. Conclusions: Our results indicate that the immune profiles of AA patients differ from CA in terms of cytokines' production; AAs expressed elevated IL-17A, whereas CA expressed elevated IFN-γ, the latter indicative of Th1 immunity that has a more favorable prognosis. As such, these differences could be used as biomarkers and to guide therapeutic strategy for these populations. The mutational sequencing will help us to elucidate the impaired tumor immune response in AAs with colon cancer when compared to CAs that we observed in terms of cell recruitment and cytokine secretion in our previous findings. Importantly, our data indicate that IL-17A and TNF-α promote the protein production of PD-L1 in an MSI AA cell line, which may result in the impairment of T cells' antitumor activity. Taken together, the differences in the immunologic profiles in AA when compared to CA suggest a deficiency of the appropriate immune defense mechanisms in this population that may contribute to the cancer health disparities among CRC patients. Citation Format: Jenny E. Paredes, Ping Ji, Jone Garai, Marzia Spagnardi, Maria Munoz-Sagastibelza, Sayed Imtiaz, Gayle Mendez, Mubarak Akadri, Raavi Gupta, Mohamed Alshal, Maksim Agaronov, Henry Talus, Ellen Li, Jovanny Zabaleta, Laura Martello-Rooney, Jennie Williams. Tumor immune response in colon cancer African American patients and its role in cancer disparities [abstract]. In: Proceedings of the Eleventh AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2018 Nov 2-5; New Orleans, LA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl):Abstract nr C118.

Background: Breast cancer is the most frequently diagnosed form of invasive cancer in women and the second leading cause of cancer death. Triple-negative breast cancer (TNBC) accounts for 10-20% of the disease. This aggressive subtype lacks receptors for estrogen, progesterone, and human epidermal growth factor receptor 2 (HER2), drastically reducing treatment options. Hispanic/Latinas, who derive ancestry from European, African, and Native origins, are at least 30% more likely to be diagnosed with TNBC compared to non-Hispanic White women. However, little is known about the genomic architecture of TNBCs in Hispanic/Latinas. The goal of our study was to test the association between genetic ancestry and gene expression in TNBC samples from Hispanics/Latinas to help identify potential therapeutic targets. Methods and Results: We obtained 41 TNBC tissues through collaboration with the National Cancer Institute in Colombia along with demographic and clinical information. Genetic ancestry proportions were estimated using a panel of 106 Ancestry Informative Markers (AIMs). RNA was extracted from FFPE tissues, qualified using Agilent chips, and used for RNA sequencing at the Translational Genomics Core at the Stanley S. Scott Cancer Center. We used an online tool (http://cbc.mc.vanderbilt.edu/tnbc/) to validate TNBC status, and 33 of the 41 samples were confirmed. Quality control procedures ensured that the samples were of acceptable quality for further analysis. Individuals were classified as having high or low genetic ancestry proportions for each of the ancestral components, based on mean ancestry proportions overall. Differential gene expression between individuals in each ancestry category was estimated utilizing DESeq2 in R-Studio. Average genetic ancestry proportions for the 33 samples analyzed were 0.52 European (18 individuals were in the high European ancestry group, 14 in the low European ancestry group), 0.36 Indigenous American (15 high, 17 low) and 0.1 African (8 high, 24 low). We observed that 91 genes were differentially expressed between high vs. low European ancestry, 21 for the Native and 12 for the African. Interestingly, 5 genes were common between the European and Native ancestries. LGALS9C and MESP1 had significant differences in normalized counts between high and low ancestry for both the Europeans and Natives, though the subgroups displayed opposite correlation patterns. These results were validated by real-time PCR. Using cBioPortal, we found that MESP1 and LGALS9C were associated with amplification in 2.8% and 1.5% of breast cancer cases, respectively. LGALS9C expression displays an inverse correlation with disease outcome in several cancers, including breast. Conclusions: Our data suggest that expression levels of several genes, most notably MESP1 and LGALS9C, may be ancestry specific in TNBC patients from Colombia. Confirmation and further understanding of these findings are of outmost importance given that they could be therapeutic targets. Citation Format: Laura Carrasquilla, Jone Garai, Silvia J. Serrano-Gomez, Maria C. Sanabria-Salas, Melody C. Baddoo, Laura Fejerman, Jovanny Zabaleta. Relationship between ancestry fractions and gene expression in triple-negative breast cancer in Hispanic/Latina women [abstract]. In: Proceedings of the Eleventh AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2018 Nov 2-5; New Orleans, LA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl):Abstract nr C035.

Single-nucleotide polymorphisms (SNPs) in cytokine genes can affect gene expression and thereby modulate inflammation and carcinogenesis. However, the data on the association between SNPs in the interleukin 1 beta gene (IL1B) and colorectal cancer (CRC) are conflicting. We found an association between a 4-SNP haplotype block of the IL1B (-3737C/-1464G/-511T/-31C) and CRC risk, and this association was exclusively observed in individuals with a higher proportion of African ancestry, such as individuals from the Coastal Colombian region (odds ratio, OR 2.06; 95% CI 1.31–3.25; p 2q14 (p = 0.03). We conclude that Colombian individuals with high African ancestry proportions at locus 2q14 harbour more IL1B-CGTC copies and are consequently at an increased risk of CRC. This haplotype has been previously found to increase the IL1B promoter activity and is the most frequent haplotype in African Americans. Despite of limitations in the number of samples and the lack of functional analysis to examine the effect of these haplotypes on CRC cell lines, our results suggest that inflammation and ethnicity play a major role in the modulation of CRC risk.

Background: Studies in Latinas have reported a high prevalence of ER/PR/HER2-negative (TNBC) and HER2-enriched breast tumors. In our ongoing study of breast cancer molecular characteristics of Colombian women (N=301), more than 20% of the tumors were classified as TNBC. Molecular analyses of this aggressive subtype of breast cancer in women of European ancestry have revealed additional molecular heterogeneity, which is associated with treatment response and survival. The goal of the present study was to explore the molecular profile of TNBC in a sample of Latin American women from Colombia and assess if genetic ancestry modified the gene expression profile. Methods: We performed whole-transcriptome RNA-seq analysis in 48 TNBC tumors that were classified into intrinsic subtypes by immunohistochemistry (IHC) following St. Gallen 2015 surrogates. Genetic ancestry was estimated from a panel of 80 ancestry-informative markers (AIM). We categorized patients with TNBC according to the median of Indigenous ancestry (low Indigenous ancestry group: ancestry proportion below the median; high Indigenous ancestry: ancestry proportion above the median). We used top differentially expressed genes from Lehmann et al. (2011) (n=128) to explore the distribution of TNBC subtypes in our samples and to explore differences in the expression of these genes according to genetic ancestry. Results: Unsupervised hierarchical clustering analysis did not show any clear clustering of the Colombian samples into defined subtypes based on expression of the 128 top differentially expressed genes reported by Lehmann et al. Subsequently, we selected genes that clustered together according to the definitions of TNBC subtypes of Lehmann, leaving a total of 35 genes for the analysis. Unsupervised hierarchical clustering using this list of genes identified two clusters, one predominantly observed for woman of high Indigenous American ancestry (average Indigenous American ancestry for this group was 45%) and another more common in women of low Indigenous ancestry (average Indigenous American ancestry for this group was 36%, p=0.04). The cluster that included most of the women with high Indigenous ancestry proportions was characterized by lower gene expression for the 35 genes compared to the cluster that included women with low Indigenous ancestry proportions. Conclusions: Our results suggest differential expression of genes within TNBC breast cancer subtype according to genetic ancestry; nevertheless, we could not identify clear TNBC subtypes previously defined by Lehmann et al. in our study. Further analyses are needed to identify TNBC subtypes in Hispanic/Latina samples. Citation Format: Silvia J. Serrano-Gomez, Maria Carolina Sanabria-Salas, Jone Garai, Melody C. Baddoo, Gustavo Hernandez-Suarez, Juan Carlos Mejia, Oscar Garcia, Lucio Miele, Laura Fejerman, Jovanny Zabaleta. Identification of triple-negative breast cancer subtypes in Colombian patients [abstract]. In: Proceedings of the Tenth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2017 Sep 25-28; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2018;27(7 Suppl):Abstract nr A38.

Obesity has reached epidemic proportions worldwide with disproportionate prevalence in different communities and ethnic groups. Recently, the American Medical Association recognized obesity as a disease, which is a significant milestone that opens the possibilities of treating obesity under standardized health plans. Obesity is an inflammatory disease characterized by elevated levels of biomarkers associated with abnormal lipid profiles, glucose levels, and blood pressure that lead to the onset of metabolic syndrome. Interestingly, inflammatory biomarkers, in particular, have been implicated in the risk of developing several types of cancer. Likewise, obesity has been linked to esophageal, breast, gallbladder, kidney, pancreatic, and colorectal cancers. Thus, there exists a link between obesity status and tumor appearance, which may be associated to the differential levels and the circulating profiles of several inflammatory molecules. For example, mediators of the inflammatory responses in both obesity and gastric cancer risk are the same: pro-inflammatory molecules produced by the activated cells infiltrating the inflamed tissues. These molecules trigger pathways of activation shared by obesity and cancer. Therefore, understanding how these different pathways are modulated would help reduce the impact that both diseases, and their concomitant existence, have on society.

Background: Differences in outcome of breast cancer according to race/ethnicity have been reported. African American women have higher breast cancer-specific mortality rates compared to non-Hispanic White women (NHW). Although these differences have been attributed to the high prevalence of basal-like subtype among African Americans, these women have also less favorable outcome in less aggressive tumors when compared to NHW. Hispanic/Latino (H/L) populations are a genetically admixed and heterogeneous group, with variable levels of European, Native American and African ancestries. Some studies suggest that breast cancer-specific mortality is higher in U.S. Latinas compared to NHW even after adjustment for socioeconomic status and education. The molecular profile of breast cancer has been widely described in NHWs but knowledge is lacking in Latinas. We have previously reported that the most prevalent breast cancer intrinsic subtype in Colombian women was Luminal B. In this study we explored ancestry-associated differences in molecular profiles of luminal B tumors among these highly admixed women. Methods: To identify ancestry-associated differentially expressed genes in Luminal B tumors, we performed a whole-transcriptome RNA-seq analysis in 42 luminal tumors (21 Luminal A and 21 Luminal B) from Colombian women. Genetic ancestry was estimated from a panel of 80 ancestry-informative markers (AIM). We categorized patients according to luminal subtype and to the proportion of European ancestry (Low European ancestry group: ancestry proportion below the median; High European ancestry: ancestry proportion above the median).. We compared luminal B against luminal A tumors according to the assigned ancestry groups. We used DESeq2 package form R to test differential gene expression between the two tumor subtypes and by ancestry category. Genes with adjusted pvalue less than 0.05 were considered statistical significant. Results: We found 30 differentially expressed genes (pvalue < 0.05) by genetic ancestry among Luminal B tumors from which 27 were differentially expressed in the less European luminal B tumors and 3 in the more European group. Tests of interaction suggest that ancestry is a statistically significant modifier of expression for the following genes for Low European Ancestry group: CENPF, SNORA54, AIF1L, TNFSF13, LRRC1, ARHGAP33, ONECUT2, CDK12, BUB1, NTRK2, HES1); and FDXACB1, RAB26, ATP8B3 for the High European Ancestry group. An exploratory analysis of these genes reveals enrichment of pathways associated to ERBB signaling in the Low European Ancestry group. Conclusions: Our results suggest that the proportion of European genetic ancestry in Colombian women might modify gene expression in luminal B tumors. Further analysis will be needed to confirm these findings. Citation Format: Silvia J. Serrano-Gomez, Carolina Sanabria, Melody C. Baddoo, Nataly Cruz-Rodriguez, Jone Garai, Gustavo Hernandez-Suarez, Juan Carlos Mejia, Lucio Miele, Chindo Hicks, Laura Fejerman, Jovanny Zabaleta. Ancestry as a potential modifier of gene expression in luminal B tumors among Colombian women. [abstract]. In: Proceedings of the Ninth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2016 Sep 25-28; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2017;26(2 Suppl):Abstract nr PR05.

Background. Acute lymphoblastic leukemia (ALL) in Hispanic populations represents a public health problem that requires priority attention because its incidence and mortality increase annually. Intensive chemotherapeutic schemes in the last decade in the United States and other countries and have led to higher rates of durable remissions. However, these schemes have shown disappointing results in Hispanic populations. In Colombia, for example, the median overall survival (OS) is less than 11.3 months, the event-free survival (EFS) is 7.34 months, and only 61% of patients achieve complete remission (CR). Therefore, it is interesting to explore the underlying molecular characteristics of the disease in Hispanic patients. Previously we identified a gene expression signature associated with response to induction treatment and outcome of adult Hispanic patients with B-ALL. These ID1, ID3 and IGJ seem to be involved in many tumorigenic processes. The goal of our study is identify the role of these genes in B-ALL and its relevance in the prognosis and progression of disease in an in vitro model. Methods. CRISPR-Cas9 Gene Engineering and expression-ready ORF cDNA clones were used to either down-regulate or over-express, respectively, the expression of ID1, ID3 and IGJ genes in the adult B-ALL cell line NALM6. We used TaqMan to quantify the levels of mRNA expression of ID1/ID3/IGJ genes after over-expression. Protein expression by Flow Cytometry was used to verify silencing and over-expression of our 3-genes signature. Cell viability and proliferation were determined by anexin V-PI and MTT assay, respectively at 0, 24, 48, 72 and 96 hours after modulation. Cell cycle analysis was performed using BrdU and 7-AAD. Separately, increasing doses of cyclophosphamide, doxorubicin and cytarabine were used to determine the role of ID1, ID3, and IGJ genes in chemo resistance at 24, 48, 72, 96, and 144 hours. Total RNA from leukemic cells was isolated using Trizol method. We used microarray analysis to identify genes differentially expressed after silencing/overexpression. We performed Immunophenotype analysis of 43 B-ALL adult Hispanic patient Bone Marrow samples using the panel of antibodies recommended and standardized by the European consortium Euroflow. Correlation analysis were used to establish the association between our 3-Gene expression signature and the expression of surface markers currently used for diagnostic and follow-up of B-ALL patients. Results. We observed that both overexpression and silencing of ID1, ID3 and IGJ modulated B-ALL cells cell cycle. ID1/ID3/IGJ overexpression resulted in a loss of S phase and arrest of the cells in the G0/G1 phase of the cell cycle. On the contrary, ID1/ID3/IGJ silencing induced a loss of G0/G1 phase and gain in the number of cells in G2/M and S phases. Gain-of-expression and loss-of-expression analyses using incremental doses of chemotherapy agents currently used in the B-ALL treatment, demonstrate that ID1/ID3/IGJ signature promotes drug resistance of the NALM6 B-ALL cell line. Clinically, patients with higher ID1/ID3/IGJ expression have high expression of CD20 and CD10 markers (aberrantly expressed in immature cells) and shorter OS and EFS. In addition, more than 2,000 genes that are differentially expressed in the cells with modulation of the expression of ID1/ID3/IGJ suggesting a complex network of biological processes implicated in the drug resistance and poor prognosis present in the patients with this 3-gene signature. Conclusions. Our data identify the ID1/ID3/IGJ signature as possible modulator of molecular events during B-ALL differentiation, proliferation and drug resistance. These results highlight the potential role of this gene signature both as a risk stratification tool and as a candidate therapeutic target in Hispanic population. Citation Format: Nataly Cruz-Rodriguez, Alba Lucia Combita, Jone Garai, Leonardo Jose Enciso, Sandra Milena Quijano, Silvia Serrano-Gomez, Li Li, Jovanny Zabaleta. Biological and clinical significance of ID1/ID3/IGJ expression signature modulation in B-Acute lymphoblastic leukemia. [abstract]. In: Proceedings of the Ninth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2016 Sep 25-28; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2017;26(2 Suppl):Abstract nr B36.

Factors responsible for invasive and metastatic progression of prostate cancer (PCa) remain largely unknown. Previously, we reported cloning of prosaposin (PSAP) and its genomic amplification and/or overexpression in several androgen-independent metastatic PCa cell lines and lymph node metastases. PSAP is the lysosomal precursor of saposins, which serve as activators for lysosomal hydrolases involved in the degradation of ceramide (Cer) and other sphingolipids. Our current data show that, in metastatic PCa cells, stable down-modulation of PSAP by RNA-interference via a lysosomal proteolysis-dependent pathway decreased β1A-integrin expression, its cell-surface clustering, and adhesion to basement membrane proteins; led to disassembly of focal adhesion complex; and decreased phosphorylative activity of focal adhesion kinase and its downstream adaptor molecule, paxillin. Cathepsin D (CathD) expression and proteolytic activity, migration, and invasion were also significantly decreased in PSAP knock-down cells. Transient-transfection studies with β1A integrin- or CathD-siRNA oligos confirmed the cause and effect relationship between PSAP and CathD or PSAP and Cer-β1A integrin, regulating PCa cell migration and invasion. Our findings suggest that by a coordinated regulation of Cer levels, CathD and β1A-integrin expression, and attenuation of “inside-out” integrin-signaling pathway, PSAP is involved in PCa invasion and therefore might be used as a molecular target for PCa therapy. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5127.