Reduced Relapse Free Survival Research Articles

Testis-specific serine kinase 6 (TSSK6) is abnormally expressed in colorectal cancer and promotes oncogenic behaviors

Cancer testis antigens (CTAs) are a collection of proteins whose expression is normally restricted to the gamete, but abnormally activated in a wide variety of tumors. The CTA, Testis specific serine kinase 6 (TSSK6), is essential for male fertility in mice. Functional relevance of TSSK6 to cancer, if any, has not previously been investigated. Here we find that TSSK6 is frequently anomalously expressed in colorectal cancer and patients with elevated TSSK6 expression have reduced relapse free survival. Depletion of TSSK6 from colorectal cancer cells attenuates anchorage independent growth, invasion and growth in vivo. Conversely, overexpression of TSSK6 enhances anchorage independence and invasion in vitro as well as in vivo tumor growth. Notably, ectopic expression of TSSK6 in semi-transformed human colonic epithelial cells is sufficient to confer anchorage independence and enhance invasion. In somatic cells, TSSK6 co-localizes with and enhances the formation of paxillin and tensin positive foci at the cell periphery, suggesting a function in focal adhesion formation. Importantly, TSSK6 kinase activity is essential to induce these tumorigenic behaviors. Our findings establish that TSSK6 exhibits oncogenic activity when abnormally expressed in colorectal cancer cells. Thus, TSSK6 is a previously unrecognized intervention target for therapy, which could exhibit an exceptionally broad therapeutic window.

A circadian rhythm-related gene signature for predicting relapse risk and immunotherapeutic effect in prostate adenocarcinoma.

Prostate adenocarcinoma (PRAD) represents the most common male carcinoma in developed countries, its high relapse risk contributes to the second-leading cause of cancer-related deaths. Therefore, it is required to develop an effective signature for predicting the relapse risk of PRAD. To identify a circadian rhythm- (CR-) related predictive signature, we analyzed RNA-seq data of patients with prostate adenocarcinoma (PRAD) from the TCGA and GEO cohort. Seven circadian rhythm- (CR-) related genes (FBXL22, MTA1, TP53, RORC, DRD4, PPARGC1A, ZFHX3) were eventually identified to develop a CR-related signature. AUCs for 3-year overall survival were 0.852, 0.856 and 0.944 in the training set, validation set and an external independent test set (GSE70768), respectively. Kaplan-Meier curve analysis showed that the high-risk group has a reduced relapse-free survival (RFS) than the low-risk group in the training set, validation set, and test set, respectively (P < 0.05). We constructed a prognostic nomogram combining the CR-related signature with T staging to precisely estimate relapse risk of PRAD patients. Finally, we observed that the CR-related gene signature was associated with tumor mutation burden, multiple immune checkpoint molecules and microsatellite instability, and thus could predict response to immune checkpoint inhibitors in PRAD. Conclusively, we developed a circadian rhythm-related gene signature for predicting RFS and immunotherapy efficacy in prostate adenocarcinoma.

HO-1 Modulates Aerobic Glycolysis through LDH in Prostate Cancer Cells.

Prostate cancer (PCa) is the second most diagnosed malignancy and the fifth leading cause of cancer associated death in men worldwide. Dysregulation of cellular energetics has become a hallmark of cancer, evidenced by numerous connections between signaling pathways that include oncoproteins and key metabolic enzymes. We previously showed that heme oxygenase 1 (HO-1), a cellular homeostatic regulator counteracting oxidative and inflammatory damage, exhibits anti-tumoral activity in PCa cells, inhibiting cell proliferation, migration, tumor growth and angiogenesis. The aim of this study was to assess the role of HO-1 on the metabolic signature of PCa. After HO-1 pharmacological induction with hemin, PC3 and C4-2B cells exhibited a significantly impaired cellular metabolic rate, reflected by glucose uptake, ATP production, lactate dehydrogenase (LDH) activity and extracellular lactate levels. Further, we undertook a bioinformatics approach to assess the clinical significance of LDHA, LDHB and HMOX1 in PCa, identifying that high LDHA or low LDHB expression was associated with reduced relapse free survival (RFS). Interestingly, the shortest RFS was observed for PCa patients with low HMOX1 and high LDHA, while an improved prognosis was observed for those with high HMOX1 and LDHB. Thus, HO-1 induction causes a shift in the cellular metabolic profile of PCa, leading to a less aggressive phenotype of the disease.

Teratomatous Elements in Orchiectomy Specimens Are Associated with a Reduced Relapse-Free Survival in Metastasized Testicular Germ Cell Tumors

Introduction: The impact of teratomatous elements in orchiectomy specimens of metastasized testicular germ cell tumors (TGCT) regarding oncological outcome is still unclear. Methods: We performed a retrospective analysis including 146 patients with metastasized TGCT analysing patient characteristics. Results: Twenty-six (18%) of all patients showed teratomatous elements in the orchiectomy specimens. TGCT with teratomatous elements showed a significantly higher frequency of clinical-stage 2C-3 disease (73 vs. 49%, p = 0.031), visceral metastases (58 vs. 32%, p = 0.015), and poor prognosis (p = 0.011) than TGCT without teratomatous elements. Teratoma-containing TGCT revealed a significantly higher rate of post-chemotherapy retroperitoneal lymph node dissection (PC-RPLND, 54 vs. 32%, p = 0.041), with teratomatous elements being more often present in the PC-RPLND specimens (43 vs. 11%, p = 0.020) than nonteratoma-containing primaries. In the Kaplan-Meier estimates, the presence of teratomatous elements in orchiectomy specimens was associated with a significantly reduced relapse-free survival (RFS) (p = 0.049) during a median follow-up of 36 months (10–115.5). Conclusions: The presence of teratomatous elements in orchiectomy specimens is associated with an advanced tumor stage, worse treatment response as well as a reduced RFS in metastasized TGCT. Consequently, the presence of teratomatous elements might act as a reliable stratification tool for treatment decision in TGCT patients.

Protein Kinase C Isozymes Associated With Relapse Free Survival in Non-Small Cell Lung Cancer Patients.

IntroductionProtein expression is deregulated in cancer, and the proteomic changes observed in lung cancer may be a consequence of mutations in essential genes. The purpose of this study was to identify protein expression associated with prognosis in lung cancers stratified by smoking status, molecular subtypes, and EGFR-, TP53-, and KRAS-mutations.MethodsWe performed profiling of 295 cancer-relevant phosphorylated and non-phosphorylated proteins, using reverse phase protein arrays. Biopsies from 80 patients with operable lung adenocarcinomas were analyzed for protein expression and association with relapse free survival (RFS) were studied.ResultsSpearman’s rank correlation analysis identified 46 proteins with significant association to RFS (p<0.05). High expression of protein kinase C (PKC)-α and the phosporylated state of PKC-α, PKC-β, and PKC-δ, showed the strongest positive correlation to RFS, especially in the wild type samples. This was confirmed in gene expression data from 172 samples. Based on protein expression, unsupervised hierarchical clustering separated the samples into four subclusters enriched with the molecular subtypes terminal respiratory unit (TRU), proximal proliferative (PP), and proximal inflammatory (PI) (p=0.0001). Subcluster 2 contained a smaller cluster (2a) enriched with samples of the subtype PP, low expression of the PKC isozymes, and associated with poor RFS (p=0.003) compared to the other samples. Low expression of the PKC isozymes in the subtype PP and a reduced relapse free survival was confirmed with The Cancer Genome Atlas (TCGA) lung adenocarcinoma (LUAD) samples.ConclusionThis study identified different proteins associated with RFS depending on molecular subtype, smoking- and mutational-status, with PKC-α, PKC-β, and PKC-δ showing the strongest correlation.

Reduced-Intensity Versus Myeloablative Conditioning for Allogeneic Hematopoietic Cell Transplantation in Patients with Myelodysplastic Syndrome: A Systematic Review and Meta-Analysis of Randomized Clinical Trials & Cohort Studies

Background: Conditioning regimen given before allogeneic hematopoietic stem cell transplantation (allo-HSCT) contributes significantly to the outcomes following transplant for myelodysplastic syndrome (MDS). Myeloablative conditioning (MAC) regimens are often associated with a lower risk of relapse; however, their use is often limited by toxicity and a higher risk of non-relapse mortality (NRM). Reduced-intensity conditioning (RIC) regimens are associated with a higher risk of relapse, a lower NRM, a lower incidence of graft versus host disease (GVHD), and hence is feasible in patients with advanced age or comorbidities. This study highlights the importance of the difference between the two interventions used in allo-HSCT for myelodysplastic syndrome (MDS). Methods: We conducted a comprehensive literature search using PubMed, Clinicaltrial.gov, Embase, Cochrane, and Web of Science on 5th May 2020 with no restriction of language or period. Initial research revealed 1698 articles. After excluding review articles, duplicates, and non-relevant articles, we included two randomized clinical trials and three cohort studies, which reported overall survival (OS), relapse-free survival (RFS), relapse incidence (RI), and incidence of acute and chronic (GVHD). Based on inclusion criteria, two randomized clinical trials (BMT CTN 0901 Trial Scott et al. 2017 &amp; EBMT RICMAC Trial Kroger et al. 2017) and three cohort studies (BBMT 55226 Park et al. 2018, BBMT Orozco et al. 2019, BMT Alatrash et al. 2019) were included. Results: Among combined MDS and acute myeloid leukemia (AML) patients (n=1188) enrolled in 5 studies, 651 patients had MDS. Patients underwent MAC (n=484) or RIC (n=692) followed by either a matched related donor (MRD), n=236, or matched unrelated donor (MUD), n=236, allo-HCT. The median age range at the time of transplant was 50-54 years The use of RIC causes statistically non-significant trend of improved overall survival (OS) (OR 1.11: 95% CI: 0.74-1.67, p-value= 0.61) along with more risk of relapse incidence (RI) (R 1.34: 95% CI: 0.56-3.18, p value= 0.51), and reduced relapse free survival (RFS) (OR 0.88, 95% CI: 0.55-1.44: p value= 0.59). Differences were found in terms of safety of both conditioning regimens. MAC allo-HCT have more acute GVHD, grade 3 and 4 (OR 0.66: 95% CI: 0.29-1.52, p value= 0.33) and chronic GVHD, grade 3 and 4 (OR 0.72: 95% CI: 0.41-1.27: p value= 0.26) and treatment-related mortality (TRM) (OR 0.83, 95% CI: 0.37-1.86, p value= 0.65). The meta-analysis results are shown in figure-1. Conclusion: In adult MDS patients undergoing allo-HSCT, RIC is associated with improved overall survival, but at the cost of reduced RFS, and a higher risk of relapse. MAC, on the other hand, is associated with more treatment-related mortality and GVHD. Results of our analysis point out these trends but these are not statistically significant Figure 1 Disclosures Anwer: Incyte, Seattle Genetics, Acetylon Pharmaceuticals, AbbVie Pharma, Astellas Pharma, Celegene, Millennium Pharmaceuticals.: Honoraria, Research Funding, Speakers Bureau.

Acute Myeloid Leukemia with Isocitrate Dehydrogenases (IDH) 1 and 2 Mutations. a Real-World Study from the European IDH Research Group

Acute Myeloid Leukemia with Isocitrate Dehydrogenases (IDH) 1 and 2 Mutations. a Real-World Study from the European IDH Research Group

African American Race Is Associated with Decreased Relapse-Free Survival in Immune Thrombotic Thrombocytopenic Purpura

African American Race Is Associated with Decreased Relapse-Free Survival in Immune Thrombotic Thrombocytopenic Purpura

Quantifying Argonaute 2 (Ago2) expression to stratify breast cancer

BackgroundArgonaute-2 (Ago2) is an essential component of microRNA biogenesis implicated in tumourigenesis. However Ago2 expression and localisation in breast cancer remains undetermined. The aim was to define Ago2 expression (mRNA and protein) and localisation in breast cancer, and investigate associations with clinicopathological details.MethodsAgo2 protein was stained in breast cancer cell lines and tissue microarrays (TMAs), with intensity and localization assessed. Staining intensity was correlated with clinicopathological details. Using independent databases, Ago2 mRNA expression and gene alterations in breast cancer were investigated.ResultsIn the breast cancer TMAs, 4 distinct staining intensities were observed (Negative, Weak, Moderate, Strong), with 64.2% of samples stained weak or negatively for Ago2 protein. An association was found between strong Ago2 staining and, the Her2 positive or basal subtypes, and between Ago2 intensity and receptor status (Estrogen or Progesterone). In tumours Ago2 mRNA expression correlated with reduced relapse free survival. Conversely, Ago2 mRNA was expressed significantly lower in SK-BR-3 (HER2 positive) and BT-20 (Basal/Triple negative) cell lines. Interestingly, high levels of Ago2 gene amplification (10–27%) were observed in breast cancer across multiple patient datasets. Importantly, knowledge of Ago2 expression improves predictions of breast cancer subtype by 20%, ER status by 15.7% and PR status by 17.5%.ConclusionsQuantification of Ago2 improves the stratification of breast cancer and suggests a differential role for Ago2 in breast cancer subtypes, based on levels and cellular localisation. Further investigation of the mechanisms affecting Ago2 dysregulation will reveal insights into the molecular differences underpinning breast cancer subtypes.

Distinct prognosis of mRNA expression of the five RecQ DNA-helicase family members - RECQL, BLM, WRN, RECQL4, and RECQL5 - in patients with breast cancer.

BackgroundFive RecQ helicase family members have a role in maintaining genome stability. However, their prognostic roles in breast cancer remain unknown. We aimed to investigate the prognostic values of the RecQ family and clinical outcomes in breast cancer.MethodsWe used the Kaplan–Meier Plotter database (http://kmplot.com/analysis) to analyze prognostic values of RecQ-family mRNA expression in all breast cancers and in different intrinsic subtypes and clinicopathological characteristics. Protein-expression levels of WRN and RECQL4 were confirmed by immunohistochemistry (IHC) in breast cancer tissues.ResultsIncreased expression of RECQL mRNA was significantly associated with reduced relapse-free survival (RFS) and postprogression survival (PPS) in all breast cancers, and improved overall survival (OS) in patients with basal-like breast cancer and in mutant-p53-type breast cancer patients. Increased expression of BLM mRNA was correlated with reduced distant metastasis-free survival (DMFS) in all patients. Increased expression of WRN mRNA was associated with improved OS and RFS in breast cancer patients. Increased expression of RECQL4 mRNA was associated with reduced OS, DMFS, and RFS in all breast cancers, and with reduced OS in patients with luminal A, HER2-positive, ER-positive, and PR-positive breast cancer. Increased expression of RECQL5 mRNA was associated with improved RFS in all patients, and with improved OS in patients with lymph-node-negative breast cancer, but with reduced OS in patients with HER2-positive breast cancer. IHC staining confirmed that high expression of WRN was correlated with increased OS and high expression of RECQL4 associated with reduced OS at protein levels.ConclusionmRNA-expression levels of RecQ members were significantly correlated with prognosis in breast cancer patients. These preliminary findings require further study to determine whether RecQ-targeting reagents might be developed for clinical application in breast cancer.

Prognostic value of tumor infiltration immune cells in pancreatic cancer

Objective: To investigate the prognostic effect of tumour-infiltrating immune cell, including CD8(+) T cell, regulatory T-cell (Treg) and myeloid-derived suppressor cells (MDSC) on pancreatic patients. Methods: This study retrospectively collected the data of 80 patients who were histologically diagnosed of pancreatic cancer and underwent classical R0 surgical resection at Tianjin Medical University Cancer Institute and Hospital from January 2010 to May 2012. All patients survival were followed up until the cut-off date of January 2015. Clinicopathological features including immunohistochemical staining of FOXP3, CD8 and CD33 were reviewed as indice for evaluating the prognosis of pancreatic patients.The prognostic effect of tumour-infiltrating immune cells were analysed by Kaplan-Meier and Log-rank test. Multiple-factor analysis was conducted with the Cox regression model. The correlation between tumour-infiltrating immune cells and clinicopathological features was analysed by χ(2) test. The C57BL/6 mouse model was used to evaluate the efficacy of Treg and MDSC depletion therapy in vivo. Student's t-test was applied to assess the difference of the tumour volume, Ki-67 positive rate and CD8(+) T-cell infiltration proportion between depletion group and control group. Results: Eventually, 80 patients were included and no patient was lost during the follow-up period. The median follow-up time was 33.2 months (7.4-59.9 months). Patients with high level of tumour-infiltrating CD8(+) T cells had longer overall survival (OS) time ((21.6±11.9)months vs. (13.6±7.4)months, χ(2)=4.647, P = 0.031) than those with low level of tumour-infiltrating CD8(+) T cells. Tumor infiltration FOXP3(+) cells were strongly associated with reduced OS((20.9±8.5)months vs.(13.4±8.8)months, χ(2)=10.528, P=0.001), reduced relapse free survival (RFS) ((15.2±9.0)months vs. (9.5±8.8)months, χ(2)=6.288, P=0.012) and larger tumor size(χ(2)=4.073, r=0.226, P=0.044). The high intratumoural MDSC group showed a significantly shorter OS((23.5±11.8)months vs. (13.8±7.6)months, χ(2)=5.724, P=0.017), RFS((17.9±11.3)months vs. (10.2±7.5)months, χ(2)=7.430, P=0.006) and more advanced N stage (χ(2)=4.714, r=0.243, P=0.030) than the low intratumoural MDSC group. Multivariate Cox analysis revealed that pTNM (P=0.008), tumour-infiltrating Treg density (P=0.009) and intratumoural MDSC density (P=0.034) were independent and negative prognostic factors for OS; pTNM(P=0.003) and tumour-infiltrating MDSC level(P=0.018) were independent and negative factors for RFS. The experiment in vivo revealed that Treg and MDSC depletion therapy significantly decreased tumour volume in the C57BL/6 mouse model of subcutaneous tumours((1 396.3±442.5)mm(3) vs. (3 356.9±533.5)mm(3), t=4.986, P=0.018). Tumour Ki-67 positive rate significantly decreased (23%±5% vs. 55%±10%, t=3.130, P=0.011) in Treg and MDSC depletion group, whereas, the proportion of tumour-infiltrating CD8(+) T cells significantly increased in depletion groups (3.25%±0.69% vs. 0.76%±0.25%, t=3.393, P=0.007). Conclusions: Tumour-infiltrating Treg, MDSC level and pTNM stage are independent prognostic factors for patients with pancreatic cancer. Treg and MDSC depletion therapy can significantly retard tumour growth and increase the level of tumour-infiltrating CD8(+) T-cells in the C57BL/6 mouse model of subcutaneous tumours.

Abstract P1-01-09: BMP4 suppresses the progression of breast cancer through altered expression of metastasis regulating genes

Abstract Metastasis is a lethal manifestation of cancer, the development of which is the major cause of death in cancer patients. During a search for metastasis-regulating elements, an inverse correlation was identified between the in vivo tumor expression of bone morphogenetic protein-4 (BMP4) and spontaneous metastasis in a panel of isogenic mammary tumors of varying metastatic capacity. BMP4 is an essential morphogen in development, regulating cellular mechanisms akin to those in metastasis, including cellular differentiation, pluripotency and apoptosis. We therefore initiated an investigation of the impact of BMP4 expression on the metastatic process. We studied the effect of enforced expression of BMP4 in a highly metastatic mammary tumour model called 4T1.2, comparing in vitro properties and tumour progression in mice. There were no differences in proliferation in vitro or when implanted into the mammary gland of immunocompetent mice. In contrast, mice bearing equivalent-sized 4T1.2-BMP4 tumors revealed dramatically reduced metastasis to lung, lymph node and bone. In a parallel study where the established orthotopic primary tumor was resected, survival was significantly extended in mice bearing 4T1.2-BMP4 tumors. Enforced BMP4 expression in tumor cells introduced intravenously resulted in a 2.5-fold decrease in lung metastatic burden, consistent with the impaired capacity of tumor cells to survive in circulation and colonize the lung. Conversely, silencing BMP4 expression in separate weakly metastatic tumours enhanced their ability to colonize the lung and shortened the survival of the mice. No changes were found in the ability of tumor cells expressing BMP4 or treated with recombinant BMP4 to migrate or invade through Matrigel in chemotactic assays but BMP4 enhanced anoikis in both mouse and human breast cancer cells, indicating that BMP4 sensitizes disseminated cells to anoikic stresses induced by cell-substrate detachment and shear flow during systemic transit. BMP4 activated canonical BMP-SMAD signaling in our mammary tumours, leading to altered expression of known metastasis-regulating genes, including SMAD7. SMAD7 depletion in metastasis-deficient 4T1.2-BMP4 tumors accelerated the onset of metastatic disease. In a meta-analysis of 3,587 breast cancer patients in publically available datasets, low BMP4 mRNA expression was significantly associated with reduced relapse-free survival (RFS) (HR = 0.85, P = 0.01). In an independent analysis using the BreastMark algorithm, low levels of BMP4 mRNA were associated with reduced RFS (HR = 0.88, P = 0.035), distant metastasis-free survival (HR = 0.83, P = 0.035) and overall survival (HR = 0.78, P = 0.006). At the protein level, in a tissue microarray from 415 treatment naïve patients, improved overall survival was observed in multivariate analysis for both BMP4 (HR = 0.66, P = 0.037) and SMAD7 expression (HR = 0.64, P = 0.035) individually. Expression of both proteins compared to neither further improved OS (HR = 0.55, P = 0.005). In summary, we found strong evidence that BMP4 is a metastasis suppressor correlating inversely with metastatic potential in preclinical breast cancer models and predicting improved relapse-free and overall survival in breast cancer patients. Citation Format: Redfern AD, Eckhardt BL, Cao Y, Sloan EK, Parker BS, Loi S, Ueno NT, Lau PK, Latham B, Anderson RL. BMP4 suppresses the progression of breast cancer through altered expression of metastasis regulating genes [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-01-09.

Immunohistochemical Localization of KRAS and BRAF and its Clinical Utility in Patients with Colorectal Cancer

Background: Colorectal cancer (CRC) is a multistep process occurring due to the accumulation of several genetic alterations. The most important ones are RAS and RAF that have been implicated as key intermediates in RAS-mediated signaling cascade. KRAS and BRAF protein expression levels and prognostic evaluation in CRC patients remains unknown. Moreover, minimal evidence exists regarding the predictive role of KRAS and BRAF for fluoropyrimidine-based adjuvant chemotherapy. Hence, present study aimed to investigate the prevalence of KRAS and BRAF protein expressions in CRC patients and further to correlate the results with clinicopathological parameters and prognosis. Methods and findings: A total 82 CRC patients were included in this retrospective study. KRAS and BRAF protein expressions were studied by immunohistochemistry technique using paraffin embedded tumor tissue blocks. The data was analyzed using SPSS software. p value = 0.05 was considered as statistically significant. Immunohistochemical localization of KRAS and BRAF showed cytoplasmic staining in 34% and 63% of patients, respectively. BRAF immunopositivity was found to be significantly higher in patients with positive nodal status (p=0.018), presence of perineural invasion (p=0.039) and = 5.0 ng/mL pre-operative serum CEA levels (p=0.008) as compared to their respective counterparts. Moreover, a trend of higher positive BRAF expression was observed in advanced stage patients as compared to early stage patients (p=0.080). Survival analysis demonstrated a significant reduced relapse-free survival (RFS) with KRAS positive expression in rectal cancer patients (p=0.045). Similar trend of reduced RFS was observed with positive KRAS expression in the subgroup of rectal cancer patients treated with adjuvant therapy (p=0.073). However, BRAF protein expression failed to show any prognostic or predictive value in CRC patients. Conclusion: KRAS and BRAF protein expression might be associated with disease aggressiveness in CRC. Further, KRAS protein expression could be useful prognostic and predictive marker in rectal cancer patients.

Abstract B03: Racial Variation in Annexin A2 (AnxA2) Gene Expression and Poor Outcome in Triple-Negative Breast Cancer

Abstract Background: Triple-negative breast cancer (TNBC) is identified by the absence of three major receptors (estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER-2)) that drive most breast cancers. The incidence of TNBC is associated with health disparity due to its disproportionate occurrence in African American (AA) women and its association with worst overall survival in AA women. Previously, our lab in breast cancer has investigated the prevalence, functionality, and mechanistic properties of one of the members of the human annexin family, Annexin A2 (AnxA2), a 36 kDa calcium-dependent phospholipid binding protein. Our previous studies and others have shown that AnxA2 is overexpressed in TNBC and has a reciprocal relationship with HER2 at mRNA and protein levels, but its association with racial variation and clinical outcomes is unknown. Methods: AnxA2 mRNA expression was evaluated in breast cancer subtypes from The Cancer Genome Atlas (TCGA) Breast Invasive Carcinoma (BRCA) mRNA database that includes 1,094 patients. Associations between clinical outcomes and AnxA2 gene expression were tested in a genome wide association study of combined publicly available microarray datasets that includes 4,147 patients (mean follow-up of 69 months). Two tissue microarrays (TMA) consisting of 154 breast cancer patient samples of various subtypes and forty archived paraffin-embedded breast tissues (TNBC and normal tissue) were collected from patients based on ethnicity and analyzed by immunohistochemistry and scored by a clinical pathologist in a blind study to provide scores (0,+1,+2,+3) based on AnxA2 staining intensity. Results: AnxA2 gene expression was significantly increased in TNBC in comparison to other breast cancer subtypes. Furthermore, AnxA2 gene expression was significantly elevated in AA (n=158) women in comparison with Caucasian (CA; n=654, P&amp;lt;0.0001) and Hispanic (HA; n=51, P&amp;lt;0.0001) women and was significantly associated with TNBC in AA women. High expression levels of AnxA2 were associated with reduced overall survival (hazard of death = 2.66; 95% confidence interval {CI] = 1.14 - 6.25, P = 0.0192), reduced relapse free survival (hazard of relapse = 1.45; 95% confidence interval {CI] = 1.12 - 1.89, P = 0.0051), and reduced distant metastasis free survival (hazard of metastasis = 1.7; 95% confidence interval {CI] = 1.00 - 2.91, P = 0.048) in TNBC (basal-like intrinsic subtype). AnxA2 gene expression was not associated with poor outcome in other intrinsic breast cancer subtypes, such as Luminal A, Luminal B, and Her-2, indicating the specific association of AnxA2 with the aggressive behavior of TNBC. Analysis of breast tumor subtypes and normal tissues confirmed that AnxA2 is highly expressed in TNBC compared to its low to negligible expression in other subtypes and normal tumors. Conclusion: AnxA2 overexpression is associated with racial variation and is a potential prognostic and diagnostic candidate for TNBC. Impact: AnxA2 has potential prognostic and diagnostic value, implicating a role for AnxA2 in the aggressive biology of TNBC in AA women. Citation Format: Lee D. Gibbs, Pankaj Chaudhary, Jamboor K. Vishwanatha. Racial Variation in Annexin A2 (AnxA2) Gene Expression and Poor Outcome in Triple-Negative Breast Cancer. [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 B03.

Mutant PIK3CA Induces EMT in a Cell Type Specific Manner.

Breast cancer is characterized into different molecular subtypes, and each subtype is characterized by differential gene expression that are associated with distinct survival outcomes in patients. PIK3CA mutations are commonly associated with most breast cancer subtypes. More recently PIK3CA mutations have been shown to induce tumor heterogeneity and are associated with activation of EGFR-signaling and reduced relapse free survival in basal subtype of breast cancer. Thus, understanding what determines PIK3CA induced heterogeneity and oncogenesis, is an important area of investigation. In this study, we assessed the effect of mutant PIK3CA together with mutant Ras plus mutant p53 on oncogenic behavior of two distinct stem/progenitor breast cell lines, designated as K5+/K19- and K5+/K19+. Constructs were ectopically overexpressed in K5+/K19- and K5+/K19+ stem/progenitor cells, followed by various in-vitro and in-vivo analyses. Oncogene combination m-Ras/m-p53/m-PIK3CA efficiently transformed both K5+/K19- and K5+/K19+ cell lines in-vitro, as assessed by anchorage-independent soft agar colony formation assay. Significantly, while this oncogene combination induced a complete epithelial-to-mesenchymal transition (EMT) in K5+/K19- cell line, mostly epithelial phenotype with minor EMT component was seen in K5+/K19+ cell line. However, both K5+/K19- and K5+/K19+ transformed cells exhibited increased invasion and migration abilities. Analyses of CD44 and CD24 expression showed both cell lines had tumor-initiating CD44+/CD24low cell population, however transformed K5+/K19- cells had more proportion of these cells. Significantly, both cell types exhibited in-vivo tumorigenesis, and maintained their EMT and epithelial nature in-vivo in mice tumors. Notably, while both cell types exhibited increase in tumor-initiating cell population, differential EMT phenotype was observed in these cell lines. These results suggest that EMT is a cell type dependent phenomenon and does not dictate oncogenesis.

Abstract 1426: Racial variation in annexin A2(AnxA2) gene expression and poor outcome in triple-negative breast cancer

Abstract Background: Triple-negative breast cancer (TNBC) is identified by the absence of three major receptors that drive most breast cancers. The incidence of TNBC is also associated with health disparity due to its disproportionate occurrence in African American (AA) women. Our previous studies and others have shown that AnxA2 is overexpressed in TNBC, but its association with racial variation and outcomes is unknown. Methods: AnxA2 gene expression was evaluated in breast cancer subtypes from The Cancer Genome Atlas (TCGA) Breast Invasive Carcinoma (BRCA) mRNA database that include 1,094 patients. Associations between clinical outcomes and AnxA2 gene expression were tested in a genome wide association study of combined publicly available datasets that include 4,147 patients. Results: AnxA2 gene expression was significantly increased in TNBC in comparison to other breast cancer subtypes. Furthermore, AnxA2 gene expression was significantly elevated in AA women in comparison with Caucasian (CA) women and was associated with the disproportionate occurrence of TNBC in AA women. High expression levels of AnxA2 was associated with reduced overall survival (hazard of death = 2.66; 95% confidence interval {CI] = 1.14 - 6.25, P = 0.0192), reduced relapse free survival (hazard of relapse = 1.45; 95% confidence interval {CI] = 1.12 – 1.89, P = 0.0051), and reduced distant metastasis free survival (hazard of metastasis = 1.7; 95% confidence interval {CI] = 1.00 – 2.91, P = 0.048). AnxA2 gene expression was not associated with poor outcome in other intrinsic breast cancer subtypes, such as Luminal A, Luminal B, and Her-2, indicating the specific association of AnxA2 with the aggressive behavior of TNBC. Conclusion: AnxA2 overexpression is associated with racial variation and is a potential prognostic candidate for TNBC. Impact: AnxA2 has potential prognostic value, implicating a role for AnxA2 in the aggressive biology of TNBC in AA women. Citation Format: Lee D. Gibbs, Jamboor K. Vishwanatha. Racial variation in annexin A2(AnxA2) gene expression and poor outcome in triple-negative breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1426.

Mutant p53 proteins counteract autophagic mechanism sensitizing cancer cells to mTOR inhibition

Mutations in TP53 gene play a pivotal role in tumorigenesis and cancer development. Here, we report that gain-of-function mutant p53 proteins inhibit the autophagic pathway favoring antiapoptotic effects as well as proliferation of pancreas and breast cancer cells. We found that mutant p53 significantly counteracts the formation of autophagic vesicles and their fusion with lysosomes throughout the repression of some key autophagy-related proteins and enzymes as BECN1 (and P-BECN1), DRAM1, ATG12, SESN1/2 and P-AMPK with the concomitant stimulation of mTOR signaling. As a paradigm of this mechanism, we show that atg12 gene repression was mediated by the recruitment of the p50 NF-κB/mutant p53 protein complex onto the atg12 promoter. Either mutant p53 or p50 NF-κB depletion downregulates atg12 gene expression. We further correlated the low expression levels of autophagic genes (atg12, becn1, sesn1, and dram1) with a reduced relapse free survival (RFS) and distant metastasis free survival (DMFS) of breast cancer patients carrying TP53 gene mutations conferring a prognostic value to this mutant p53-and autophagy-related signature. Interestingly, the mutant p53-driven mTOR stimulation sensitized cancer cells to the treatment with the mTOR inhibitor everolimus. All these results reveal a novel mechanism through which mutant p53 proteins promote cancer cell proliferation with the concomitant inhibition of autophagy.

A new role of the Rac-GAP β2-chimaerin in cell adhesion reveals opposite functions in breast cancer initiation and tumor progression.

β2-chimaerin is a Rac1-specific negative regulator and a candidate tumor suppressor in breast cancer but its precise function in mammary tumorigenesis in vivo is unknown. Here, we study for the first time the role of β2-chimaerin in breast cancer using a mouse model and describe an unforeseen role for this protein in epithelial cell-cell adhesion. We demonstrate that expression of β2-chimaerin in breast cancer epithelial cells reduces E-cadherin protein levels, thus loosening cell-cell contacts. In vivo, genetic ablation of β2-chimaerin in the MMTV-Neu/ErbB2 mice accelerates tumor onset, but delays tumor progression. Finally, analysis of clinical databases revealed an inverse correlation between β2-chimaerin and E-cadherin gene expressions in Her2+ breast tumors. Furthermore, breast cancer patients with low β2-chimaerin expression have reduced relapse free survival but develop metastasis at similar times. Overall, our data redefine the role of β2-chimaerin as tumor suppressor and provide the first in vivo evidence of a dual function in breast cancer, suppressing tumor initiation but favoring tumor progression.

Improved Detection of Minimal Residual Disease By Flow Cytometry in AML By Combining Manual Gating and Visne Clustering

Background: In acute myeloid leukemia (AML), detection of minimal residual disease (MRD) by flow cytometry is an adverse prognostic factor besides pre-treatment risk classifications, including cytogenetic and molecular aberrations. High dimensional multiparameter flow cytometry (MPFC) offers improved sensitivity and specificity, however manual analysis is increasingly challenging. In this study, we explore the value of the recently proposed viSNE algorithm to quantify MRD levels in patients with AML achieving complete remission (CR) after intensive induction chemotherapy.Methods: Bone marrow samples from patients with AML (excluding patients with acute promyelocytic leukemia) were analyzed by 8-10 MPFC using a NAVIOS flow cytometer (Beckman Coulter, Brea, CA, USA). Only patients achieving a CR or CR with incomplete blood count recovery (CRi) post-induction were included in this analysis. Manual gating of MRD flow data was performed as described previously (Köhnke et al., Leukemia 2014) using a cutoff for MRD positivity of 0.1%. The viSNE algorithm was performed as described previously (Amir et al., Nat. Biotech. 2013) and MRD positivity was defined as the presence of a distinct cluster of >100 cells which consisted of >90% patient cells. Kaplan-Meier estimator and log-rank test as well as Cox's proportional hazards regression model were used to analyze survival data.Results: Post-induction flow cytometry and clinical data of 38 patients with AML achieving CR (n=34) or CRi (n=4) were available for analysis (median age 53 years; de-novo AML n=32, tAML n=1, sAML n=5). Most patients belonged to the intermediate cytogenetic risk group (MRC favorable n=5, intermediate n=22, adverse n=11).19/38 patients were MRD positive post-induction by manual gating. 12/19 patients deemed MRD positive relapsed, whereas 3/19 patients deemed MRD negative relapsed. Therefore, MRD positivity by manual gating correlated with reduced relapse free survival (median RFS for MRD positive patients: 7.5 months vs. median not reached for MRD negative patients, log-rank test p=0.017). For overall survival (OS), no significant impact of MRD positivity could be detected so far (p=0.3), however follow-up was short (median follow-up 9.3 months). MRD positivity by manual gating remained an independent risk factor for RFS (HR 4.8, p=0.021) when compared to genetic risk and age.MRD positivity by viSNE clustering was seen in 19/38 patients. 10/19 patients deemed MRD positive by viSNE relapsed, while 5/19 deemed MRD negative by viSNE experienced a relapse. This resulted in a trend towards shorter RFS for MRD positivity by viSNE (median RFS 9.9 months vs. 19.0 months for MRD negative patients, p=0.185). Among the patients deemed MRD positive by viSNE who did not relapse, i.e. false-positive patients, follow-up was very short (<3.5 months) in 4/9 cases and final judgment whether these patients are truly negative should be withheld. However, differentiation between healthy monocytes and potential MRD cells by viSNE seems to be especially challenging and warrants optimization.Nevertheless, within the group of patients deemed MRD negative by manual gating, viSNE was able to detect all patients who experienced a subsequent relapse. To maximize sensitivity for MRD detection, we therefore combined the results of both methods and defined MRD positivity as positivity by manual gating and/or viSNE clustering. Using this combined strategy, 28/38 patients were defined as MRD positive and 10/38 as MRD negative. Within the MRD positive group, 15/28 patients relapsed, whereas none of the patients in the MRD negative group relapsed (Figure 1, median RFS for MRD positive patients: 7.7 months, log-rank test p=0.028). Of note, within the MRD negative group only 2 patients (both with intermediate genetic risk) went on to receive a allogeneic stem cell transplantation whereas 8 remained in remission with chemotherapy alone.Conclusion: In summary, viSNE clustering used in combination with manual gating improves the sensitivity of MRD detection by flow cytometry. Importantly, viSNE is able to detect MRD positivity within the manual MRD negative patient population. This is the first report of MRD detection in AML using this method. However, improvements in the algorithm as well as further studies are needed to validate the prognostic value of viSNE clustering in AML. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Gain of function mutant p53 proteins cooperate with E2F4 to transcriptionally downregulate RAD17 and BRCA1 gene expression.

Genomic instability (IN) is a common feature of many human cancers. The TP53 tumour suppressor gene is mutated in approximately half of human cancers. Here, we show that BRCA1 and RAD17 genes, whose derived proteins play a pivotal role in DNA damage repair, are transcriptional targets of gain-of-function mutant p53 proteins. Indeed, high levels of mutp53 protein facilitate DNA damage accumulation and severely impair BRCA1 and RAD17 expression in proliferating cancer cells. The recruitment of mutp53/E2F4 complex onto specific regions of BRCA1 and RAD17 promoters leads to the inhibition of their expression. BRCA1 and RAD17 mRNA expression is reduced in HNSCC patients carrying TP53 mutations when compared to those bearing wt-p53 gene. Furthermore, the analysis of gene expression databases for breast cancer patients reveals that low expression of DNA repair genes correlates significantly with reduced relapse free survival of patients carrying TP53 gene mutations. Collectively, these findings highlight the direct involvement of transcriptionally active gain of function mutant p53 proteins in genomic instability through the impairment of DNA repair mechanisms.