Basal Tumors Research Articles

Abstract P3-14-11: Mammaprint and Blueprint identify genomic differences in HR+ HER2- breast cancers from young Black and White women

Abstract Introduction: Hormone receptor positive (HR+), human epidermal growth factor receptor 2-negative (HER2-) breast cancer, the most common immunohistochemical subtype, remains the dominant contributor to annual breast cancer deaths worldwide across all racial and ethnic groups. Black women are 41% more likely to die from breast cancer compared to White women, predominantly among women diagnosed ≤ 50 years of age. Yet, Black women remain underrepresented in clinical trials and population-based studies. Thus, it is critical to better characterize tumor molecular features from young Black women to identify factors contributing to the existing racial survival disparity. In the current study, we compared risk of distant recurrence signature, MammaPrint (MP), molecular subtyping signature, BluePrint (BP), and whole transcriptome differences between young Black women with HR+ HER2- breast cancer compared to matched White controls. Methods: This study included 156 Black women aged ≤ 50 with stage I-III, HR+ HER2- breast cancer of whom, 68 were recruited from 2009-2014 as part of the BEST study (5R01CA204819-04) with follow-up data available (median 114.5 months). The remaining 88 Black women were enrolled in the ongoing FLEX Study (NCT03053193) from 2017. White women (n=156) were randomly selected from FLEX and matched by age, tumor stage, and receptor status. Tumors were classified through MP as Low Risk (MP-LR) versus High Risk (MP-HR), with MP-HR further stratified into HR1 and HR2; HR2 tumors exhibit superior chemosensitivity as demonstrated in a prior large clinical trial of breast cancer patients (ISPY2). All women had MP and BP to classify tumors as Luminal A (MP-LR), Luminal B (MP-HR), HER2, or Basal, and full transcriptomic analyses. Differential gene expression analysis was performed with R package ‘limma’ to compare Black and White women and further compare within each molecular subtype. Differentially expressed genes (DEGs) with a false discovery rate <0.05 were significant. Results: Of 312 young women with localized, HR+ HER2- breast cancer, high grade tumors were more frequent among Black compared to White women (34.6% vs 25.6%; p=0.08). MP-HR tumors were significantly more frequent among Black compared to White women (67.3% vs. 50.0%; p=0.002). Among MP-HR tumors, more HR2 tumors were seen in Black (25.6%) compared to White women (14.1%). Among women with MP-HR tumors and known treatment information, most Black women (94.3%) and all White women received chemotherapy. There were more Luminal B tumors in Black compared to White women (51.9% vs. 41.7%; p=0.07). BP reclassified a larger proportion of ER+ tumors as Basal in Black compared to White women (14.1% vs. 8.3%). Of 68 Black women with available survival data, 7 had death and/or distant recurrence events, of whom 6 (85.7%) had MP-HR tumors (4 Luminal B, 1 HER2, and 1 Basal) and 1 had MP-LR Luminal A tumor. Compared to White women, Black women with: 1) Luminal B tumors had 192 DEGs with upregulation of suspected poor prognosis genes, PSPH and IGHG1; 2) Luminal A tumors had upregulation of PSPH; and 3) Basal tumors had downregulation of POTEH. Conclusion: Among young women with localized HR+ HER2- breast cancer, MP and BP molecular signatures more robustly identified racial disparities in risk and subtype distribution beyond that identified through clinical factors adjusted for age and tumor characteristics. The transcriptomic differences among Black compared to White women across all BP subtypes provide novel insights about tumor biological differences. These findings have tremendous translational potential to identify etiologic underpinnings of racial survival disparities which may guide therapeutic strategies to improve outcomes. Citation Format: Sonya Reid, Tuya Pal, Ingrid A. Mayer, Xiao-Ou Shu, Ann L. Tezak, Kent Hoskins, Dipali Sharma, Patricia Robinson, Jennifer Wei, Jake Ruby, Shiyu Wang, Josien Haan, Andrea Menicucci, William Audeh, FLEX Investigators Group. Mammaprint and Blueprint identify genomic differences in HR+ HER2- breast cancers from young Black and White women [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-14-11.

Abstract P2-08-06: Defining transcriptomic profiles of breast cancer with early lymph node metastases: A FLEX database sub-study

Abstract Introduction: Early lymph node (LN) metastasis often precedes systemic metastasis and corresponds with a 35% decrease in 10-year survival compared to patients without LN metastasis. Understanding biologic pathways involved in early LN metastasis could identify promising drug targets for early-stage breast cancer (EBC) treatment. While tumor size modestly correlates with the probability of clinical or occult axillary metastases, small T1 tumors are often found to have LN metastases, while many large T3 tumors are often LN negative. We compared large tumors without evidence of LN metastasis (pT2-3N0) and small tumors with LN metastasis (pT1N1-3) by whole transcriptome analysis to elucidate molecular biological differences associated with the presence or absence of early LN metastasis at diagnosis. Methods: The FLEX study enrolls patients with EBC who undergo standard of care MammaPrint (MP) and BluePrint (BP) testing, and consent to clinically annotated full transcriptome data collection. MP classifies tumors as having High Risk (HR) or Low Risk (LR) of distant recurrence. MP combined with BP subtype tumors as either Luminal A-type (MP LR; 56.8%), Luminal B-type (MP HR; 36.4%), HER2-type (1.5%), or Basal-type (5.3%). In our primary comparison, 355 patients had small, LN-positive tumors (pT1N1-3), and 235 had large, LN-negative tumors (pT2-3N0). Control groups consisted of small, LN-negative (pT1N0; n=1384) and large, LN-positive tumors (pT2-3N1-3; n=375). R package ‘limma’ was used for quantile normalization and differential gene expression (DGE) analyses. Differentially expressed genes (DEGs) with a false discovery rate < 0.05 and fold change > 1.4 were considered significant for this exploratory analysis. Gene set enrichment analysis (GSEA) was performed using the Hallmark gene sets (MSigDB). Results: Principal component analysis of each sample’s transcriptional profile revealed more gene profile differences between MP risk and BP subtype than by pathological stage. Overall, DGE analysis comparing pT2-3N0 to pT1N1-3 identified 30 DEGs; of which MUCL1 had a > 2-fold decrease. Within MP LR and BP Luminal A-type tumors, no DEGs were identified comparing pT2-3N0 to pT1N1-3. HR tumors had 73 DEGs (33 upregulated and 40 downregulated); two of 40 downregulated genes, including MUCL1, had > 2-fold change. Within Luminal B tumors, 34 DEGs were identified when comparing pT2-3N0 with pT1N1-3, although none had > 2-fold change. Basal and HER2 tumors were not analyzed due to small numbers. GSEA showed upregulated proliferation and oxidative phosphorylation pathways, and downregulated epithelial to mesenchymal transition (EMT) and immune signatures in pT2-3N0 relative to pT1N1-3 tumors overall, within HR, and Luminal B tumors. Conclusion: This study provides a foundation for understanding the mechanisms that promote LN metastasis. Studies have previously correlated LN metastasis with EMT, immune function, and gene overexpression (BCL2L1, AURKA, CDKN2A, MCL1, MYC). While we did not find significant differences in these genes, our results indicated similar differentially regulated pathways. Overall, we found most biological differences within MP HR and Luminal B tumors. Proliferation-related pathways were upregulated and EMT and immune-related pathways were downregulated in pT2-3N0 tumors compared with pT1N1-3 tumors, suggesting these pathways distinguish small, node-positive tumors from large, node-negative tumors and are involved in early LN metastasis. Interestingly, MUCL1 which has been reported to promote migration and invasion in breast cancer, was consistently downregulated in pT2-3N0 relative to pT1N1-3 tumors. Future studies will investigate antineoplastic therapies for EBC that modulate these dysregulated pathways to reduce early LN metastasis and subsequent systemic metastasis. Citation Format: Nibash Budhathoki, Faisal Fa'ak, Nina D'Abreo, Shubhada Dhage, Phyu Phyu Soe, Dennis Holmes, Josien Haan, Shiyu Wang, Patricia Dauer, Andrea Menicucci, William Audeh, Douglas K Marks. Defining transcriptomic profiles of breast cancer with early lymph node metastases: A FLEX database sub-study [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-08-06.

Abstract P5-07-01: Proteogenomic analysis of differential chemotherapy responses in patient-derived xenografts of triple-negative breast cancer

Abstract Background: Chemotherapy is essential for the management of patients with triple-negative breast cancer (TNBC). Identification of biomarkers that may indicate treatment efficacy will be critical to improve patient stratification prior to treatment. To elucidate molecular determinants underlying chemotherapy response, we conducted a proteogenomic study using TNBC patient-derived xenografts (PDXs) treated with chemotherapy. Methods: Orthotopic TNBC PDXs were treated with four weekly cycles of docetaxel, carboplatin, or the combination. Changes in tumor volume after 4 weeks of treatment were evaluated. Genomic, transcriptomic, and mass-spectrometry-based proteomic profiling were performed on baseline tumors prior to treatments to identify associations with chemotherapy response (https://pdxportal.research.bcm.edu). Fisher’s exact test was used to identify significant associations between mutation and copy number events and dichotomized treatment response. For gene level analyses, Spearman’s correlation was calculated between mRNA or protein abundance and log2 fold change in tumor volumes after treatment. Signed -log10 p-values from Spearman’s correlation analysis were used as input for Gene Set Enrichment Analysis. Validation in external datasets was performed using METABRIC (PMID: 22522925) and BrighTNess clinical trial (PMID: 29501363) human datasets, an independent PDX dataset (PMID: 32546838), and shRNA screen data from DepMap (PMID: 30389920). Results: Combination carboplatin and docetaxel was largely ineffective at generating enhanced responses over the best single agent, suggesting de-escalation of chemotherapy may be possible. Genomic aberrations in BRCA2 and BCL9 were enriched in carboplatin-responsive PDXs while aberrations in RAF1 were enriched in docetaxel-resistant PDXs. Genes with gene-drug response correlations supported by both mRNA and protein measurements, but not mRNA or protein alone, for both carboplatin and docetaxel treatment in PDXs were associated with prognosis from basal human breast tumors receiving any chemotherapy from the METABRIC dataset. These data suggest that the combination of mRNA and protein data provided increased accuracy in identifying genes associated with clinical outcome in TNBC. Some of the top genes with genomic aberrations and/or overexpression at both mRNA and protein levels in chemoresistant PDXs, many of which have not been evaluated for their ability to augment response to taxane- or platinum-based chemotherapies, were validated in independent datasets with PDXs and TNBC patients receiving carboplatin and taxane combination. Further, some were found to be dependencies in TNBC cell lines from a publicly available genetic perturbation dataset. At the pathway level, both mRNA and protein data associated models resistant to both agents with enhanced oxidative phosphorylation and proteostatic pathways including proteosome degradation and the unfolded protein response (UPR) pathway related to endoplasmic reticulum stress. Pharmacological targeting the UPR pathway in combination with docetaxel showed activity in PDX models resistant to single-agent docetaxel. These results suggest targeting the UPR pathway as a novel therapeutic strategy to overcome chemotherapy resistance in TNBC. Conclusion: Proteogenomic analysis of PDX tumors identified diverse genes and pathways associated with chemotherapy resistance and response and further suggests potential therapeutic opportunities in TNBC. Citation Format: Jonathan T Lei, Chen Huang, Ramakrishnan R Srinivasan, Suhas Vasaikar, Lacey E Dobrolecki, Alaina N Lewis, Christina Sallas, Susan G Hilsenbeck, C. Kent Osborne, Mothaffar F Rimawi, Matthew J Ellis, Varduhi Petrosyan, Alexander B Saltzman, Anna Malovannaya, Gerburg Wulf, Daniel C Kraushaar, Tao Wang, Xi Chen, Gloria V Echeverria, Meenakshi Anurag, Bing Zhang, Michael T Lewis. Proteogenomic analysis of differential chemotherapy responses in patient-derived xenografts of triple-negative breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-07-01.

Permissive role of Na+/H+ exchanger isoform 1 in migration and invasion of triple-negative basal-like breast cancer cells.

In breast cancer, it is the resulting metastasis that is the primary cause of fatality. pH regulatory proteins and the tumor microenvironment play an important role in metastasis of cancer cells and acid-extruding proteins are critical in this process. There are several types of breast cancer and triple-negative breast cancer tends to be more metastatic and invasive and is itself is composed of several types. MDA-MB-468 are a triple-negative breast cancer cell line and are classified as basal-like and basal tumors account for up to 15% of breast cancers. Here we examined the effect of removal of the acid-extruding protein, the Na+/H+ exchanger isoform one, from MDA-MB-468 cells. NHE1 was deleted from these cells using the CRISPR/Cas9 system. Western blotting and measurement of activity confirmed the absence of the protein. In wounding/cell migration experiments, deletion of NHE1 reduced the rate of cell migration in the presence of low- or high-serum concentrations. Anchorage-dependent colony formation was also greatly reduced by deletion of the NHE1 protein. Cell proliferation was not affected by knockout of NHE1. The results demonstrate that NHE1 has an important role in migration and invasion of basal-like triple-negative breast cancer cells.

Alteration in glycolytic/cholesterogenic gene expression is associated with bladder cancer prognosis and immune cell infiltration

BackgroundOncogenic metabolic reprogramming contributes to tumor growth and immune evasion. The intertumoral metabolic heterogeneity and interaction of distinct metabolic pathways may determine patient outcomes. In this study, we aim to determine the clinical and immunological significance of metabolic subtypes according to the expression levels of genes related to glycolysis and cholesterol-synthesis in bladder cancer (BCa).MethodsBased on the median expression levels of glycolytic and cholesterogenic genes, patients were stratified into 4 subtypes (mixed, cholesterogenic, glycolytic, and quiescent) in an integrated cohort including TCGA, GSE13507, and IMvigor210. Clinical, genomic, transcriptomic, and tumor microenvironment characteristics were compared between the 4 subtypes.ResultsThe 4 metabolic subtypes exhibited distinct clinical, molecular, and genomic patterns. Compared to quiescent subtype, mixed subtype was more likely to be basal tumors and was significantly associated with poorer prognosis even after controlling for age, gender, histological grade, clinical stage, and molecular phenotypes. Additionally, mixed tumors harbored a higher frequency of RB1 and LRP1B copy number deletion compared to quiescent tumors (25.7% vs. 12.7 and 27.9% vs. 10.2%, respectively, both adjusted P value< 0.05). Furthermore, aberrant PIK3CA expression level was significantly correlated with those of glycolytic and cholesterogenic genes. The quiescent subtype was associated with lower stemness indices and lower signature scores for gene sets involved in genomic instability, including DNA replication, DNA damage repair, mismatch repair, and homologous recombination genes. Moreover, quiescent tumors exhibited lower expression levels of pyruvate dehydrogenase kinases 1-3 (PDK1-3) than the other subtypes. In addition, distinct immune cell infiltration patterns were observed across the 4 metabolic subtypes, with greater infiltration of M0/M2 macrophages observed in glycolytic and mixed subtypes. However, no significant difference in immunotherapy response was observed across the 4 metabolic subtypes.ConclusionThis study proposed a new metabolic subtyping method for BCa based on genes involved in glycolysis and cholesterol synthesis pathways. Our findings may provide novel insight for the development of personalized subtype-specific treatment strategies targeting metabolic vulnerabilities.

Lineage-specific silencing of PSAT1 induces serine auxotrophy and sensitivity to dietary serine starvation in luminal breast tumors.

SUMMARYA major challenge of targeting metabolism for cancer therapy is pathway redundancy, in which multiple sources of critical nutrients can limit the effectiveness of some metabolism-targeted therapies. Here, we analyze lineage-dependent gene expression in human breast tumors to identify differences in metabolic gene expression that may limit pathway redundancy and create therapeutic vulnerabilities. We find that the serine synthesis pathway gene PSAT1 is the most depleted metabolic gene in luminal breast tumors relative to basal tumors. Low PSAT1 prevents de novo serine biosynthesis and sensitizes luminal breast cancer cells to serine and glycine starvation in vitro and in vivo. This PSAT1 expression disparity preexists in the putative cells of origin of basal and luminal tumors and is due to luminal-specific hypermethylation of the PSAT1 gene. Our data demonstrate that luminal breast tumors are auxotrophic for serine and may be uniquely sensitive to therapies targeting serine availability.

Keyhole surgery for giant basal brain tumors. Theoretical explanation with an example of trans-eyebrow subraorbital approach

Improvement of modern neurosurgical technology has given rise to keyhole surgery for a wide range of pathologies. Supraorbital trans-eyebrow approach has been described in detail for small tumors of anterior cranial fossa and suprasellar neoplasms. Can we use this small and less traumatic approach for a more complex tumor extending beyond the suprasellar region? To analyze own experience of surgical treatment of large and giant parasellar tumors via supraorbital approach, to identify the principles of such operations, indications and possible limitations. There were 58 resections of parasellar tumors via mini-supraorbital approach had been performed between 2016 and 2021 at the 2nd pediatric neurosurgical department. We selected 42 patients with large and giant tumors extending beyond the chiasmal-sellar zone and causing occlusive hydrocephalus (mean volume 44.2 cm3). Preoperative status of patients, postoperative clinical data and logistics of in-hospital treatment were analyzed. Total resection was performed in all cases. No redo surgeries or revisions were required. There were no cases of wound infection, CSF leakage and damage to large vessels. We standardized planning of surgeries for large neoplasms considering available knowledge about the nature of diseases, analysis of clinical and X-ray data, goals and objectives of surgery. The reader is offered a theoretical substantiation of applicability of keyhole surgery for chiasmal-sellar tumors on the example of well-known mini-supraorbital approach. We gave certain advices on planning such operations and illustrated clinical examples.

Distinct Oncogenic Transcriptomes in Human Mammary Epithelial Cells Infected With Cytomegalovirus.

Human cytomegalovirus is being recognized as a potential oncovirus beside its oncomodulation role. We previously isolated two clinical isolates, HCMV-DB (KT959235) and HCMV-BL (MW980585), which in primary human mammary epithelial cells promoted oncogenic molecular pathways, established anchorage-independent growth in vitro, and produced tumorigenicity in mice models, therefore named high-risk oncogenic strains. In contrast, other clinical HCMV strains such as HCMV-FS, KM, and SC did not trigger such traits, therefore named low-risk oncogenic strains. In this study, we compared high-risk oncogenic HCMV-DB and BL strains (high-risk) with low-risk oncogenic strains HCMV-FS, KM, and SC (low-risk) additionally to the prototypic HCMV-TB40/E, knowing that all strains infect HMECs in vitro. Numerous pro-oncogenic features including enhanced expression of oncogenes, cell survival, proliferation, and epithelial-mesenchymal transition genes were observed with HCMV-BL. In vitro, mammosphere formation was observed only in high-risk strains. HCMV-TB40/E showed an intermediate transcriptome landscape with limited mammosphere formation. Since we observed that Ki67 gene expression allows us to discriminate between high and low-risk HCMV strains in vitro, we further tested its expression in vivo. Among HCMV-positive breast cancer biopsies, we only detected high expression of the Ki67 gene in basal tumors which may correspond to the presence of high-risk HCMV strains within tumors. Altogether, the transcriptome of HMECs infected with HCMV clinical isolates displays an “oncogenic gradient” where high-risk strains specifically induce a prooncogenic environment which might participate in breast cancer development.

Outcomes of intracranial germinoma-A retrospective multinational Asian study on effect of clinical presentation and differential treatment strategies.

This multinational study was conducted to report clinical presentations and treatment strategies in patients with intracranial germinomas across selected Asian centers, including failure patterns, risk factors, and outcomes. A retrospective data collection and analysis of these patients, treated between 1995 and 2015 from eight healthcare institutions across four countries was undertaken. From the results, 418 patients were analyzed, with a median follow-up of 8.9 years; 79.9% of the patients were M0, and 87.6% had β-human chorionic gonadotropin values <50 mIU/mL. The 5/10-year overall survival (OS) and recurrence-free survival (RFS) rates were 97.2%/96.2% and 89.9%/86.9%, respectively. RFS was predicted by the radiotherapy (RT) field, with focal RT having the worst outcome, whereas chemotherapy usage had no impact on survival. Among patients who received chemotherapy, response to chemotherapy did not predict survival outcomes. In M0 patients, primary basal ganglia tumors predicted a worse RFS. In patients with bifocal tumors, an extended field RT was associated with better outcomes. In multivariable analysis, only RT fields were associated with RFS. In relapsed patients, salvage rates were high at 85.7%. Additionally, patients who received salvage RT had a better outcome (91.6% vs. 66.7%). Survival outcomes of patients with germinoma were excellent. Thus, the focus of treatment for intracranial germinoma should be on survivorship. Further studies are warranted to find the optimal intensity and volume of radiation, including the role of chemotherapy in the survival of patients with intracranial germinomas, considering age, primary tumor location, and extent of disease.

Abstract PO-021: Targeting the mitochondrial pyruvate complex to alter metabolic programming in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) can be stratified into distinct transcriptome subtypes, with the ‘basal-like’ or ‘squamous’ subtype being associated with worse prognosis, compared to the ‘classical’ subtype. Our group recently demonstrated that PDAC tumors have unique metabolic transcriptome profiles, and that genes involved in glycolysis and cholesterol synthesis pathways are positively correlated with basal-like and classical gene expression patterns, respectively. The mitochondrial pyruvate complex (MPC) mediates the transport of pyruvate into the mitochondria which attenuates the effect of glycolysis on tumor progression. The mitochondrial pyruvate carrier 1 (MPC1) gene, which encodes one of two subunits of MPC, is deleted in over 60% of metastatic PDAC and PDAC glycolytic tumors have lowest levels of MPC1 expression. Using PDAC tissue microarrays, we also found that reduced MPC1 protein expression correlates with reduced survival in patients. We hypothesized that targeting MPC1 will alter metabolic reprogramming and may modulate tumor aggressiveness and therapeutic vulnerability in PDAC tumor cells. Genomically and clinically annotated patient-derived tumor organoids (PDOs) were generated from metastatic biopsies from patients enrolled in the PanGen study (NCT02869802). PDOs from both basal and classical tumors were used in the study. In order to investigate glycolysis in PDOs, we adapted the Seahorse Glycolytic Stress Test. Glycolysis, glycolytic capacity and reserve were analyzed in PDOs under basal and treated conditions. To alter MPC1 activity, PDOs were treated for 48 hours with 5uM of UK-5099, an MPC1 inhibitor, or 2.5-5uM SRT1720. SRT1720 is an activator of sirtuin 1 (SIRT1) and the transcriptional coactivator peroxisome proliferator-activated receptor γ coactivator-1α (PGC1-α), which regulates the expression of MPC1. An unpaired t-test with an alpha of 0.05 was used for all statistical analysis. Glycolysis analysis revealed distinct glycolytic profiles in PDOs with differences in glycolytic capacity and reserves trending with different tumor subtypes. Treatment with UK-5099 resulted in an increase in both glycolytic rate and reserve in PDOs from basal and classical tumors. Treatment with SRT1720 resulted in significantly reduced glycolytic rate and capacity. These data suggest that PDAC PDOs exhibit distinct metabolic profiles and that targeting MPC1 can modulate glycolysis in PDOs. Our ongoing efforts aim to further characterize the subtype-specific effect of MPC1 modulators on glycolysis and chemotherapy response in PDAC PDOs. Citation Format: Hassan A. Ali, Andrew Metcalfe, James T. Topham, Cassia S. Warren, Joanna M. Karasinska, David F. Schaeffer, Daniel J. Renouf. Targeting the mitochondrial pyruvate complex to alter metabolic programming in pancreatic cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-021.

Diagnostic and prognostic implications of a three-antibody molecular subtyping algorithm for non-muscle invasive bladder cancer.

Intrinsic molecular subtypes may explain marked variation between bladder cancer patients in prognosis and response to therapy. Complex testing algorithms and little attention to more prevalent, early‐stage (non‐muscle invasive) bladder cancers (NMIBCs) have hindered implementation of subtyping in clinical practice. Here, using a three‐antibody immunohistochemistry (IHC) algorithm, we identify the diagnostic and prognostic associations of well‐validated proteomic features of basal and luminal subtypes in NMIBC. By IHC, we divided 481 NMIBCs into basal (GATA3−/KRT5+) and luminal (GATA3+/KRT5 variable) subtypes. We further divided the luminal subtype into URO (p16 low), URO‐KRT5+ (KRT5+), and genomically unstable (GU) (p16 high) subtypes. Expression thresholds were confirmed using unsupervised hierarchical clustering. Subtypes were correlated with pathology and outcomes. All NMIBC cases clustered into the basal/squamous (basal) or one of the three luminal (URO, URO‐KRT5+, and GU) subtypes. Although uncommon in this NMIBC cohort, basal tumors (3%, n = 16) had dramatically higher grade (100%, n = 16, odds ratio [OR] = 13, relative risk = 3.25) and stage, and rapid progression to muscle invasion (median progression‐free survival = 35.4 months, p = 0.0001). URO, the most common subtype (46%, n = 220), showed rapid recurrence (median recurrence‐free survival [RFS] = 11.5 months, p = 0.039) compared to its GU counterpart (29%, n = 137, median RFS = 16.9 months), even in patients who received intravesical immunotherapy (p = 0.049). URO‐KRT5+ tumors (22%, n = 108) were typically low grade (66%, n = 71, OR = 3.7) and recurred slowly (median RFS = 38.7 months). Therefore, a simple immunohistochemical algorithm can identify clinically relevant molecular subtypes of NMIBC. In routine clinical practice, this three‐antibody algorithm may help clarify diagnostic dilemmas and optimize surveillance and treatment strategies for patients.

Pparg signaling controls bladder cancer subtype and immune exclusion

Pparg, a nuclear receptor, is downregulated in basal subtype bladder cancers that tend to be muscle invasive and amplified in luminal subtype bladder cancers that tend to be non-muscle invasive. Bladder cancers derive from the urothelium, one of the most quiescent epithelia in the body, which is composed of basal, intermediate, and superficial cells. We find that expression of an activated form of Pparg (VP16;Pparg) in basal progenitors induces formation of superficial cells in situ, that exit the cell cycle, and do not form tumors. Expression in basal progenitors that have been activated by mild injury however, results in luminal tumor formation. We find that these tumors are immune deserted, which may be linked to down-regulation of Nf-kb, a Pparg target. Interestingly, some luminal tumors begin to shift to basal subtype tumors with time, down-regulating Pparg and other luminal markers. Our findings have important implications for treatment and diagnosis of bladder cancer.

Gray matter structural plasticity in patients with basal ganglia germ cell tumors: A voxel-based morphometry study

BackgroundBasal ganglia germ cell tumors (BGGCTs) are rare intracranial germ cell tumors (iGCTs) that often presents with cognitive impairment. ObjectiveTo assess structural brain plasticity in the presence of unilateral basal ganglia germ cell tumors (BGGCTs), and the correlation between gray matter volume (GMV) changes and cognitive tests. Materials and methodsWe applied voxel-based morphometry (VBM) to structural magnetic resonance imaging (MRI) scans to compare a sample of 41 patients with BGGCTs in the left (n = 22) or right (n = 19) and a sample of 16 patients as control group using a two-sample t-test, correcting for family-wise-errors. A battery of cognitive tests was administered to all BGGCTs patients prior to MRI. We used Pearson correlation analysis to assess the correlation between cognitive test scores and GMV changes. ResultsIn patients with left BGGCTs, whole-brain VBM analysis revealed a large cluster of voxels reflecting an increase in GMV in the left parahippocampal region (k = 529 voxels, T = 4.18, p < 0.01), right middle cingulate cortex (k = 172 voxels, T = 3.96, p < 0.01), and a decrease in volume in the left thalamus (k = 527 voxels, T = −4.88, p < 0.01), right inferior frontal gyrus (k = 495 voxels, T = −4.29, p < 0.01). Pearson correlation analysis showed that the GMV were significantly correlated with the Integrated Visual and Auditory continuous performance test (IVA-CPT) scale (r = 0.637, P = 0.002), abstract reasoning (r = 0.597, P = 0.011), Self-rating Depression Scale (SAS) scale (r = −0.623, P = 0.004) and memory recall (r = 0.648, P = 0.003). ConclusionThese results demonstrate that slow growing but destructive BGGCTs markedly and asymmetrically effect the GMV in left parahippocampal, left thalamus, right middle cingulate cortex, right inferior frontal gyrus and GMV changes were significantly associated with cognitive test.

Opposing effects of BRCA1 mRNA expression on patient survival in breast and colorectal cancer and variations among African American, Asian, and younger patients

Breast cancer (BC) and colorectal cancer (CRC) are common and show poor survival in advanced stages. Using The Cancer Genome Atlas (TCGA) computational tool cBioPortal, we evaluated overall patient survival in BRCA1 mRNA-low versus -high cohorts (<−1.29 versus >1.05 SD from mean BRCA1 expression, respectively). Analysis included 1082 BC patients with mRNA data (PanCancer Atlas), 382 CRCs (Firehose Legacy) and 592 CRCs (PanCancer Atlas). As previously reported, BRCA1 mRNA-low tumor expression positively correlated with BC patient survival but was negatively associated in CRC. We observed a correlation between BRCA1 mRNA-high and age <45 years at CRC diagnosis using a Fisher’s exact test [Firehose Legacy database (p-value = 0.0091); CRC PanCancer Atlas (p-value = 0.0778)]. We correlated BRCA1 mRNA-low expression and basal BC (p-value = 0.0016) and BRCA1 mRNA-low tumors and frequency of African American patients (p-value = 0.0448) with BC. Other trends included higher frequency of advanced lymph node stage and mucinous adenocarcinoma among BRCA1 mRNA-low CRC and higher frequency of males in BRCA1 mRNA-high BC and CRC. African Americans more frequently had BRCA1 mRNA-low BC and BRCA1 mRNA-high CRC and the opposite was observed among Asians. Using a gene co-expression tool (cBioPortal), we observed TOP2A and ATAD5 levels correlate (Spearman’s correlation>0.6) with BRCA1 in BC and CRC, whereas LMNB2 correlates with BRCA1 in CRC, suggesting tissue-specific BRCA1 interactions. Our results indicate potential for BRCA1 mRNA expression levels as a prognostic biomarker in BC and CRC, suggest tissue-specificity in BRCA1 molecular interactions, and point to BRCA1 mRNA-high levels as a characteristic of CRC tumors in younger versus older individuals.

Prognosis Associated With Luminal and Basal Subtypes of Metastatic Prostate Cancer

Luminal and basal subtypes of primary prostate cancer have been shown to be molecularly distinct and clinically important in predicting response to therapy. These subtypes have not been described in metastatic prostate cancer. To identify clinical and molecular correlates of luminal and basal subtypes in metastatic castration-resistant prostate cancer (mCRPC) and investigate differences in survival, particularly after treatment with androgen-signaling inhibitors (ASIs). In this cohort study, a retrospective analysis was conducted of 4 cohorts with mCRPC (N = 634) across multiple academic centers. Treatment was at the physicians' discretion. Details of the study cohorts have been published elsewhere between 2016 and 2019. Data were analyzed from March 2018 to February 2021. The primary clinical end point was overall survival from the date of tissue biopsy/molecular profiling. Luminal and basal subtypes were also stratified by postbiopsy ASI treatment. The primary molecular analyses included associations with small cell/neuroendocrine prostate cancer (SCNC), molecular pathways, and DNA alterations. In the 634 patients, 288 (45%) had tumors classified as luminal, and 346 (55%) had tumors classified as basal. However, 53 of 59 (90%) SCNC tumors were basal (P < .001). Similar to primary prostate cancer, luminal tumors exhibited overexpression of AR pathway genes. In basal tumors, a significantly higher rate of RB1 loss (23% basal vs 4% luminal; P < .001), FOXA1 alterations (36% basal vs 27% luminal; P = .03) and MYC alterations (73% basal vs 56% luminal; P < .001) were identified. Patients with basal tumors had worse overall survival compared with those with luminal tumors only in patients treated with an ASI postbiopsy (East Coast Dream Team: hazard ratio [HR], 0.39; 95% CI, 0.20-0.74; P = .004; West Coast Dream Team: HR, 0.57; 95% CI, 0.33-0.97; P = .04). Among patients with luminal tumors, those treated with an ASI had significantly better survival (HR, 0.27; 95% CI, 0.14-0.53; P < .001), whereas patients with basal tumors did not (HR, 0.62; 95% CI, 0.36-1.04, P = .07). The interaction term between subtype and ASI treatment was statistically significant (HR, 0.42; 95% CI, 0.20-0.89; P = .02). These findings represent the largest integrated clinical, transcriptomic, and genomic analysis of mCRPC samples to date, and suggest that mCRPC can be classified as luminal and basal tumors. Analogous to primary prostate cancer, these data suggest that the benefit of ASI treatment is more pronounced in luminal tumors and support the use of ASIs in this population. In the basal tumors, a chemotherapeutic approach could be considered in some patients given the similarity to SCNC and the diminished benefit of ASI therapy. Further validation in prospective clinical trials is warranted.

Hedgehog Inhibitor Induction with Addition of Concurrent Superficial Radiotherapy in Patients with Locally Advanced Basal Cell Carcinoma: A Case Series.

Locally advanced basal cell cancer is a rare and challenging clinical problem. Historically, these patients were treated with aggressive surgery or radiotherapy. Most sporadic basal cell carcinomas have somatic mutations in the hedgehog pathway. Oral hedgehog inhibitors induce rapid and often complete clinical responses in locally advanced basal cell tumors. Unfortunately, these responses are usually transient. We hypothesized that treatment failure represents persistence of drug resistant cells that could be eradicated by addition of localized radiotherapy. We performed a retrospective review of our patients with locally advanced basal cell cancer treated with sonidegib or vismodegib induction therapy who were treated with added superficial radiotherapy at the time of maximal response. Twelve patients met inclusion criteria. All patients achieved a complete response following hedgehog inhibitor therapy with addition of radiotherapy. Progression-free survival at 40 months was 89%, with a median follow-up of 40 months. Relapses occurred in only 2 of 12 patients (16.6%). Nine patients experienced grade I-II toxicity from hedgehog inhibitor induction therapy (taste changes [3], weight loss [3], muscle cramps [3]). Eight patients experienced mild radiotherapy-induced skin toxicity during concurrent therapy. No patients had to discontinue treatment. Induction therapy with hedgehog inhibitors followed by addition of concurrent radiation therapy resulted in an extremely high clinical response rate with relatively minor and reversible toxicity. This gave a high rate of progression-free survival and a low disease-specific progression rate. Further prospective evaluation of this treatment approach is needed to confirm the apparent clinical activity. Locally advanced basal cell cancers are challenging to treat. Previously, aggressive surgical resection or radiotherapy represented the best treatment options. Most basal cell cancers have somatic mutations in the hedgehog pathway. Oral inhibitors of this pathway produce rapid but transient clinical responses. This study reports 12 patients treated with hedgehog inhibitor induction therapy to near-maximal response. Addition of concurrent involved field radiotherapy resulted in a very high complete response rate with minimal toxicity. There was prolonged progression-free survival in 90% of patients. This study identified a novel treatment approach for patients with advanced basal cell carcinoma.

Integrative Transcriptomic, Lipidomic, and Metabolomic Analysis Reveals Potential Biomarkers of Basal and Luminal Muscle Invasive Bladder Cancer Subtypes.

Muscle invasive bladder cancer (MIBC) is a heterogeneous disease with a high recurrence rate and poor clinical outcomes. Molecular subtype provides a new framework for the study of MIBC heterogeneity. Clinically, MIBC can be classified as basal and luminal subtypes; they display different clinical and pathological characteristics, but the molecular mechanism is still unclear. Lipidomic and metabolomic molecules have recently been considered to play an important role in the genesis and development of tumors, especially as potential biomarkers. Their different expression profiles in basal and luminal subtypes provide clues for the molecular mechanism of basal and luminal subtypes and the discovery of new biomarkers. Herein, we stratified MIBC patients into basal and luminal subtypes using a MIBC classifier based on transcriptome expression profiles. We qualitatively and quantitatively analyzed the lipids and metabolites of basal and luminal MIBC subtypes and identified their differential lipid and metabolite profiles. Our results suggest that free fatty acids (FFAs) and sulfatides (SLs), which are closely associated with immune and stromal cell types, can contribute to the diagnosis of basal and luminal subtypes of MIBC. Moreover, we showed that glycerophosphocholine (GCP)/imidazoles and nucleosides/imidazoles ratios can accurately distinguish the basal and luminal tumors. Overall, by integrating transcriptomic, lipidomic, and metabolomic data, our study reveals specific biomarkers to differentially diagnose basal and luminal MIBC subtypes and may provide a basis for precision therapy of MIBC.

Discovery and 3D imaging of a novel ΔNp63-expressing basal cell type in human pancreatic ducts with implications in disease

ObjectiveThe aggressive basal-like molecular subtype of pancreatic ductal adenocarcinoma (PDAC) harbours a ΔNp63 (p40) gene expression signature reminiscent of a basal cell type. Distinct from other epithelia with basal tumours,...

Oncogenic Hedgehog-Smoothened Signaling Depends on YAP1‒TAZ/TEAD Transcription to Restrain Differentiation in Basal Cell Carcinoma

Disruption of the transcriptional activity of the Hippo pathway members YAP1 and TAZ has become a major target for cancer treatment. However, detailed analysis of the effectiveness and networks affected by YAP1/TAZ transcriptional targeting is limited. In this study, we utilize TEAD inhibitor, an inhibitor of the binding of YAP1 and TAZ with their main transcriptional target TEAD in a mouse model of basal cell carcinoma, to unveil the consequences of YAP1/TAZ transcriptional blockage in cancer cells. Both TEAD inhibitor and YAP1/TAZ knockdown lead to reduced proliferation and increased differentiation of mouse basal cell carcinoma driven by oncogenic hedgehog-smoothened (SmoM2) activity. Although TEAD-transcriptional networks were essential to inactivate differentiation, this inactivation was found to be indirect and potentially mediated through the repression of KLF4 by SNAI2. By comparing the transcriptional effects of TEAD inhibition with those caused by YAP1/TAZ depletion, we determined YAP1/TAZ‒TEAD‒independent effects in cancer cells that impact STAT3 and NF-κB. Our results reveal the gene networks affected by targeting YAP1/TAZ‒TEAD in basal cell carcinoma tumors and expose the potential pitfalls for targeting TEAD transcription in cancer.

Abstract 608: Molecular features of breast cancer involved in classification and prognosis of a multi-country Latin American cohort: The US-LACRN-MPBCS breast cancer cohort

Abstract Although gene expression-derived PAM50 intrinsic subtypes (LumA, LumB, HER2E and Basal) were reported in Latin American breast cancer, most studies did not adequately represent the unique and diverse genetic admixture of the Latin American population and/or included a small number of individuals. As a result of these limitations, confirmation of the prognostic value of available intrinsic subtype classification signatures in a diverse cohort of Latin American women is of utmost importance. We assessed the general distribution and prognostic performance of PAM50-based intrinsic and immunohistochemistry (IHC)-based surrogate subtype classifications in Latin American women included in the Molecular Profile of Breast Cancer Study (MPBCS), an initiative of the US-Latin America Cancer Research Network (US-LACRN) comprising institutions of Argentina, Brazil, Chile, Mexico and Uruguay. MPBCS focused on stage II-III breast cancer in Latin American women. Eligible enrolled patients (n=1300) were characterized clinically, pathologically and epidemiologically and followed-up for 5 years. IHC subtypes were assessed according to St Gallen's 2013 criteria, using Ki67 to discriminate LumB from LumA tumors. A total of 1071 tumors were characterized by gene-expression microarrays. PAM50 classification defined 45% of tumors as LumA, 19.7% as LumB, 13.8% as HER2E and 17.5% as Basal. Normal-like tumors (6.3%) were excluded from the analysis. The 5-year prognostic ability of PAM50 and IHC classifications, both at the cancer-specific (OS) and progression-free survival (PFS), was tested. The prognosis for LumA tumors was significantly better than for other subtypes, while Basal-like tumors had the worst prognosis. The prognostic power of IHC-based subtypes (C-index 0.698 for OS, 0.635 for PFS) was very similar to that of PAM50 (C-index 0.678 for OS, 0.639 for PFS), indicating that in US-LACRN-MPBCS, contrary to other cohorts, surrogate subtypes are as useful as PAM50 for discriminating recurrence risk. PAM50-derived risks of recurrence (RORs), in particular ROR-S (C-index 0.699 for OS, 0.649 for PFS), clearly discriminated risk into low, intermediate and high-risk groups. Transcriptomic pathway analysis showed high proliferation (i.e. cell cycle control and DNA damage repair) associated with LumB, HER2E and Basal tumors, and a strong dependency on the estrogen pathway for LumA. Overall, a general concordance of the molecular features of US-LACRN-MPBCS breast cancer tumors with those of other cohorts was confirmed. The shift towards non-luminal subtypes could be partly attributable to the recruitment bias towards advanced stages. Further refinement of analyses using molecular ancestry assignation may help to reveal more subtle differences in this heterogeneously admixed population. Citation Format: Andrea S. Llera, Eliana Abdelhay, Osvaldo Podhajcer, Nora Artagaveytia, Adrián Daneri-Navarro, Bettina Müller, Carlos Velázquez Contreras, Darío Rocha, Juan Martín Sendoya, Renata Binato, Elmer Fernández, Elsa Alcoba, Isabel Alonso, Alicia I. Bravo, Natalia Camejo, Dirce Carraro, Mónica Castro, Juan M. Castro-Cervantes, Sandra Cataldi, Alfonso Cayota, Mauricio Cerda, Susanne Crocamo, Raul Delgadillo-Cisterna, Lucía Delgado, Alicia del Toro Arreola, Marisa Dreyer Breitenbach, Jorge Fernández, Wanda Fernández, Ramon A. Franco-Topete, Fancy Gaete, Jorge Gómez, Gonzalo Greif, Marisol Guerrero, Marianne Marianne Henderson, Andres de J Moran-Mendoza, María Aparecida Nagai, Antonio Oceguera-Villanueva, Antonio Quintero-Ramos, Rui Reis, Javier Retamales, Robinson Rodríguez, Cristina Rosales, Efrain Salas-González, Laura Segovia, Araceli Silva-García, Vidya Vedham, Livia Zagame, The US-Latin American Cancer Research Network. Molecular features of breast cancer involved in classification and prognosis of a multi-country Latin American cohort: The US-LACRN-MPBCS breast cancer cohort [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 608.