Sinnwell, Jason P.

Abstract Background: Triple-negative breast cancers (TNBC) exhibit high rates of recurrence and mortality. However, recent studies suggest that a subset of patients (pts) with early-stage TNBC enriched in tumor-infiltrating lymphocytes (TILs) have excellent clinical outcomes even in the absence of systemic therapy. Additional histological biomarkers that could identify pts for future systemic therapy escalation/de-escalation strategies are of great interest. TNBC are frequently highly proliferative with abundant mitoses. However, classic markers of proliferation (manual mitosis counting and Ki-67) appear to offer no prognostic value. Here, we evaluated the prognostic effects of automated mitotic spindle hotspot (AMSH) counting on RFS in independent cohorts of systemically untreated early-stage TNBC. Methods: AMSH counting was conducted with a state-of-the-art deep learning algorithm trained on the detection of mitoses within 2 mm2 areas with the highest mitotic density (i.e. hotspots) in digital H&E images. Details of the development, training and validation of the algorithm were published previously [1] in a cohort of unselected TNBC. We obtained AMSH counts in a centrally confirmed TNBC cohort from Mayo Clinic [2] and focused our analysis on pts who received locoregional therapy but no systemic therapy. Using a fractional polynomial analysis with a multivariable proportional hazards regression model, we confirmed the assumption of linearity in the log hazard for the continuous variable AMSH and evaluated whether AMSH counts were prognostic of RFS. We corroborated our findings in an independent cohort of systemically untreated TNBC pts from the Radboud University Medical Center in the Netherlands (Radboud Cohort). Results are reported at a median follow-up of 8.1 and 6.7 years for the Mayo and Netherlands cohorts, respectively. Results: Among 182 pts with who did not receive systemic therapy in the Mayo Cohort, 140 (77%) with available AMSH counts were included. The mean age was 61 (range: 31-94), 71% were postmenopausal, 67% had tumors ≤ 2cm, and 83% were node-negative. As expected, most tumors were Nottingham grade 3 (84%) and had a high Ki-67 proliferation index (54% with Ki-67 >30%). Most tumors (73%) had stromal TILs ≤ 30%. The median AMSH count was 18 (IQR: 8, 42). AMSH counts were linearly associated with grade and tumor size, with the proportion of pts with grade 3 tumors and size > 2 cm increasing as the AMSH counts increased (p=0.007 and p=0.059, respectively). In a multivariate model controlling for nodal status, tumor size, and stromal TILs, AMSH counts were independently associated with RFS (p< 0.0001). For every 10-point increase in the AMSH count, we observed a 17% increase in the risk of experiencing an RFS event (HR 1.17, 95% CI 1.08-1.26). We corroborated our findings in the Radboud Cohort (n=126). The mean age was 68 (range: 40-96), and 81% were node-negative. While the median AMSH count was 36 (IQR: 16-63), higher than in the Mayo Cohort (p=0.004), the prognostic impact was similar, with a significant association between AMSH count and RFS (p=0.028) in a multivariate model corrected for nodal status, tumor size, and stromal TILs. For every 10-point increase in the AMSH count in the Netherlands cohort, we observed a 9% increase in the risk of experiencing an RFS event (HR 1.09, 95% CI 1.01-1.17). RFS rates according to AMSH counts for both cohorts are shown in the Table. Conclusions: AMSH counting is a new proliferation biomarker that provides prognostic value independent of nodal status, tumor size, and stromal TILs in systemically untreated early-stage TNBC. Plans are underway to evaluate AMSH counts in additional cohorts of systemically untreated TNBC, and in other disease settings such as prior to neoadjuvant systemic therapy. If validated, this biomarker should be prospectively evaluated as a potential selection biomarker in clinical trials of systemic therapy de-escalation. References: 1. PMID: 29994086 2. PMID: 28913760 Table RFS according to AMSH counts in the Mayo and Radboud Cohorts Citation Format: Roberto A. Leon-Ferre, Jodi M. Carter, David Zahrieh, Jason P. Sinnwell, Roberto Salgado, Vera Suman, David Hillman, Judy C. Boughey, Krishna R. Kalari, Fergus J. Couch, James N. Ingle, Maschenka Balkenkohl, Francesco Ciompi, Jeroen van der Laak, Matthew P. Goetz. Mitotic spindle hotspot counting using deep learning networks is highly associated with clinical outcomes in patients with early-stage triple-negative breast cancer who did not receive systemic therapy [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-11-34.

Abstract Signaling networks and the protein interactions that define them are of great interest, but techniques to assess these networks in unmanipulated primary human cells are limited. We have developed and applied a novel technological and analytical platform to measure proteins in shared complexes detected by exposed surface epitopes (PiSCES). Immunoprecipitation reactions are performed in multiplex using microsphere beads to measure 20+ proteins and 210+ distinct pair-wise protein associations that are important for T cell antigen receptor signaling. Jurkat T cells stimulated with superantigen generated an extensive PiSCES signature using this method and provided a dataset from which we optimized an analytical package. The system is currently being utilized to address the hypothesis that T cells isolated from skin lesions of autoimmune alopecia areta (AA) patients will have a unique PiSCES signature compared to those isolated from non-AA donors. To investigate this, we have isolated non-engineered primary cells from AA patient skin lesions and healthy controls, and have begun to identify differences in their PiSCES signatures, either directly after isolation or after stimulation with anti-CD3 and anti-CD28. We conclude that this novel PiSCES approach is capable of generating protein-protein interaction signatures that provide a unique view of TCR signalosome activity solicited from physiologic non-engineered primary human patient samples.

Abstract We identified resistance mechanisms to abiraterone acetate/prednisone (AA/P) in patients with metastatic castration-resistant prostate cancer (mCRPC) in the Prostate Cancer Medically Optimized Genome-Enhanced Therapy (PROMOTE) study. We analyzed whole-exome sequencing (WES) and RNA-sequencing data from 83 patients with metastatic biopsies before (V1) and after 12 weeks of AA/P treatment (V2). Resistance was determined by time to treatment change (TTTC). At V2, 18 and 11 of 58 patients had either short-term (median 3.6 months; range 1.4–4.5) or long-term (median 29 months; range 23.5–41.7) responses, respectively. Nonresponders had low expression of TGFBR3 and increased activation of the Wnt pathway, cell cycle, upregulation of AR variants, both pre- and posttreatment, with further deletion of AR inhibitor CDK11B posttreatment. Deletion of androgen processing genes, HSD17B11, CYP19A1 were observed in nonresponders posttreatment. Genes involved in cell cycle, DNA repair, Wnt-signaling, and Aurora kinase pathways were differentially expressed between the responder and non-responder at V2. Activation of Wnt signaling in nonresponder and deactivation of MYC or its target genes in responders was detected via SCN loss, somatic mutations, and transcriptomics. Upregulation of genes in the AURKA pathway are consistent with the activation of MYC regulated genes in nonresponders. Several genes in the AKT1 axis had increased mutation rate in nonresponders. We also found evidence of resistance via PDCD1 overexpression in responders. Implications: Finally, we identified candidates drugs to reverse AA/P resistance: topoisomerase inhibitors and drugs targeting the cell cycle via the MYC/AURKA/AURKB/TOP2A and/or PI3K_AKT_MTOR pathways.

Neoadjuvant chemotherapy (NAC) remains the cornerstone of the treatment for triple negative breast cancer (TNBC), with the goal of complete eradication of disease. However, for patients with residual disease after NAC, recurrence and mortality rates are high and the identification of novel therapeutic targets is urgently needed. We quantified tyrosine phosphorylation (pTyr)-mediated signaling networks in chemotherapy sensitive (CS) and resistant (CR) TNBC patient-derived xenografts (PDX), to gain novel therapeutic insights. The antitumor activity of SFK inhibition was examined in vivo. Treated tumors were further subjected to phosphoproteomic and RNAseq analysis, to identify the mechanism of actions of the drug. We identified Src Family Kinases (SFKs) as potential therapeutic targets in CR TNBC PDXs. Treatment with dasatinib, an FDA approved SFK inhibitor, led to inhibition of tumor growth in vivo. Further analysis of post-treatment PDXs revealed multiple mechanisms of actions of the drug, confirming the multi-target inhibition of dasatinib. Analysis of pTyr in tumor specimens suggested a low prevalence of SFK-driven tumors, which may provide insight into prior clinical trial results demonstrating a lack of dasatinib antitumor activity in unselected breast cancer patients. Taken together, these results underscore the importance of pTyr characterization of tumors, in identifying new targets, as well as stratifying patients based on their activated signaling networks for therapeutic options. Our data provide a strong rationale for studying SFK inhibitors in biomarker-selected SFK-driven TNBC.

AbstractBackground:Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype. Patients with TNBC are primarily treated with neoadjuvant chemotherapy (NAC). Response to NAC is prognostic, with reductions in low overall survival and disease-free survival rates in those patients who do not achieve pathological complete response (pCR). Based on this premise, we focused on patients with residual disease and hypothesized that paired analysis of primary and residual TNBC tumors following NAC could identify unique biomarkers associated with post-NAC recurrence.Methods and Results: We investigated 24 TNBC tumors from 12 patients with pre- and post-NAC data, including four patients who recurred shortly after surgery(<24 months) and eight who remained recurrence-free (> 48 months). These tumors were collected from a prospective NAC breast cancer study (BEAUTY) conducted at the Mayo Clinic. Differential expression analysis of pre-NAC biopsies showed minimal gene expression differences between early recurrent and non-recurrent TNBC tumors; however, post-NAC samples demonstrated a significant alteration in expression patterns in response to intervention. Topological-level differences associated with early recurrence were implicated in 251 gene sets, and an independent assessment of microarray gene expression data from the paired NAC I-SPY1 trial confirmed 56 genesets. Underlying these 56 gene sets, 113 genes were observed to be differentially expressed in the I-SPY1 and BEAUTY post-NAC studies. An independent (n=392) breast cancer data set with relapse-free survival (RFS) data was used to refine our gene list to a 17 gene signature. A three-fold cross-validation analysis of the gene signature with the combined BEAUTY and I-SPY1 data yielded an average AUC of 0.88 for six machine learning models. Due to the limited studies with pre-and post-NAC TNBC tumor data, further validation of the signature is needed.Conclusion:Analysis of multi-omics data from post-NAC TNBC chemoresistant tumors showed downregulation of mismatch repair, purin, and tubulin pathways. Additionally, weidentified a 17 gene signature in TNBC associated with post-NAC recurrence enriched with down-regulation of immune genes.

Abstract Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype with low overall survival rates and high molecular heterogeneity; therefore, few targeted therapies are available. The luminal androgen receptor (LAR) is the most consistently identified TNBC subtype, but the clinical utility has yet to be established. Here, we constructed a novel genomic classifier, LAR-Sig, that distinguishes the LAR subtype from other TNBC subtypes and provide evidence that it is a clinically distinct disease. A meta-analysis of seven TNBC datasets (n = 1086 samples) from neoadjuvant clinical trials demonstrated that LAR patients have significantly reduced response (pCR) rates than non-LAR TNBC patients (odds ratio = 2.11, 95% CI: 1.33, 2.89). Moreover, deconvolution of the tumor microenvironment confirmed an enrichment of luminal epithelium corresponding with a decrease in basal and myoepithelium in LAR TNBC tumors. Increased immunosuppression in LAR patients may lead to a decreased presence of cycling T-cells and plasma cells. While, an increased presence of myofibroblast-like cancer-associated cells may impede drug delivery and treatment. In summary, the lower levels of tumor infiltrating lymphocytes (TILs), reduced immune activity in the micro-environment, and lower pCR rates after NAC, suggest that new therapeutic strategies for the LAR TNBC subtype need to be developed.

Abstract Background: Genome-wide association studies (GWAS) of multiple myeloma in populations of European ancestry (EA) identified and confirmed 24 susceptibility loci. For other cancers (e.g., colorectum and melanoma), risk loci have also been associated with patient survival. Methods: We explored the possible association of all the known risk variants and their polygenic risk score (PRS) with multiple myeloma overall survival (OS) in multiple populations of EA [the International Multiple Myeloma rESEarch (IMMEnSE) consortium, the International Lymphoma Epidemiology consortium, CoMMpass, and the German GWAS] for a total of 3,748 multiple myeloma cases. Cox proportional hazards regression was used to assess the association between each risk SNP with OS under the allelic and codominant models of inheritance. All analyses were adjusted for age, sex, country of origin (for IMMEnSE) or principal components (for the others) and disease stage (ISS). SNP associations were meta-analyzed. Results: SNP associations were meta-analyzed. From the meta-analysis, two multiple myeloma risk SNPs were associated with OS (P &lt; 0.05), specifically POT1-AS1-rs2170352 [HR = 1.37; 95% confidence interval (CI) = 1.09–1.73; P = 0.007] and TNFRSF13B-rs4273077 (HR = 1.19; 95% CI = 1.01–1.41; P = 0.04). The association between the combined 24 SNP MM-PRS and OS, however, was not significant. Conclusions: Overall, our results did not support an association between the majority of multiple myeloma risk SNPs and OS. Impact: This is the first study to investigate the association between multiple myeloma PRS and OS in multiple myeloma.

Abstract Mass-spectrometry (MS) assays detect lower levels of monoclonal proteins and result in earlier detection of monoclonal gammopathy of undetermined significance (MGUS). We examined heavy chain MGUS prevalence using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS among 3 risk groups, ages 50 or older: 327 African Americans (AA) and 1223 European Americans (EA) from a clinical biobank and 1093 unaffected first-degree relatives (FDR) of patients with hematologic disorders. Age- and sex-adjusted prevalence rates were directly standardized to 2010 United States population. Prevalence ratios were estimated for comparisons of AA and FDR to the EA group using the Poisson distribution. Results were also compared with population-based prevalence using conventional gel-based methods. Risk groups had similar sex and age distributions. MALDI-TOF MGUS prevalence was higher in the AA (16.5% [95% confidence interval (CI), 12.2%, 20.8%]) and FDR (18.3% [95% CI, 16.6%, 21.6%]) than in EA (10.8% [95% CI, 8.8%, 12.7%]), translating to prevalence ratios of 1.73 (95% CI, 1.31, 2.29) and 1.90 (95% CI, 1.55, 2.34), respectively. MALDI-TOF EA prevalence was over threefold higher than conventional estimates but showed similar age trends. Thus, the MALDI-TOF assay found greater numbers with MGUS but similar relative differences by race, family history, and age as prior studies.

Approximately one‐third of patients with metastatic castration‐resistant prostate cancer (CRPC) exhibited primary abiraterone resistance. To identify alternative treatment for abiraterone nonresponders, we performed drug discovery analyses using the L1000 database using differentially expressed genes identified in tumor biopsies and patient‐derived xenograft (PDX) tumors between abiraterone responders and nonresponders enrolled in PROMOTE trial. This approach identified 3 drugs, including topoisomerase II (TOP2) inhibitor mitoxantrone, CDK4/6 inhibitor palbociclib, and pan‐CDK inhibitor PHA‐793887. These drugs significantly suppressed the growth of abiraterone‐resistant cell lines and PDX models. Moreover, we identified 11 genes targeted by all 3 drugs that were associated with worse outcomes in both the PROMOTE and Stand Up To Cancer cohorts. This 11‐gene panel might also function as biomarkers to select the 3 alternative therapies for this subgroup of patients with CRPC, warranting further clinical investigation.

Abstract Background: Cyclin dependent 4/6 kinase inhibitors (CDK4/6i) and endocrine therapy (ET) have improved progression-free survival (PFS) and overall survival in HR+ MBC, but progression of disease ultimately occurs. Apart from HR+ status, there are no clinically available biomarkers that enable oncologists to determine prognosis and predict response to CDK4/6i. An emerging biomarker is serum thymidine kinase 1 (TK1), a secreted marker of proliferation that is prognostic in pts with HR+ HER2- MBC. High levels of TKa are associated with inferior PFS, whereas pts with low TKa levels pretreatment, or TKa levels that decrease on ET and a CDK4/6i, have superior PFS. Notably, TKa levels rebound ≥ 5 days off Pb, with resumption of cell cycling. PROMISE (NCT0281902) is a prospective study that enrolled women with HR+ MBC starting Pb + letrozole (L) in the 1st line [FL] or Pb + fulvestrant in the 2nd line [SL] setting. The trial includes a comprehensive “omic” assessment of blood, tumor, urine and the fecal microbiome to identify novel genomic variants and pathways associated with an early decline in TKa (measured after 2 months or end of cycle [C]2) and PFS. Here, we report the association between i) pre-treatment TKa (pre-TKa) levels and PFS (i.e. from registration to the 1st disease event) and ii) TKa levels at the end of C2 (C2-TKa) and PFS-2 (i.e. from the start of C3 to the 1st disease event).Methods: TKa testing was performed using the DiviTum assay (Biovica). TKa+ disease was defined as ≥ 200 Du/L and TKa- disease as below limit of detection to 200 Du/L. Log-rank test and univariate Cox modeling were used to assess the association between pre-TKa levels and PFS and between end of C2-TKa levels and PFS-2. The database was locked on June 28, 2021. Results: Of 68 pts enrolled, 4 were ineligible and pre-TKa data was unavailable for 4. Of the remaining 60 pts (45 FL, 15 SL), the percentage of pts with pre-TKa+ disease was 33.3% in FL (15/45, 95% CI: 20.0-49.0%), and 46.7% (7/15, 95% CI: 21.4-71.9%) in the SL. The median follow-up time for pts on study was 24 months (range: 2-42 months). There were 22 disease events (13 in FL, 9 in SL). In the FL setting, PFS was significantly shorter for preTKa+ pts compared to preTKa- pts (HR: 4.15, 95% CI:1.35-12.74; p=0.007), but not for SL pts (HR: 1.11, 95% CI: 0.30-4.18, p=0.875). End of C2 TKa data was obtained for pts while on Pb (n=5), or after stopping Pb as follows: 1-4 days (n=9), 5-8 days (n=28) and 9-36 days (n=11). PFS-2 was not associated with C2-TKa in the FL (p=0.834) or SL (p=0.454) settings. An analysis of TKa levels by metastatic site will be presented at the meeting.Conclusions: A secreted biomarker of proliferation (TK1) obtained prior to initiating CDK4/6i and ET for the treatment of HR+ MBC is associated with PFS in pts receiving 1st line Pb + L, but not in those receiving 2nd line Pb + fulvestrant. While the end of C2 TKa levels were not associated with PFS, the interpretability of these data are limited, given treatment delays (0-36 days) prior to starting C3 that may result in TKa rebound. Future studies evaluating the predictive nature of TKa and Pb response should focus on earlier timepoints while on drug. Citation Format: Ciara C O'Sullivan, Jun He, Jason Sinnwell, Vera J Suman, Krishna R Kalari, Peter T Vedell, Ann M Moyer, Xiaojia Tang, Kevin J Thompson, Abe Eyman Casey, Alvaro Moreno-Aspitia, Donald W Northfelt, Minetta C Liu, Tufia C Haddad, Saranya Chumsri, Brendan McMenomy, Prema Peethambaram, Kathryn J Ruddy, Karthik V Giridhar, Roberto A Leon-Ferre, Mattias Bergqvist, Adrian Nordmark, Richard M Weinshilboum, Liewei Wang, Matthew P Goetz. Serum thymidine kinase 1 activity (TKa) levels and progression-free survival (PFS) in patients (pts) with hormone receptor positive (HR+) HER2-negative metastatic breast cancer (MBC) on palbociclib (Pb) and endocrine therapy (ET) [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 P5-13-22.