Primary Samples Research Articles

The HER2 flip-HER2 amplification of tumor cells in the cerebrospinal fluid of breast cancer patients with leptomeningeal disease: implications for treating the LM tumor with anti-HER2 therapy

IntroductionCNSide is a platform that detects and characterizes tumor cells in the cerebrospinal fluid (CSF) of patients with leptomeningeal disease (LMD). The platform was validated per College of American Pathologists (CAP) and Clinical Laboratories Improvement Amendment (CLIA) guidelines and run as a commercial Laboratory Developed Test (LDT) at Biocept in San Diego, CA. The platform allows CSF tumor cell (CSF-TC) enumeration and biomarker characterization by fluorescent in situ hybridization (FISH). MethodsWe performed a multicenter retrospective chart review of HER2 FISH CNSide test results that were commercially ordered on 26 patients by physicians for LMD breast cancer patients between April 2020 and October 2022.ResultsWe show that HER2 is amplified on CSF tumor cells in 62% (16/26) of LMD breast cancer patients. 10/26 (38%) patients had discordant HER2-positivity between the primary tumor tissue and CSF-TC; of these, 35% (9/26) of the patients displayed HER2 amplification on the CSF-TCs, however were categorized as HER2 negative on the primary tumor. Of the 27% (7/26) patients with a HER2 positive primary tumor, one patient showed a HER2 negative LMD tumor. Two patients, 8% (2/26) had a HER2 equivocal primary tumor; of these, one demonstrated a HER2 negative, and one a HER2 positive LMD tumor. Serial analysis (at least 4 longitudinal tests) of HER2 status of the CSF-TC throughout therapy was available for 14 patients and demonstrated that HER2 status of the LMD changed in 29% (4/14) during their treatment course and impacted care decisions. ConclusionsOur data suggests that CSF-TC HER2 FISH analysis in LMD breast cancer patients may be discordant to the primary tumor sample and the discovery of HER2 positivity in the CSF may open doors to anti-HER2 targeted therapy options for LMD patients.

Seasonal variation in D2/3 dopamine receptor availability in the human brain.

Brain functional and physiological plasticity is essential to combat dynamic environmental challenges. The rhythmic dopamine signaling pathway, which regulates emotion, reward and learning, shows seasonal patterns with higher capacity of dopamine synthesis and lower number of dopamine transporters during dark seasons. However, seasonal variation of the dopamine receptor signaling remains to be characterized. Based on a historical database of healthy human brain [11C]raclopride PET scans (n = 291, 224 males and 67 females), we investigated the seasonal patterns of D2/3 dopamine receptor signaling. Daylength at the time of scanning was used as a predictor for brain regional non-displaceable binding of the radiotracer, while controlling for age and sex. Daylength was negatively correlated with availability of D2/3 dopamine receptors in the striatum. The largest effect was found in the left caudate, and based on the primary sample, every 4.26h (i.e., one standard deviation) increase of daylength was associated with a mean 2.8% drop (95% CI -0.042 to -0.014) of the receptor availability. Seasonally varying D2/3 receptor signaling may also underlie the seasonality of mood, feeding, and motivational processes. Our finding suggests that in future studies of brain dopamine signaling, especially in high-latitude regions, the effect of seasonality should be considered.

MYC Inhibition Potentiates CD8+ T Cells Against Multiple Myeloma and Overcomes Immunomodulatory Drug Resistance

Abstract Purpose: Immunomodulatory drugs (IMiDs), such as lenalidomide and pomalidomide, are cornerstone therapies in Multiple Myeloma (MM), yet patients inevitably become refractory. IMiDs exert cytotoxicity through inducing Cereblon-dependent proteasomal degradation of IKZF1 and IKZF3, resulting in downregulation of the oncogenic transcription factors IRF4 and MYC. To date, clinical IMiD resistance independent of CRBN or IKZF1/3 has not been well-explored. Here, we investigated the roles of IRF4 and MYC in this context. Experimental Design: Using bone marrow aspirates from patients with IMiD naïve or refractory MM, we examined IKZF1/3 protein levels and IRF4/MYC gene expression following ex vivo pomalidomide treatment via flow cytometry and qPCR. We also assessed ex vivo sensitivity to the MYC inhibitor, MYCi975, using flow cytometry. Results: We discovered that while pomalidomide frequently led to IKZF1/3 degradation in MM cells, MYC gene expression was unaffected by pomalidomide in most IMiD refractory samples. We subsequently demonstrated that MYCi975 exerted strong anti-MM effects in both IMiD naïve and refractory samples. Unexpectedly, we identified CD8+ T cells from patients with MM as crucial effectors of MYCi975-induced cytotoxicity in primary MM samples, and we discovered MYCi975 enhanced the cytotoxic functions of memory CD8+ T cells. We lastly observed synergy between MYCi975 and pomalidomide in IMiD refractory samples, suggesting restoring MYC downregulation can re-sensitize refractory MM to IMiDs. Conclusion: Our study supports the concept that MYC represents an Achille’s heel in MM across disease states and that MYCi975 may be a promising therapeutic for patients with MM, particularly in combination with IMiDs.

Claudin-18 status and its correlation with HER2 and PD-L1 expression in gastric cancer with peritoneal dissemination

Abstract Background Gastric cancer with peritoneal dissemination (PD) has a dismal prognosis, and current treatments have shown little efficacy. CLDN18.2-targeted therapies have shown promising efficacy against gastric cancers that express high levels of CLDN18. Because of the limited information regarding CLDN18.2 status in PD, we analyzed PD-positive gastric cancers for CLDN18 status in both primary and PD, along with HER2 and PD-L1 combined positive score (CPS). Methods Immunohistochemical analyses were performed on 84 gastric cancer cases using paired primary and PD tissue samples. Results At 40% cut-off, CLDN18 was positive in 57% (48/84) primary tumors and in 44% (37/84) PDs. At 75% cut-off, 28.6% (24/84) primary tumors and 20.2% (17/84) PDs were CLDN18-positive. The concordance rate between primary tumors and PD was 79.8% at 40% cut-off and 75% at 75% cut-off. When comparing biopsy and surgical specimens, the concordance rates were 87.5% at 40% cut-off and 81.3% at 75% cut-off. Within a tumor, the superficial area tended to have a higher CLDN18-positive rate than the invasive front (P = 0.001). Although HER2 -positivity was only 11.9% in this cohort, CLDN18 positivity in HER2-negative tumors (n = 74) was relatively high: 60.8% at 40% cut-off and 28.4% at 75% cut-off. Among double-negative (HER2 − and PD-L1 CPS < 1) tumors, CLDN18 positivity was 67.6% at 40% cut-off and 26.5% at 75% cut-off. Conclusions CLDN18 expression is generally maintained in PD and is relatively high even in double-negative tumors, making it a promising therapeutic target for PD-positive gastric cancer.

AML/T cell interactomics uncover correlates of patient outcomes and the key role of ICAM1 in T cell killing of AML.

T cells are important for the control of acute myeloid leukemia (AML), a common and often deadly malignancy. We observed that some AML patient samples are resistant to killing by human-engineered cytotoxic CD4+ T cells. Single-cell RNA-seq of primary AML samples and CD4+ T cells before and after their interaction uncovered transcriptional programs that correlate with AML sensitivity or resistance to CD4+ T cell killing. Resistance-associated AML programs were enriched in AML patients with poor survival, and killing-resistant AML cells did not engage T cells in vitro. Killing-sensitive AML potently activated T cells before being killed, and upregulated ICAM1, a key component of the immune synapse with T cells. Without ICAM1, killing-sensitive AML became resistant to killing by primary ex vivo-isolated CD8+ T cells in vitro, and engineered CD4+ T cells in vitro and in vivo. While AML heterogeneity implies that multiple factors may determine their sensitivity to T cell killing, these data show that ICAM1 acts as an immune trigger, allowing T cell killing, and could play a role in AML patient survival in vivo.

Blockage of BCL-XL overcomes venetoclax resistance across BCL2+ lymphoid malignancies irrespective of BIM status

AbstractVenetoclax (VEN), a B-cell lymphoma 2 (BCL2) inhibitor, has a promising single-agent activity in mantle cell lymphoma (MCL), acute lymphoblastic leukemia (ALL), and large BCLs, but remissions were generally short, which calls for rational drug combinations. Using a panel of 21 lymphoma and leukemia cell lines and 28 primary samples, we demonstrated strong synergy between VEN and A1155463, a BCL-XL inhibitor. Immunoprecipitation experiments and studies on clones with knockout of expression or transgenic expression of BCL-XL confirmed its key role in mediating inherent and acquired VEN resistance. Of note, the VEN and A1155463 combination was synthetically lethal even in the cell lines with lack of expression of the proapoptotic BCL2L11/BIM and in the derived clones with genetic knockout of BCL2L11/BIM. This is clinically important because BCL2L11/BIM deletion, downregulation, or sequestration results in VEN resistance. Immunoprecipitation experiments further suggested that the proapoptotic effector BAX belongs to principal mediators of the VEN and A1155463 mode of action in the BIM-deficient cells. Lastly, the efficacy of the new proapoptotic combination was confirmed in vivo on a panel of 9 patient–derived lymphoma xenografts models including MCL (n = 3), B-ALL (n = 2), T-ALL (n = 1), and diffuse large BCL (n = 3). Because continuous inhibition of BCL-XL causes thrombocytopenia, we proposed and tested an interrupted 4 days on/3 days off treatment regimen, which retained the desired antitumor synergy with manageable platelet toxicity. The proposed VEN and A1155463 combination represents an innovative chemotherapy-free regimen with significant preclinical activity across diverse BCL2+ hematologic malignancies irrespective of the BCL1L11/BIM status.

Therapeutic Potential of Antibody Targeting Neuronal Pentraxin Receptor in Esophageal Squamous Cell Carcinoma.

Esophageal squamous cell carcinoma (ESCC) has a poor prognosis despite advances in multidisciplinary treatments and immune checkpoint inhibitors. We previously reported that neural pentraxin receptor (NPTXR), a transmembrane protein mainly expressed in the brain and involved in synaptic transmission, is implicated in gastric cancer malignancy. This study evaluated the expression and function of NPTXR in ESCC, the therapeutic potential of monoclonal antibody (mAb) against NPTXR, and its prognostic value in ESCC patients. The study involved analyzing the NPTXR expression in 21 ESCC cell lines and total 371 primary ESCC tissue samples using quantitative reverse-transcription polymerase chain reaction and immunohistochemistry. The impact of NPTXR on the malignant behavior of ESCC was examined using small interfering RNA-mediated knockdown and a subsequent assessment of cell proliferation, apoptosis, and adhesion. This study further investigated the efficacy of anti-NPTXR mAb in vitro and associations between the expression of NPTXR messenger RNA (mRNA) and protein with clinicopathological factors and the prognosis. NPTXR was overexpressed in several ESCC cell lines and primary ESCC tissues. Knockdown of NPTXR in ESCC cells resulted in reduced proliferation, increased apoptosis, and decreased cell adhesion. The mAb against NPTXR significantly inhibited ESCC cell proliferation in vitro. A high NPTXR expression in patient tissues was correlated with a worse overall survival, suggesting its potential as a prognostic biomarker. NPTXR influences the malignant behavior of ESCC cells. Anti-NPTXR mAb may be a promising therapeutic agent, and its expression in ESCC tissues may serve as a prognostic biomarker.

Extreme-emission-line galaxies in the MUSE Hubble Ultra Deep Field Survey

We aim to apply a methodology to build a sample of extreme-emission-line galaxies (EELGs) using integral field spectroscopy data. In this work, we followed the spectroscopic criteria corresponding to EELG selection and used the MUSE Hubble Ultra-Deep field survey, which includes the deepest spectroscopic survey ever performed. Objects in the primary (extended) sample were detected requiring a rest-frame equivalent width of EWo geqslant 300 leq EWo leq 300 ) in any of the emission lines of OII OIII or Halpha . A detailed closer inspection of the spectra of the candidates selected has been performed on a one-by-one basis in order to confirm their classification. For this sample, the line fluxes, physical properties, and chemical abundances of the EELGs have been derived, as well as their spatially resolved structure and kinematics. Four (five) of the galaxies in the primary (extended) sample, sim 57$<!PCT!>$ (sim 83$<!PCT!>$), were spatially resolved. Three (none) of them present a clear pattern compatible with rotation. We show how our entire EELG sample shares the same loci defined by high-redshift galaxies (z approx 6-8) for the mass-metallicity relation, illustrating their role as local analogs.

Accounting for sample overlap in economics meta‐analyses: The generalized‐weights method in practice

AbstractMeta‐analyses in economics frequently exhibit considerable overlap among primary samples. If not addressed, sample overlap leads to efficiency losses and inflated rates of false positives at the meta‐analytical level. In previous work, we proposed a generalized‐weights (GW) approach to handle sample overlap. This approach effectively approximates the correlation structure between primary estimates using information on sample sizes and overlap degrees in the primary studies. This paper demonstrates the application of the GW method to economics meta‐analyses, addressing practical challenges that are likely to be encountered. We account for variations in data aggregation levels, estimation methods, and effect size metrics, among other issues. We derive explicit covariance formulas for different scenarios, evaluate the accuracy of the approximations, and employ Monte Carlo simulations to demonstrate how the method enhances efficiency and restores the false positive rate to its nominal level.

Mutation characteristics and molecular evolution of ovarian metastasis from gastric cancer and potential biomarkers for paclitaxel treatment

Ovarian metastasis is one of the major causes of treatment failure in patients with gastric cancer (GC). However, the genomic characteristics of ovarian metastasis in GC remain poorly understood. In this study, we enroll 74 GC patients with ovarian metastasis, with 64 having matched primary and metastatic samples. Here, we show a characterization of the mutation landscape of this disease, alongside an investigation into the molecular heterogeneity and pathway mutation enrichments between synchronous and metachronous metastasis. We classify patients into distinct clonal evolution patterns based on the distribution of mutations in paired samples. Notably, the parallel evolution group exhibits the most favorable prognosis. Additionally, by analyzing the differential response to chemotherapy, we identify potential biomarkers, including SALL4, CCDC105, and CLDN18, for predicting the efficacy of paclitaxel treatment. Furthermore, we validate that CLDN18 fusion mutations improve tumor response to paclitaxel treatment in GC with ovarian metastasis in vitro and vivo.

Drivers of Urban Agriculture and their Influence on the Socio-Economic Status of the Urban Farmers in Uganda, A Case Study of Kampala and Mbarara Cities

The study analyzed the influence of forms and drivers of urban Agriculture on the socio-economic status of the farmers in Kampala and Mbarara Cities. The specific objective of the study was to examine the influence drivers of urban agriculture on the socio-economic status of farmers. The study adopted a cross-sectional research design using multiple data collection methods. A multistage cluster sampling technique involving simple random sampling to get both primary and secondary sampling units, snowball to get target respondents and purposive sampling for key informants and focus group members was used. Data were collected using social survey questionnaires for households, focus group discussions checklist (FGDs), key informant interview guides and observations, from a sample of 384 respondents. The study found a strong association between drivers of UA and the socioeconomic status of the urban farmers in Kampala and Mbarara Cities. Household socio-economic status has substantially correlated with the drivers of the urban farmers and out of 9 items considered only 5 was significant which include access to capital (x2 = 1.175, p< 0.047), access to agriculture extension services (x2 = 3.634, p< 0.026), access to financial grants (x2 = 5.190, p<0.001), access to markets (x2 = 6.993, p< 0.001), value addition(x2 = 3.718, p<0.014). The study confirmed a significant relationship between drivers of urban agriculture and the socio-economic status of urban farmers. Urban agriculture drivers like access to capital and financial grants, nature of market access and value addition had a significant influence on household socio-economic status.

Abstract PO2-16-02: RBsig gene-expression signature in patients with endocrine resistant metastatic breast cancer treated with palbociclib and fulvestrant in the PYTHIA trial

Abstract Background: We have previously identified RBsig, a gene-expression signature of resistance to CDK4/6 inhibitors composed of E2F1/E2F2 dependent genes, which is associated with genetic loss of RB1. Although RBsig has been previously tested in vitro and in clinical trials with CDK4/6 inhibitors, further clinical validation is needed. Additionally, data on the correlation of RBsig with other prognostic/predictive biomarkers for CDK4/6 inhibitors such as serum Thymidine Kinase activity (sTKa), is lacking. Here, we aimed to investigate RBsig in patients (pts) treated with palbociclib (P) + fulvestrant (F) within the PYTHIA trial (IBCSG 53- 14/BIG 14-04; NCT02536742), a downstream trial of the AURORA molecular program (BIG 14-01; NCT02102165). Methods: PYTHIA is a biomarker discovery phase II trial which enrolled 122 pts with endocrine-resistant ER+ and HER2 - metastatic breast cancer (MBC) who received P+F at standard schedule and dose. We analyzed a subset of 38 pts enrolled in PYTHIA and successfully included in AURORA who had available RNAseq from tumor tissue samples (24 primary, 24 metastatic, 10 both). Ki67 by IHC was also available. The PYTHIA biomarker assessment included sTKa at baseline and during treatment (D15 and D28) measured using DiviTumÒ TKa (Biovica International). Evaluation of the prognostic role of RBsig was a pre-specified primary objective of PYTHIA. RBsig is composed of 87 genes and was calculated as previously described. Wilcoxon rank sum test was used to compare continuous RBsig values between groups defined by sTKa. Median progression free survival (mPFS) was estimated using Kaplan-Meier method for groups defined by dichotomizing RBsig at zero. Results: Median RBsig was higher in metastatic than in primary samples; in paired samples, RBsig increased in metastatic for 7/10 pts vs primary. When evaluated as continuous variables, RBsig correlated with tissue Ki67 both in primary (Spearman R 0.8) and in metastatic (R 0.68) samples. Additionally, RBsig was higher in Basal and Luminal B intrinsic subtypes in metastatic samples. RBsig was significantly higher in pts with high baseline sTKa (median cut-off) (Wilcoxon test, p=0.04 and p=0.03 in primary and metastatic samples respectively), with a similar direction, albeit not statistically significant, observed in the group of pts with no sTKa clearance at D15 (below limit of detection cut-off). When evaluated in the combined cohort, where RBSig values were taken from metastatic samples when available and from primary samples otherwise, higher RBsig values were associated with poorer PFS as compared to lower RBsig values [mPFS 5.5 months (95% CI 3.7, 14) vs 13 months (95% CI 8.6, 28); Log-rank p-value=0.004]. Conclusions: Higher RBsig was associated with higher tumor proliferation (measured by both tissue ki67 and sTKa), luminal B status, and shorter PFS on treatment with P+F. This analysis reinforces prior data on RBsig prognostic significance in pts treated with CDK4/6 inhibitors. Further investigation on the added value of RBsig as a novel biomarker for patients’ stratification, including in the early setting, is warranted. Citation Format: Matteo Benelli, Svitlana Tyekucheva, Thayane Crestani, Michail Ignatiadis, Patrick Neven, Marco Colleoni, Stéphanie Henry, Konstantinos Papadimitriou, Antonio Bernardo, Elena Seles, François Duhoux, Iain Macpherson, Alastair Thomson, David Mark Davies, Ilenia Migliaccio, Gabriele Zoppoli, Roswitha Kammler, Heidi De Swert, Barbara Ruepp, Philippe Aftimos, Jose Seoane, Dario Romagnoli, David Venet, Theodora Goulioti, David Cameron, Sherene Loi, Martine Piccart, Meredith Regan, Luca Malorni. RBsig gene-expression signature in patients with endocrine resistant metastatic breast cancer treated with palbociclib and fulvestrant in the PYTHIA trial [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-16-02.

Abstract PO3-13-02: Genomic and intrinsic subtype correlates of serum thymidine kinase activity in patients with metastatic breast cancer treated with palbociclib and fulvestrant in the PYTHIA trial

Abstract Background: Serum thymidine kinase activity (sTKa) is a novel non-invasive proliferation biomarker providing prognostic, predictive and monitoring information in patients (pts) with metastatic breast cancer (MBC). We have shown in PYTHIA that high baseline sTKa and lack of sTKa clearance at day 15 of the first cycle of treatment with palbociclib (P)+ fulvestrant (F) are poor prognostic factors in terms of PFS. Here we characterized the molecular features of tumor samples from PYTHIA, and their relation with sTKa. Methods: PYTHIA (IBCSG 53- 14/BIG 14-04; NCT02536742) is a phase II biomarker discovery trial downstream of the AURORA molecular program (BIG 14-01; NCT02102165) which enrolled 122 pts with endocrine-resistant, ER+ and HER2 - MBC receiving P+F. Intrinsic subtypes of metastatic samples from 24 pts were estimated using RNA-Seq. Somatic mutations (mut) from 66 pts were identified using an NGSpanel of 411 cancer-related genes (36 primary and 48 metastatic samples). sTKa was measured using DiviTumÒ TKa (Biovica International) before treatment (D0) and at day 15 (D15) of cycle 1 of P+F. sTKa clearance at D15 was defined as sTKa below the limit of detection (LOD). Intrinsic subtypes and ESR1 mut were assessed in metastatic samples, PIK3CA and TP53 mut were derived from a combined cohort, wheremetastatic samples were used when available and primary samples otherwise. Association of mut and continuous sTKa with PFS was assessed using Cox regression. Results: In metastatic samples, sTKa levels at D0 tended to be higher in luminal B and HER2-enriched subtypes. Lack of sTKa clearance at D15 was observed in 14% of the cases, all classified as luminal B. PIK3CA and TP53 were the most frequently altered genes (38% and 27%, respectively). ESR1 mut were found in 8% of primary and 25% of metastatic samples. Only TP53 was associated with worse PFS. sTKa levels at D0 tended to be higher in pts with TP53 mut vs wild-type (wt) and lower in PIK3CA mut vs wt. Interestingly, lack of sTKa clearance at D15 was more frequent in TP53 mut vs wt and less frequent in PIK3CA mut vs wt. Levels of sTKa were similar in pts with ESR1 mut vs wt both at D0 and at D15 (Table 1). The inclusion of TP53 as a predictor of PFS in bivariate Cox models with sTKa at either D0 (likelihood ratio test p=0.44) or D15 (p=0.21) did not add to the prognostic value provided by sTKa alone. Conclusions: Tumors with high proliferation and poor prognostic features (luminal B, HER2-enriched and TP53 mut) tend to have higher baseline sTKa and less sTKa response on-treatment, in line with sTKa being a circulating dynamic biomarker of tumor cell proliferation. After accounting for sTKa values, TP53mutational status did not contribute to prognosis. This suggests that high sTKa at D0 and lack of sTKa clearance at D15 might be associated with a worse prognosis independently of TP53 status. Further validation in larger datasets is warranted. Table 1: Results summary Citation Format: Svitlana Tyekucheva, Thayane Crestani, Michail Ignatiadis, Patrick Neven, Marco Colleoni, Stéphanie Henry, Konstantinos Papadimitriou, Antonio Bernardo, Elena Seles, Francois Duhoux, Iain Macpherson, Alastair Thomson, David Mark Davies, Mattias Bergqvist, Ilenia Migliaccio, Gabriele Zoppoli, Roswitha Kammler, Heidi De Swert, Barbara Ruepp, Angel Guerrero, Arnau Llinas, Danai Fimereli, Amal Arahmani, David Cameron, Sherene Loi, Martine Piccart, Meredith Regan, Matteo Benelli, Luca Malorni. Genomic and intrinsic subtype correlates of serum thymidine kinase activity in patients with metastatic breast cancer treated with palbociclib and fulvestrant in the PYTHIA trial [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-13-02.

Abstract GS03-06: Genomic and transcriptomic profiling of primary tumors from patients with HR+, HER2-, node-positive, high-risk early breast cancer in the monarchE trial

Abstract Background Two years of adjuvant abemaciclib combined with endocrine therapy (ET) resulted in significant and clinically meaningful improvement in invasive disease-free survival (IDFS) and distant relapse-free survival (DRFS) in patients (pts) with HR+, HER2-, node-positive, high-risk early breast cancer in the monarchE trial (NCT03155997). Abemaciclib benefit was sustained beyond the completion of treatment (tx) with deepening magnitude of absolute benefit in IDFS and DRFS at 5 years. Here, we evaluate comprehensive molecular profiling of archived primary tumor tissue and association with clinical outcomes. Methods For biomarker analysis, a proportionally stratified random sampling case-cohort design was utilized to include all patients who experienced an IDFS event at a pre-specified interim analysis with a median follow up of 54 months. A cohort of 895 pts (189 with IDFS event) in the abemaciclib + ET arm was matched 1:1 with 903 pts (270 with IDFS event) in the ET arm. Baseline primary tumor samples underwent exome-capture RNA sequencing (RNAseq; n=1324, 23% intent-to-treat (ITT) population) and paired tumor-normal (germline blood samples) whole-exome sequencing (WES; n=1234, 22% ITT population). Expression-based intrinsic subtypes (i.e., luminal A (LumA), luminal B (LumB), HER2-enriched (HER2E), basal- and normal-like) were characterized using the Absolute Intrinsic Molecular Subtyping model. The 21-gene expression signature score (Oncotype DX test) was inferred from RNAseq; samples were categorized into lower (0-25) and high (26-100) risk groups. To investigate associations of biomarkers with abemaciclib benefit, WES genomic events including oncogenic and hotspot mutations by OncoKB, and copy number events, of incidence >9% were pre-selected. Results The biomarker subset of monarchE was reflective of the ITT population. A total of 1190 tumors (abemaciclib+ET n= 605; ET alone n=585) yielded adequate RNAseq results. Intrinsic subtype distribution was consistent across tx arms (Table A). Low tumor purity limited assessment of the normal-like subtype. The 4-year IDFS benefit of abemaciclib was consistent across all subtypes. LumA cancers had the lowest risk of recurrence while HER2E and basal-like subtypes had the highest. Inferred 21-gene expression signature score showed similar benefits from abemaciclib in both lower and high-risk groups (Table B). A total of 1173 tumors yielded adequate WES results (abemaciclib+ET n=580; ET alone n=593). Consistent abemaciclib benefit was observed across the most frequently altered genes (Table C). In exploratory analysis, lower benefit from abemaciclib was seen in the subset of focal-high level MYC amplified tumors (n=176, HR 1.30, 95% CI, 0.77, 2.20) compared to MYC non-amplified tumors (n=997, HR 0.62, 95% CI, 0.47, 0.80, nominal interaction p=0.014). The treatment benefit of abemaciclib was observed across all subpopulations of altered genes based on gene expression data. Conclusions Adjuvant abemaciclib+ET maintained IDFS benefit compared to ET alone across all molecular subtypes as measured by RNAseq. Benefit was consistent across most altered genes assessed by WES, except for the subset of tumors with MYC amplification. Additional research is necessary to confirm these findings. Table. Citation Format: Nicholas Turner, Jorge Reis-Filho, Matthew Goetz, Christine Desmedt, Sarat Chandarlapaty, Hironobu Sasano, Carlos Arteaga, Sherene Loi, Stephanie Graff, Deli Liu, Vanessa Rodrik-Outmezguine, Anthony Sireci, Cynthia Sandoval Rubenstein, Helen Won, Lacey M. Litchfield, Maria Munoz, Stephen Johnston. Genomic and transcriptomic profiling of primary tumors from patients with HR+, HER2-, node-positive, high-risk early breast cancer in the monarchE trial [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr GS03-06.

Abstract PS03-08: Spatial biomechanics determines fate in breast cancer

Abstract Background: With the recent advancements of spatial omics, it has become increasingly recognized that spatial analysis the of tumor architecture is key to decipher the heterogenous intratumoral relationships between different tumor components and provide better understanding of cancer therapy resistance, as well as help to identify potential targets for personalized therapy. The challenge of the discovery and implementation of such biomarkers still lays in the technological and methodological difficulties, and their translation into clinical practice. In parallel a major advancement in cancer research has been the recognition of the importance of cancer biomechanical properties in cancer progression, metastasis, and therapy response. Methods: We developed a new computational platform to identify biomechanical drivers of cancer outcome from spatial omics data. We used it to identify recurring spatial patterns of these markers within the tumor microenvironment, and define the spatial scale of heterogeneity of such patterns. Our dataset consists in 700 primary breast cancer baseline samples analyzed with two 30-marker imaging mass cytometry panels to identify cancer, immune, and stromal cells and structures and their biomechanical states. Our patient cohort includes 20+ years of follow up, with up to 6 samples per patient from 64 patients alive 12 years post diagnosis, and 49 patients lost to breast cancer deaths. Patients were treated with surgery, radiation, chemotherapy, endocrine therapy, or combinations of these post-surgery. Based on this approach, we derived a new three-parameter quantitative metric of preferential spatial co-localization between different cells or structures, and we use this metric to stratify patients into responders and non-responders with the aid of single and multivariate survival analysis. Finally, we include a correlation with Atomic Force Microscopy to identify the mechanical signature of these driving patterns in breast cancer. Results: Our work enabled the identification of tumor-immune-stromal spatial patterns that drive breast cancer outcome and their spatial scale. Among our results, we were able to further define spatial regions of hypoxia-driven EMT. We found these regions to be usually on the scale of 50 mm radius, and enriched in presence of unspecified immune cells. They are also more frequent in ER+ areas, with high NaKATPase activity. Our analysis also shed light on the controversy on the role of fibroblasts. We have found that the presence of a strong, spatially structured stroma, in fibroblast-rich regions is a strong predictor of positive outcome. Similarly, in our analysis collagen cross-linking emerges as a positive control factor in Vimentin-rich microenvironments, offering new interpretation to the role of the ECM structure. Importantly, our first principle approach allows for the identification of driving structures beyond heterogeneity, and thus enables easy extension to additional datasets. Finally, when correlating these patterns with Atomic force microscopy measurements, we can clearly see that patterns predictive of poor survival present a clear heterogeneous stiffness signature, as opposed to a very homogenous good survival pattern signature. Conclusion: These results confirm the importance of spatial distribution of biomechanical drivers in cancer, offering new avenues of physics-based therapeutic targets. Our results also offer a solid base to inform machine learning algorithms on how to significantly parametrize spatial patterns in breast cancer for clinical significance. Furthermore, we demonstrate for the first time the use of Atomic Force Microscopy as a single biomarker for these patterns in breast cancer with a direct correlation based on pathology matching. Citation Format: Sara Nizzero, Maria Pelaez Soni, Gregory Zaugg, Mariam Gachechiladze, Yitian Xu, Licheng Zhang, Junjun Zheng, Brian Menegaz, Lee B Jordan, Colin A Purdie, Philip R Quinlan, Chandandeep Nagi, Karla A Sepulveda, Philipp Oertle, Tobias A Appenzeller, Marko Loparic, Zhihui Wang, Shu-Hsia Chen, Vittorio Cristini, Marija Plodinec, Alastair Thompson. Spatial biomechanics determines fate in breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PS03-08.

Abstract PO2-05-13: HER2 status presented an unstable switching from primary to recurrent breast cancer

Abstract Background: Breast cancer patients with HER2-2+ expression had heterogeneous characteristic. Treatment decision is of great importance after progression. Objective: We explored patients with HER2-2+ in matched primary or recurrent/metastatic tumor samples from January 2010 to June 2022 in our institute were included to evaluate the evolution from or to HER2-2+ expression. Meanwhile, with an emerging of novel entity as HER2-low expression breast cancer, we included this category when analysis as well. Results: In the cohort of a total of 159 breast cancer patients with HER2-2+ expression in either primary tumor or locoregional/distant metastases samples, HER2-2+ breast cancer accounted for 44.0% in primary tumor and 88.8% in recurrent diseases. There were 18.5% and 15.2% patients with HER2 gene amplification on ISH assay among primary and recurrent/metastatic HER2-2+ breast cancers, respectively. The overall rate of HER2 IHC discordance was 67.1%. Among primary HER2-2+ cases, 74.6% maintained in HER2-2+ when disease relapsed. Discordance was mostly driven by cases switching from HER2-2+ to HER2-1+ (64.7%). In HER2-2+ recurrent/metastatic cases, discordance was mostly driven by cases switching from HER2-0 to HER2-2+ (47.1%). The proportion of HER2 discordant cases got a higher statistically trend among HR-positive patients in compared with HR-negative patients (44.1% vs. 21.7%, p = 0.062). There were 35.0% in primary HR-positive/HER2-negative patients and 11.8% in primary triple-negative patients switching from HER2-0 to HER2-low when disease progressed, respectively. Meanwhile, HER2 discordance rate had a significant difference across different metastatic sites (p = 0.026). All contralateral breast and skin metastasis presented with discordant HER2 results. While, all central nervous system metastasis maintained the same HER2-low expression. HER2-positive phenotype in primary breast cancer samples loss their positivity in 42.1% cases. There was a total of 6.6% cases gained HER2 positivity in recurrent breast cancer samples. Conclusion: HER2 expression from primary to relapse breast cancer samples were highly unstable. Biopsy of recurrent sites was necessary to find novel treatment. Citation Format: Anjie Zhu, Hui-Ping Li. HER2 status presented an unstable switching from primary to recurrent breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-05-13.

Abstract PO5-25-03: Immune cell infiltrate profiles in primary Triple Negative Breast Cancer and co-relation with treatment response and survival outcomes

Abstract Introduction Tumor microenvironment (TME) in breast cancer is a heterogeneous landscape with multiple cell types, each modulating the tumor progression and the disease prognosis. These cell types include lymphocytes, RBCs, stromal cells, macrophages, cancer-associated fibroblasts (CAFs), and cancer stem cells. Each cell type in the TME influences tumor progression and clinical outcomes for patients in a good or bad way, depending on the interaction with tumor cells. Tumor-infiltrating lymphocytes (TILs) are shown to be prognostic for therapy response in many cancers in the western cohorts. Our recent study showed that a higher proportion of Indian TNBC patients had intra-tumoral TILs compared to Western cohorts, and these are predictive of better response to NACT and longer disease-free survival in patients, making it a distinct finding from Western reports. In the follow-up study, we are characterizing infiltrating immune cell subtypes and their comparative association with treatment response in TNBC patients. Results from preliminary analysis and comparative analysis between responders and non-responders will be presented here. We speculate that the pro- and anti-tumorigenic immune cell population and its spatial information would help us understand TME and its precise role in response to therapy. Methods A retrospective cohort of breast cancer patients from a single one-surgeon breast cancer clinic with a uniform treatment strategy was evaluated for this study. FFPE primary tissue samples with variable TILs scores (moderate to high), taken from patients who showed complete response to the NACT treatment (n=6) and non-responding group (n=8) determined by H&E stained slides from our cohort, were further stained for immune cells (Pan-B cell, Pan-T cells, Pan-macrophages) and T cells (CD4; Helper T cell marker, CD8; Cytotoxic T cell marker), FOXP; Regularly CD4+ cell marker) and PanCK (Pan-tumor marker) and counterstained with DAPI for multiplex imaging. Multispectral images for these slides were obtained using the Leica Aperio Versa system. Parameters such as individual cell types of identification and scoring, spatial proximity to tumor cells, and individual cell types will be determined by HALO software using the High plex module. Results A total of 14 TNBC samples were stained for the T-cell subtypes panel and Immune cell subtype s panel to determine the TIME. DAPI stain was used for nuclear counting based the parameters such as threshold intensity, nuclear size, roundness, and aggressiveness. The cytoplasmic size was selected as 2um beyond the outside nuclear boundary. Using Halo-Indica software, around 50000-300000 cells/tissue were evaluated across all samples from mIF whole slide scans of biopsy samples. Cell count was validated by cross-checking individual ROIs and manual cell count using Fiji. A Highplex module was used to identify individual signal positivity for each marker based on threshold intensity and cell completeness percent. Preliminary data showed a higher proportion of CD8+ cells and macrophages population are spatially closer to the tumor cells in Responders compared to non-responders. Conclusion Immune profiles are distinct among responding and non-responding patients in the Indian TNBC cohort. The association of these profiles with long-term survival in patients and various clinical and pathological parameters will help us determine the clinical relevance of these profiles and identify distinct immune subgroups as prognostic in Indian TNBCs. Citation Format: Pooja Vaid, Devaki Kelkar, LS Shashidhara, C B Koppiker, Madhura Kulkarni. Immune cell infiltrate profiles in primary Triple Negative Breast Cancer and co-relation with treatment response and survival outcomes [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-25-03.

Abstract PO5-24-03: Breast cancer intrinsic subtypes predict outcomes in primary and metastatic samples

Abstract Background: The prognostic and predictive value of the PAM50 intrinsic subtypes, namely Luminal A, Luminal B, Her2, and Basal-like subtypes, is well-studied in primary as well as metastatic breast cancer settings. Prosigna has emerged as a rapid PAM50 subtype predictor based on the NanoString nCounter assay. However, assay reproducibility across various RNASeq or qRT-PCR platforms can be challenging, especially when applying the predictor on metastatic breast cancer tumors. Here, we used SpinAdapt to create an intrinsic subtype predictor that works on RNA sequencing data, and validates on multiple tumor-sites. We evaluate real-world outcomes for our intrinsic subtype predictions across various immunohistochemical (IHC) labels and metastatic sites. Methods: We trained the subtype predictor on a cohort of 2,497 breast cancer patients using the PAM50 genes, profiled using Nanostring RNA nCounter assay (GSE148426). Approximately 5,423 de-identified records of breast cancer patients sequenced using whole-exome capture RNA-seq were included in our reference dataset. The reference dataset contained samples collected from various sites including breast (n=2440), liver (n=936), lymph node (n=577), lung (n=540), and bone (n=304). For subtype classification, we first batch-corrected the external dataset to Tempus RNA-seq reference dataset using SpinAdapt, then trained a Support-Vector Classifier (SVC) on the corrected data. A 10-fold CV experiment was performed on the corrected dataset to analytically validate the intrinsic subtype predictions. We retrospectively analyzed 7,021 de-identified breast cancer patients with known hormone receptor (HR) or HER2 status and a matched RNASeq sample. The concordance between HR/HER2 status and PAM50 prediction was analyzed, and these patients were excluded from training. Real-world overall survival (rwOS) was evaluated from the time of first diagnosis. The outcomes across intrinsic subtypes were further assessed according to tumor collection site and HR/HER2 IHC status. Results: The 10-fold CV experiment on the Tempus-adapted GSE148426 dataset achieved F-1 scores of 0.97, 0.86, 0.94, and 0.87 on Basal, HER2-like, Luminal A, and Luminal B PAM50 subtypes, respectively. On the Tempus evaluation dataset, 85.3% of HR+/HER2- patients (n=4,366), 65.4% of HR-/HER2+ patients (n=240), and 75.4% of HR-/HER2- patients (n=1,930) were classified as Luminal, HER2-like, and Basal, respectively. Evaluating outcomes on Tempus patients for each PAM50 group, the rwOS for the basal group was significantly shorter than patients not predicted to be basal (n=5,845, p< 2e-90). The rwOS for the predicted PAM50 basal patients remained significantly shorter than non-basal patients even when stratified by site of metastasis: breast (n=2,405; p< 1e-27), lymph node (n=577, p< 1e-7), liver (n=936; p< 1e-25), lung (n=540, p< 1e-13), and bone (n=304, p< 1e-3). Interestingly, within both HR+/HER2- (n=3,653) and HR-/HER2- (n=1,664) IHC cohorts with available outcomes data, the predicted PAM50 basal subtype could further stratify each of these populations with basal-subtype showing significantly worse prognosis than the non-basal subtype (p< 1e-27 and p< 1e-7, respectively). Conclusions: We retrospectively analyzed Tempus multimodal RWD to validate an in-house breast intrinsic subtype predictor that is agnostic to the site of metastasis. The prognostic value of the basal subtype was significant for breast cancer patients across various sites of metastasis including lymph node, liver, lung, and bones and IHC groups. For patients in each of the HR+/HER2- and triple negative IHC groups, the intrinsic molecular subtypes provided an additional level of prognostic detail with statistical significance. These data emphasize the importance of combining molecular subtypes with IHC-based diagnostics to fully characterize clinically relevant subpopulations and risk. Citation Format: Talal Ahmed, Mark Carty, Kaveri Nadhamuni, Raphael Pelossof. Breast cancer intrinsic subtypes predict outcomes in primary and metastatic samples [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-24-03.

Abstract PO3-23-11: Multiomics analysis of ER+ breast cancer with matched primaries, lymph node metastases and recurrent cancers on adjuvant endocrine therapy

Abstract Background: 80% of all breast cancers (BCs) are ER-positive (ER+). A significant number develop endocrine therapy resistance (ETR) . Clones develop in lymph nodes (LNs) which differ from the primary and these are implicated in ETR. A multiomics analysis of primary ER+ breast cancers, matched LN metastasis (LN+), if present, and matched recurrences (primary, nodal or distant) has been performed. Patients and Methods: The patients included are: Cohort A: 95 patients with matched primary and recurrent ER+ BC on different endocrine therapies. 35% had involved LNs at diagnosis. Recurrences: local in 59/95, concurrent local and nodal in 33/95, LN-only in 3/95. Median time to recurrence 4.5 years. Cohort B: 18 patients with ER+ LN+ BC who developed multiple recurrences (local and/or nodal, >3 consecutive recurrences/patient) on different lines of sequential endocrine therapy. Cohort C: 7 patients with ER+ breast cancer with multiple involved LNs treated with 3-6 months of neoadjuvant ET before surgery. Biopsies of primary and matched LNs performed at diagnosis and throughout treatment (3-4 timepoints/patient). Clinical response by serial ultrasound. Primary and LN samples (mean 12 samples/patient) were analysed together with normal DNA. Targeted RNAseq and DNA exome sequencing and whole-genome expression analysis was performed. Somatic mutations and copy number alterations (CNA) were determined, and differential gene expression analysis was performed. Validation of pathways implicated in ETR was by spatial NanoString GeoMx. All patients have long term follow-up. Results: Cohort A: A mean of 6 potential driver mutations identified by DNA sequencing in each primary BC (range 1-16). Most commonly mutated genes were PIK3CA, TP53, ERBB4, MAP3K1 and ERBB3. Multiple aberrations and a highly diverse mutational landscape were observed in all recurrences. In the majority of recurrences significant changes in variant allele frequency (VAF) were identified across several ETR-implicated genes and in over 50% new gains/losses were identified. Most frequently, losses were seen in ERBB3, ARID1A, HRAS and FOXA1 with gains in CDH1, NF1, PIK3CA, ESR1, TBX3 and GATA3 in recurrences vs primaries. Transcriptomic analysis revealed differentially expressed pathways including ER, HER2, GATA3, AKT, RAS and p63 signalling. Integrated analysis revealed several driver mutations with corresponding changes in upstream pathway activity. 33 patients had concurrent local and nodal recurrences and 52% had similar somatic mutation profiles in both with only minor variance in VAF. In 48%, 1 or more new potential driver mutations was identified in axillary nodes compared to recurrent primary cancers with losses in AKT1, CDH1 and TP53 being most common. Multiomics profiling of Cohorts B and C is complete and analysis underway. Discussion: This multiomics study is the largest cohort to-date of matched early and recurrent ER+/HER2- cancers. It sheds new light on the complex somatic and transcriptomic changes in local and nodal recurrent disease. The mechanisms of ETR are diverse and underlying mechanisms and clinically meaningful biomarkers including potentially actionable mutations and targets have been identified. Cohort C will uniquely capture and compare clonal evolution of breast cancer in primary and multiple matched lymph during neoadjuvant ET. Citation Format: Carlos Martinez-Perez, Charlene Kay, Rebecca Swan, Lorna Renshaw, J Michael Dixon, Arran K Turnbull. Multiomics analysis of ER+ breast cancer with matched primaries, lymph node metastases and recurrent cancers on adjuvant endocrine therapy [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-23-11.

Immunotherapy drives mesenchymal tumour cell state shift and TME immune response in glioblastoma patients.

Glioblastoma is a highly aggressive type of brain tumour for which there is no curative treatment available. Immunotherapies have shown limited responses in unselected patients, and there is an urgent need to identify mechanisms of treatment resistance to design novel therapy strategies. Here we investigated the phenotypic and transcriptional dynamics at single-cell resolution during nivolumab immune checkpoint treatment of glioblastoma patients. We present the integrative paired single-cell RNA-seq analysis of 76 tumour samples from patients in a clinical trial of the PD-1 inhibitor nivolumab and untreated patients. We identify a distinct aggressive phenotypic signature in both tumour cells and the tumour microenvironment in response to nivolumab. Moreover, nivolumab-treatment was associated with an increased transition to mesenchymal stem-like tumour cells, and an increase in TAMs and exhausted and proliferative T cells. We verify and extend our findings in large external glioblastoma dataset (n = 298), develop a latent immune signature and find 18% of primary glioblastoma samples to be latent immune, associated with mesenchymal tumour cell state and TME immune response. Finally, we show that latent immune glioblastoma patients are associated with shorter overall survival following immune checkpoint treatment (p = 0.0041). We find a resistance mechanism signature in a quarter of glioblastoma patients associated with a tumour-cell transition to a more aggressive mesenchymal-like state, increase in TAMs and proliferative and exhausted T cells in response to immunotherapy. These patients may instead benefit from neuro-oncology therapies targeting mesenchymal tumour cells.