HER2-enriched Tumors Research Articles

The LDHC-STAT3 Signaling Network Is a Key Regulator of Basal-like Breast Cancer Cell Survival.

Breast cancer treatment has evolved drastically with the addition of immunotherapy and novel targeted drugs to the current treatment options. However, achieving long-term responses with minimal adverse events remains challenging. Cancer testis antigens (CTAs) offer novel opportunities for drug development thanks to their tumor specificity, immunogenicity, pro-tumorigenic functions, and negative prognostic connotations. We previously reported that lactate dehydrogenase C (LDHC) plays a key role in regulating genomic stability and that targeting LDHC significantly improved treatment response to DNA damage response drugs in breast cancer. Here, we explored the molecular mechanisms associated with LDHC silencing in two basal-like breast cancer cell lines, MDA-MB-468 and BT-549, and a Her2-enriched breast cancer cell line, HCC-1954. Transcriptomic analyses identified the cell line-dependent differential activation of the pro-survival STAT3 pathway following LDHC depletion. While LDHC silencing significantly compromised cell survival in basal-like breast cancer cells in conjunction with a downregulation of STAT3 signaling, the opposite effect was observed in Her2-enriched breast cancer cells, which demonstrated the enhanced activation of the pro-survival STAT3 signaling pathway. The inhibition of STAT3 not only reversed the unfavorable effect of LDHC silencing in the Her2-enriched cancer cells but also demonstrated significant anti-cancer activity when used as a single agent. Our findings suggest that the LDHC-STAT3 signaling axis plays a role in regulating breast tumor cell survival in a subtype-dependent manner. Thus, LDHC-targeted therapy could be a viable therapeutic approach for a subset of breast cancer patients, particularly patients with basal-like breast cancer, whereas patients carrying Her2-enriched tumors may likely benefit more from monotherapy with STAT3 inhibitors.

Molecular and immunological characterization of androgen receptor expression in different breast cancer subtypes.

1114 Background: While estrogen receptor (ER) is well-studied in breast cancer (BC), the role of androgen receptor (AR) in prognosis and therapy response is less understood. Here, we aimed to characterize the clinicopathologic and molecular features of AR gene expression in BC subtypes. Methods: 10,728 BC samples were tested by NGS (592, NextSeq; WES, NovaSeq) and WTS (NovaSeq; Caris Life Sciences, Phoenix, AZ). MSI was tested by IHC and NGS. TMB-high was designated as >10 somatic mutations/MB. Tumors with AR-high(H) and AR-low(L) RNA expression were classified by top and bottom quartile, respectively. Real world overall survival (OS) and treatment-associated survival was obtained from insurance claims and calculated from treatment start to last contact using Kaplan-Meier estimates. Statistical significance was determined by chi-square and Mann-Whitney U test with p-values adjusted for multiple comparisons ( q<.05). Results: Lobular carcinoma had higher AR expression (2.37-fold) and AR positivity by IHC (93.6% vs 63.6%) compared to ductal carcinoma ( q<.05). Luminal A (LumA) had higher AR expression and IHC positivity compared to HER2-enriched, basal-like, and normal-like tumors ( q<.05). AR-H tumors were significantly ( p<.05) enriched for PIK3CA mutations in LumA (60.3% vs 29.8%), LumB (48.1% vs 23.2%), HER2-enriched (51.2% vs 39.3%), and basal-like (23.8% vs 8.3%); CDH1 mutations in LumA (26% vs 10.3%) and normal-like (55.5% vs 4.8%); MAP3K1 in LumA (17.1% vs 5.3%); ESR1 in LumB (17.9% vs 10.7%); and NF1 in HER2-enriched (19.3% vs 7.1%). AR-H had higher immune infiltration for LumA (B cells, M2 Mφ, neutrophils, NK cells, DC), LumB (B cells, M2 Mφ, neutrophils, NK cells, CD4+ T cells, DC), basal-like (NK cells, CD4+ T cells), and normal-like (NK cells, DC; q<.05) than AR-L. Conversely, AR-H HER2-enriched tumors had lower immune infiltration (B cells, M1 and M2 Mφ, NK cells, CD8+ T cells, DC) and fewer TMB-high (15.1% vs 23.2%), dMMR/MSI-high (0.3% vs 2%), and PD-L1+ tumors (15% vs 40.8%; p<.05) compared to AR-L (all q<.05). Post-doxorubicin survival was longer for patients with AR-H tumors; however, opposing trends were observed for patients with LumB (75.5 vs 56.3 m; p=.081) and HER2-enriched tumors (47.9 vs 65.2 m; p.071). AR-H was associated with longer post-dox survival in patients with LumB PIK3CA-mt tumors (81.7 vs 39.3 m; p=.036), but shorter post-dox survival in HER2-enriched PIK3CA-wt (50.4 vs 70.4 m) and PIK3CA-mt cohorts (40.8 vs 58.0 m; p=.18). Conclusions: Findings from our updated analysis based on PAM50 designation suggest a strong association between AR and PIK3CA mutations across subtypes and suggest that distinct AR-associated alterations in the immune microenvironment require further study. Exploration of specific mutations and immune-oncology markers associated with AR-H may aid in molecularly selected clinical trial design for advanced BC patients.

RNA-Seq based gene expression profiling of baseline and on-treatment breast tumors to predict response to HER2-directed therapy, without chemotherapy (TBCRC026).

530 Background: Robust biomarkers are needed to personalize treatment in early stage HER2-positive breast cancer. The TBCRC026 trial (n=83) demonstrated that early metabolic changes on FDG-PET/CT [>40% decline in SULmax at day 15 post treatment initiation (C1D15); C1D15 SULmax] were associated with response to neoadjuvant trastuzumab/pertuzumab (HP), without chemotherapy, in an ER-negative, HER2-positive cohort (NCT01937117). We investigated the association of RNA sequencing (RNAseq)-based gene expression signatures (GES) at baseline and on-treatment (C1D15), with early metabolic change on PET/CT and clinical outcomes. Methods: RNAseq (664 GES) was performed on baseline (n=56) and C1D15 (n=31) tumor biopsies (25 paired samples available). Association with pCR following HP (secondary), PET/CT parameters and survival (exploratory) was studied. Gene expression changes between baseline and C1D15 samples were tested by Wilcoxon signed-rank test. Association between GES and PET/CT parameters and GES with pCR were evaluated using logistic regression models. Cox regressions were performed to study the association between PET/CT SULmax at C1D15 and a B cell signature and relapse-free survival (RFS). The likelihood ratio test was used to compare the prognostic ability of two nested Cox models. Results: Both RNAseq and clinical outcomes were available in 53/83 patients. HER2-enriched (HER2-E) intrinsic subtype comprised 71% (40/56) of cases at baseline and 61% (19/31) at D15; basal subtype for 27% (15/56) and 39% (12/31), respectively. pCR rate was 19% (10/53) with all but one exhibiting HER2-E subtype. One basal-like sample underwent pCR, with downregulation of HER2 amplicon and IgG signatures slightly more prominent. Gene expression changes from baseline to C1D15 were analyzed; ERBB2was downregulated after HP (p = 0.007), B cell signatures, CD4, CD8, natural killer cells and Treg cells were significantly upregulated at C1D15. GES associated with 40% decline in SULmax at C1D15 included ERBB2 amplicon genes ( ERBB2, GRB7) and correlation to the HER2-E subtype, whereas basal-like signatures were significantly associated with lack of response on PET at C1D15. Univariate logistic regression analysis suggested that FOS-JUN, fibroblast and B cell GES at baseline were significantly associated with pCR (p=<0.05). Finally, the addition of a B cell signature improved the prognostic value of C1D15 SULmax on PET/CT for RFS (p < 0.05). Conclusions: In this unique cohort with ER-negative, HER2-positive breast cancer treated with neoadjuvant HP without chemotherapy, HER2-E tumors and those with increased immune signaling were more likely to respond to HER2-directed therapy. Combining C1D15 SULmax on PET/CT and a B cell signature provided improved prognostic information and should be further explored as candidate biomarkers in this setting. Clinical trial information: NCT01937117 .

Facilitating Culturally Competent Breast Imaging Care in South Asian Patients.

South Asians are a rapidly growing subset of the Asian population in the United States. They comprise people from multiple countries with diverse beliefs, languages, and cultural identities and values. The incidence of breast cancer is rising in South Asian women in the United States, with earlier onset and predilection for HER2-enriched tumors. Despite the rising incidence of breast cancer, participation in screening remains lower than other populations. Health care inequities in South Asian women are multifactorial and may be due to traditional health beliefs and practices, language barriers, cultural differences, and lack of overall awareness. Developing a culturally sensitive environment in breast imaging clinic practice can lead to improved patient care and adherence. Given the scarcity of data specific to the South Asian population in United States, there is a need for health service researchers and practice leaders to obtain more high-quality data to understand the needs of South Asian patient populations.

Abstract PO2-01-06: Prognostic value of residual disease (RD) biology and gene expression changes during the neoadjuvant treatment in HER2+ early-breast cancer (EBC)

Abstract Title Prognostic value of residual disease (RD) biology and gene expression changes during the neoadjuvant treatment in HER2+ early-breast cancer (EBC). Background In HER2+ EBC, neoadjuvant trastuzumab-based therapy is the standard of care, with adjuvant therapy determined by whether residual disease (RD) is present. Patients (pts) with RD have significantly higher relapse rates than pts with pathologic complete response. Differences in tumor and immune biology between pre-treated and post-treated tumors after HER2-blockade have been described, but little is known about how these changes can affect long-term outcomes or can be leveraged to tailor adjuvant treatment. Here we evaluate the biology of RD using gene expression analyses of pre- and post-treated HER2+ tumors, and their prognostic value to predict event-free survival (EFS) in a pooled analysis of 4 neoadjuvant studies in which single-agent trastuzumab or combined with lapatinib was explored, with and without chemotherapy: CALGB 40601 (Alliance), PAMELA, NeoALTTO, and NSABP B-41. Methods Gene expression profiling by RNA sequencing was assessed on 497 pre-treated samples from pts with RD, and on pre-treated and RD in 171 tumor pairs. Intrinsic subtypes and 22 tumor and immune gene expression signatures (GESs) were evaluated. We studied and compared the association of gene expression biomarkers with EFS in different cohorts and time points, including when GESs were calculated at baseline (N=497), in the 171 paired baseline and RD samples, and the nature of biomarker changes during neoadjuvant therapy. Association between GESs and EFS was explored by uni- and multivariable Cox regression models adjusted by clinical parameters (i.e., study, treatment arm, hormone receptor status, clinical tumor size, and clinical node involvement), and the performance of the models was assessed by the c-index. Results In 497 baseline untreated samples from pts with RD after neoadjuvant therapy (N=497), intrinsic subtypes were significantly associated with EFS (log-rank [LR] P 0.007), with Luminal A showing the best prognosis and Basal-like the worst. Among 171 paired tumors, HER2-Enriched tumors were more common in baseline (50.3%) with change to Normal-like (49.7%) and Luminal A (18.7%) in RD (chi-square P < 0.001). This luminal phenotypic change was also confirmed by a significant reduction in the correlation to the HER2-Enriched centroid and a significant increase in the correlation to the Luminal A centroid between pre- and post-treated samples; a significant decrease in ERBB2 gene expression and the HER2 amplicon gene expression signature was also observed (Wilcoxon P < 0.001). At an immune GES level, we found a significant increase in B cell, CD8+ T cell, and NK cell signatures in RD specimens. In contrast, T reg GES was significantly downregulated (Wilcoxon P < 0.05). In uni- and multivariable models, immune GESs provided more prognostic information when assessed in RD samples. In contrast, subtype-related biomarkers provided more prognostic information at baseline. From all the multivariable models, the best (c-index= 0.78) included the IgG signature assessed in RD (adjusted hazard ratio 0.46, 95% CI 0.30-0.70, adjusted P 0.005). IgG signature levels (tertiles) in RD could also stratify pts into different groups based on prognostic information (EFS at 7 years: 69% in IgG low vs. 91% in IgG high, LR P 0.02). Conclusions In pts with HER2+ EBC and RD after neoadjuvant treatment with HER2-blockade, tumor-related biomarkers seem to provide more prognostic information when calculated at baseline, whereas immune biomarkers in the RD perform better for EFS prognostication. This finding could be relevant for implementing RD biomarkers in the clinic to stratify pts into different prognostic groups and tailor the post-operative treatment strategy. Supported in part by U10 CA180821, U10 CA180882, R01-CA229409 https://acknowledgments.alliancefound.org Citation Format: Aranzazu Fernandez-Martinez, Maki Tanioka, Sung Gwe Ahn, Paola Zagami, Tomás Pascual, Mattia Rediti, Gong Tang, Katherine A Hoadley, David Venet, Naim U. Rashid, Patty Spears, Judith Bliss, Jens Huober, Priya Rastogi, Md N. Islam, Javier Cortés, Antonio Llombart-Cussac, Sandra Swain, Christos Sotiriou, Aleix Prat, Charles M Perou, Lisa Carey. Prognostic value of residual disease (RD) biology and gene expression changes during the neoadjuvant treatment in HER2+ early-breast cancer (EBC) [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 PO2-01-06.

Abstract PO3-05-07: SOLTI-1718 NEREA Trial: Neratinib in hormone receptor (HR)-positive/HER2-negative HER2-enriched (HER2-E) advanced breast cancer (BC)

Abstract Background Breast cancer (BC) is a clinically and biologically heterogenous disease where intrinsic subtypes play a role. Non-luminal subtypes within HR+/HER2-negative disease do not benefit to the same extent from standard of care treatments as the luminal subtypes. Thus, other strategies are needed. HER2-E subtype represents approximately 15.0% of HR+/HER2-negative tumors in metastatic setting. According to an exploratory analysis of EGF30008 trial, HER2-E advanced BC patients, despite presenting poor outcomes across treatments, showed benefit from anti-HER2 therapy with lapatinib. Here, we report the efficacy and safety of the NEREA trial, the first study designed to evaluate neratinib and endocrine therapy in HR+/HER2-negative, PAM50 HER2-E ABC. Methods SOLTI-1718 NEREA (NCT04460430) is a single-arm, multicenter phase II study evaluating neratinib in combination with endocrine therapy (ET) in patients with HR+/HER2-, PAM50 HER2-E ABC. Key inclusion criteria include progression to prior endocrine therapy, ≤ 1 line of chemotherapy for advanced breast cancer (ABC), ECOG 0-1, and HER2-E disease by PAM50 on a metastatic tumor biopsy. The availability of a metastatic tumor sample was mandatory in the pre-screening phase to assess PAM50 subtype. Included patients received neratinib 240 mg daily in combination with ET, with either exemestane, fulvestrant or tamoxifen (as per investigator´s decision). All patients received prophylactic loperamide with an established dosing scheme during the first cycle and on-demand in subsequent cycles. The primary endpoint was progression-free survival rate at 6 months (PFS6). Secondary endpoints include other efficacy endpoints and safety. The study was based on a Simon two-stage design. Stage I of the trial would be considered successful if at least 14 of 33 patients achieved PFS > 6m. In that case, the trial would recruit up to 56 patients for a target PFS6 ≥ 50%. Results. Between July 2020 and June 2022, molecular subtyping was performed on tumors from 136 patients, and 18 HER2-E tumors were identified (7.6%). 12 evaluable patients were enrolled. Baseline characteristics were as follows: median age 60 years, 91.7% of pts were postmenopausal, 75% had visceral disease and 59% received (neo)adjuvant treatment. The median number of lines for ABC was 3 (1-5). Neratinib was combined with exemestane (41.7%), fulvestrant (33.8%) and tamoxifen (25%). At the time of data cut-off (June 2023), 9 patients (75%) had stopped their treatment because of PD and 2 (16.7%) due to toxicity. The PFS6 months occurred in one patient (8.3%, CI: 0.2% - 38.5%). Median PFS was 1.7 months (95% CI 1.6-1.9) with a patient still on treatment after 27.1 months. The incidence of treatment-related adverse events (trAEs) was 25%; diarrhea (25%), asthenia (16.7%), nausea (8.3%), vomiting (8.3%) and rash (8.3%) were most common and were predominantly grade 1/2. 25% of patients experienced grade 3 trAEs. Conclusions.Due to slow enrollment, the study was stopped early. At the time of study close, the hypothesis that neratinib has efficacy in HR+/HER2-, PAM50 HER2-E tumors was not confirmed. We thank PUMA BIOTECHNOLOGY, INC for their provision of Neratinib and financial contribution to the study. Citation Format: Eva Ciruelos, Cristina Saura, Xavier Gonzalez-Farré, Francisco Javier Salvador Bofill, Maria Vidal, Isabel Blancas, Elena López-Miranda, Maria Iglesias, Míriam Arumí, Mireia Margelí, Catarina Pulido, Serafin Morales Murillo, Fernando Henao, Pilar Sanchez, Sara Alves, Ana Godoy Ortiz, José Passos Coelho, Santiago Escrivá-de-Romaní, Alejandra Espinosa, Tomás Pascual, Aleix Prat. SOLTI-1718 NEREA Trial: Neratinib in hormone receptor (HR)-positive/HER2-negative HER2-enriched (HER2-E) advanced breast cancer (BC) [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 PO3-05-07.

Abstract PO3-05-06: Targeting PAM50 HER2-Enriched intrinsic subtype with enzalutamide in hormone receptor-positive/HER2-negative (HR+/HER2-) advanced breast cancer: results of the SOLTI-1502 ARIANNA trial

Abstract Background Pre-clinical evidence and retrospective studies suggest that PAM50 HER2-Enriched (HER2-E), HR+/HER2- tumors have estrogen receptor (ER) independence and poor prognosis but seem to have androgen receptor (AR)-addiction. Enzalutamide (ENZ) is a potent inhibitor of AR signaling. We hypothesized that ENZ may induce a significant proliferative arrest in PAM50 HER2-E, HR+/HER2-negative advanced breast cancer (ABC), leading to clinical benefit in this poor prognosis population. Methods Eligible patients (pts), males or pre/post-menopausal women with advanced HR+/HER2- ABC resistant to endocrine therapy, received ENZ 160 mg/day until disease progression. Pts were assigned to different cohorts according to PAM50 intrinsic subtypes identified in the pre-treatment tumor biopsy: Cohort A included pts with HER2-E and Cohort B pts with Luminal A/B tumors. Centralized molecular assessment of pre-treatment and C1D15 tumor biopsies were required for the primary endpoint evaluation. After C1D15 biopsy, exemestane 50mg QD could be added to ENZ at physician’s discretion. Tumor assessments were performed every 8 weeks. Primary efficacy endpoint was the relative change in the PAM50 11-gene proliferation-related signature in Cohort A. Secondary objectives included anti-proliferative effect of ENZ in Cohort B, overall response rate (ORR), Clinical benefit rate (CBR; partial response [PR] + stable disease ≥ 24 weeks) progression-free survival (PFS), and safety. Results From June 2020 to October 2022, 47 tumors were screened, and 6 PAM50 HER2-E tumors were identified (13%). A total of 34 pts were enrolled: 6 pts in Cohort A and 28 pts in Cohort B. Among them, 4 pts from Cohort A received ENZ plus exemestane and 23 pts in Cohort B. Mean age was 59 (range 39-76), 89% of pts were postmenopausal, 74% had liver disease, and 59% received (neo)adjuvant treatment. The median number of lines for ABC was 4 (1-8). Mean Ki67 (central assessment) in Cohort A was 37% (5%-70%), while in Cohort B it was 30% (3%-90%). Mean central AR in Cohort A was 66% (30-100), while in Cohort B it was 55% (1-100). The mean suppression of PAM50 11-gene proliferation-signature after 2 weeks of treatment with ENZ was 3.7% (p=0.848) in Cohort A and 1.2% (p=0.909) in Cohort B. The median change in Ki67 levels in Cohort A was -0.43% (p=0.812), while in Cohort B it was +3.86% (p=0.081). No statistically significant differentially expressed genes or changes in intrinsic subtypes were observed. Overall, the median PFS was 1.8 months (95% CI 1.6-1.8). In Cohort A, median PFS was 1.6 months (95% CI 1.0-1.9), and in Cohort B, it was 1.8 months (95% CI 1.6-1.8). CBR was 0% and 11% (n=3, 95% CI 2.8-25.9%) in Cohorts A and B, respectively. A PR with 16 months duration in cohort B was observed in a patient with an AR-mutated tumor (AR c. 689C >T). Regarding safety, grade 1-2 and 3-4 toxicities occurred in 94.1% and 35.3% of patients, respectively. ENZ dose reduction and discontinuation occurred in 2 (5.9%) and 3 (8.8%) pts, respectively. The most common toxicities related to ENZ were nausea (n=8, 24%), decreased appetite (n=8, 24%), AST increase (n=6, 18%) and vomiting (n=6, 18%). Conclusions The hypothesis that ENZ could induce proliferation arrest in HR+/HER2-, PAM50 HER2-E tumors was not confirmed, and ARIANNA was prematurely closed due to the lack of efficacy. Additional research is needed to explore if ENZ may benefit specific populations of pts with breast cancer (e.g. AR-mut tumors) and whether alternative AR modulators are more effective in blocking AR signaling in HR+/HER2-, PAM50 HER2-E tumors Citation Format: Mafalda Oliveira, Tomás Pascual, Sonia Pernas, Mireia Margelí, Salvador Blanch, Barbara Adamo, Francisco Javier Salvador Bofill, Xavier Gonzalez-Farré, Samyukta Chillara, Patricia Galván, Esther Sanfeliu, Paula Blasco, Valeria Sirenko, Alejandra Espinosa, Charles M Perou, Aleix Prat, Luis Manso. Targeting PAM50 HER2-Enriched intrinsic subtype with enzalutamide in hormone receptor-positive/HER2-negative (HR+/HER2-) advanced breast cancer: results of the SOLTI-1502 ARIANNA 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 PO3-05-06.

Abstract PO2-13-05: Deciphering pregnancy-associated breast cancer: distinctive molecular profile and clinical implications from GEICAM/2017-07 EMBARCAM study

Abstract Background: Pregnancy-Associated Breast Cancer (PABC) is defined as BC diagnosed during pregnancy, breastfeeding, or within the first year postpartum. This challenging entity has a distinct biology and worst prognosis and presents an increased risk for metastasis and death compared to non-PABC patients. Identification of molecular pathways that define this malignancy is crucial to explain its biological characteristics and potential association with pregnancy as well as serve to identify new biomarkers with preventive and clinical implications. This study assesses the gene expression profile of a PABC cohort from the Registry Study of Pregnancy and Breast Cancer (GEICAM/2017-07 EMBARCAM study, NCT04603820) to identify distinct molecular signatures and altered pathways for this entity. Methods: This was a multicenter, observational and ambispective age-matched cohort of PABC patients (n=46) and non-PABC patients (n=49). We analyzed the expression of 776 genes involved in 23 key breast cancer pathways, using the nCounter® Breast Cancer 360™ Panel (NanoString Technologies) on BC tumor samples, including molecular subtypes by PAM50 gene signature. Associations between individual differential genes and epidemiological and clinical features of these patients were explored. Normalized differential expression values were log2-transformed for statistical analysis in nSolver Software (NanoString Technologies). P-values were adjusted within each gene/signature and on the grouping variable level difference t-test using the Benjamini and Yekutieli False Discovery Rate (FDR) adjustment. Results: PABC patients’ clinical subtypes were distributed as 43,5% HR-positive/HER2-negative, 17,4% HER2-positive and 39,1% triple negative BC. These patients reported higher family history of BC/ovarian cancer compared to non-PABC (52.17% vs 26.53%; p=0.0124). Differential gene expression showed distinct molecular profiles in PABC and non-PABC patients: significantly genes enrichment involved in cell proliferation and p53 signature for PABC patients and higher expression of apoptosis, TGF-β, and PTEN signatures for non-PABC (adj p< 0.05). Furthermore, CDK4 expression signature (adj p=0.124) and DNA damage repair signatures Homologous Recombination Deficiency (HDR) (adj p=0.083) and BRCAness (adj p=0.220) were also upregulated in PABC tumors. The most significantly upregulated genes in PABC patients (CCNA2, DEPDC1, FAM83D, CDC20, CDKN3, TRIP13, TTK, MAD2L1, KIF2C, and UBE2C) (adj p< 0.05) were involved in cell cycle regulation and DNA damage repair. PABC basal-like tumors were found to be significantly more prevalent than non-PABC, as per the PAM50 classification (45.7% vs 20.4%; p=0.020). Interestingly, basal-like PABC showed higher expression of ESR1 than basal-like non-PABC (logFC=1.486; p=0.022). In contrast, luminal A PABC tumors exhibited lower ESR1 expression vs non-PABC (logFC=-1.5; p=0.021). Additionally, HER2-enriched PABC tumors displayed lower B7-H3/CD276 expression compared to HER2-enriched non-PABC (adj p=0.031). Conclusions: Our study shows that PABC is potentially a clinical and molecular different entity with predominance of the basal-like subtype. Moreover, our results suggest the activation of oncogenic pathways related to cell proliferation, DNA damage repair and p53 mutations which may lead to a clinically more aggressive phenotype for PABC patients. Likewise, the enrichment of BRCAness and HRD signatures found in PABC patients in our study may suggest an increased genetic instability due to a breakdown in the DNA damage repair. These findings may be clinically relevant and translate to new treatment options in PABC patients, who may benefit from therapies targeting DNA repair or cell-cycle checkpoints, such as PARP inhibitors. Citation Format: Juan de la Haba-Rodríguez, Regina Peña, Marina Pollan, Yolanda Jerez Gilarranz, Jose Ponce, Antonio Fernández Aramburu, Blanca Cantos, Ana Santaballa Bertrán, Elena Galve, María Eva Pérez, Susana de la Cruz, María Helena López-Ceballos, Yolanda Fernández, Fernando Moreno, Sonia González, Manuel Ruíz - Borrego, Isabel Blancas, José L Alonso-Romero, Álvaro Jiménez-Arranz, Raúl Rincón, Silvia Guil, Alejandra Díaz-Chacón, Rosalía Caballero, Begoña Bermejo, Angel Guerrero. Deciphering pregnancy-associated breast cancer: distinctive molecular profile and clinical implications from GEICAM/2017-07 EMBARCAM study [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 PO2-13-05.

Abstract PR05: Spatial transcriptomics of ductal carcinoma in situ reveal subtype specific differences in tumoral and stromal cell compartments

Abstract Up to 50% of patients diagnosed with ductal carcinoma in situ (DCIS) never experience progression to invasive disease, even if left untreated. Current knowledge of what makes DCIS become invasive is limited and we lack diagnostic tools to predict which patients can be spared treatment. Escape of tumor cells from the breast ducts is influenced by characteristics of the tumor cells, the microenvironment surrounding the ducts, and the interplay between the two. Intraductal tumor cells in DCIS are not physically in contact with extraductal stromal cells. Until now, analyses of DCIS tissue have mainly been performed on bulk tissue, however, this approach does not take into consideration the unique morphology of DCIS. We used the Nanostring GeoMX® digital spatial profiling platform to determine the transcriptome of DCIS tumor cells and the surrounding stromal cells separately. From a large cohort of >500 DCIS cases from Akershus and Oslo University Hospitals, we selected 23 pure DCIS cases, grade 3, of different molecular subtypes: triple negative (TN), Luminal A (LumA) and HER2-enriched. At least four tumor-stroma pairs were selected from each case. Bioinformatic analyses were used to explore both tumor and stroma expression data and the interplay between the two cellular compartments. There were distinct gene expression differences between DCIS of different subtypes. Intertumoral heterogeneity was larger than intratumoral heterogeneity in all subtypes. This was also apparent in stromal cell compartments, although less pronounced. Gene ontology analyses of DCIS tumor cells from each of the three subtypes showed several overlapping biological processes, suggesting common mechanisms for regulating tumor cell growth in DCIS. We also found subtype specific differences: Tumor cells of the TN subtype showed upregulation of protein synthesis. Processes associated with RNA splicing were upregulated in tumor cells of the LumA subtype, whereas the HER2-enriched subtype showed increased DNA repair activity. The immune microenvironment differed between the subtypes: LumA DCIS were characterized by lower immune cell infiltration than TN and HER2-enriched, however, there was variation between and within the cases. Using in silico cell deconvolution, we found that B-cells were more abundant in HER2-enriched tumors, while T-cells were more common in TN and LumA. T-regulatory cells were found in all subtypes. The GeoMX® digital spatial profiling platform is well suited for exploring the transcriptome of DCIS samples. We found biologically relevant differences between tumor cells of the different molecular subtypes already at the DCIS stage in breast cancer progression. The immune cell composition surrounding DCIS lesions also differed between the subtypes. Further studies of the tumor-stroma interplay are required to understand the processes involved in progression of DCIS to invasive disease. Citation Format: Helga Bergholtz, Jens Henrik Norum, Tonje G Lien, Hossein Schandiz, Torill Sauer, Jürgen Geisler, Therese Sørlie. Spatial transcriptomics of ductal carcinoma in situ reveal subtype specific differences in tumoral and stromal cell compartments [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr PR05.

Breast Carcinoma: Correlation of Molecular Subtypes with Histopathologic Characteristics

Context: Breast carcinoma is a heterogenous disease and similar histological subtypes may exhibit different behavior and treatment response. The molecular classification may provide better prognostic, predictive and therapeutic information than histological classification. The study aimed at evaluating the distribution of molecular subtypes of breast cancer and its association with prognostic factors. Method: This prospective cross-sectional study was conducted on breast cancer cases, over a period of 2 years, at M.S. Ramaiah Medical College and Hospitals, Bengaluru. The resection specimens were evaluated for histopathological features. Immunohistochemical analysis for ER, PR, Her2neu and Ki-67 expression was performed to identify molecular subtypes. Correlation of molecular subtypes with histopathological prognostic factors was done by Chi- square test or Fischer’s exact test. Results: A total of 85 cases were evaluated and classified into Luminal A (50.6%), Basal like (27%), Luminal B (11.8%) and HER-2 enriched (10.6%) molecular subtypes. Invasive ductal carcinoma was the commonest histologic type across all the molecular subtypes. Majority of low-grade tumors were Luminal A and most of high-grade tumors were Basal like (p= 0.004). HER-2 enriched and Basal like tumors were relatively more frequent in ≤ 50 years of age and Luminal A tumors exhibited less frequent lymph node metastasis. However, these associations fell short of statistical significance. Conclusion: Luminal A tumors are the most common molecular subtype. Statistically significant correlation exists between histologic grade and molecular subtype. There is no significant correlation between molecular subtype and age, tumor size, nodal metastasis, pathological T stage and TNM stage. Keywords: Basal like, Breast carcinoma, HER2­Enriched, Luminal A, Luminal­ B, Molecular subtypes

Abstract B083: Association of socioeconomic deprivation with breast cancer incidence, survival and ipsilateral recurrence by molecular subtype in Scotland, United Kingdom

Abstract Cancer disparities reflect the interplay of biological, environmental, and socioeconomic determinants. In the United States, disentangling racial and ethnic disparities in breast cancer (BC) including the effects of health care access and socioeconomic deprivation is challenging, given the latter highly correlated with race and ethnicity. In Scotland, United Kingdom (UK), the population is 96% White and there is a single payer National Health Service (NHS). The Scottish Index of Multiple Deprivation (SIMD) measures the extent to which a geographic area is deprived across seven domains: income, employment, education, health, access to services, crime and housing. Scotland is renowned for high-quality electronic health records including breast tumor estrogen receptor (ER) status collected since 1997 and Progesterone Receptor and HER2 since 2009, which can be linked to hospital data and mortality. To gain a better understanding of individual patients’ SIMD status (measured in quintiles) at BC diagnosis with incidence and survival, we used data on 62,378 women diagnosed with invasive BC between 2000 and 2016. For ipsilateral breast recurrence (IBR) outcomes, we used a subset of data for BC diagnosed 2007-2008 where manual case-note review was performed for 3495 patients from 10 years post-diagnosis. Screen-detected ER+ tumor incidence increased over time, particularly in the least deprived quintile [Average Annual Percentage Change = 2.9% with 95% CI from 1.2 to 4.7]. No marked differences were observed for non-screen-detected ER+ tumors or ER- tumors by SIMD. Most versus least deprived SIMD-quintile was associated with significantly higher mortality for luminal A and HER2-enriched tumors in multivariable Cox proportional hazard models adjusted for mode of detection and confounders (aHR = 1.46 [1.13, 1.88] and 2.10 [1.23, 3.59] respectively) but weaker non-significant associations for luminal B and triple negative BC tumors (aHR <1.20). Among 2,819 ER+ tumors, 423 patients had IBR (15%) and 438 died: SIMD was related to death (p=0.018) with the most deprived more likely to have died in the first decade of follow-up (17.7% vs 12.9%). We found no significant differences by SIMD in prognostic tumor characteristics (grade, TNM stage, treatment, screen-detection) or risk of IBR. Among 676 patients diagnosed with ER- tumors, 185 had IBR (27%) and 105 died: 50% had mastectomy and the most deprived had an increased 5-year IBR risk compared to the least deprived (aHR 3.03 [1.41-6.53]). Our analysis shows socioeconomic deprivation is associated with differential BC incidence trends for screen-detected ER+ tumors and higher mortality and IBR for selected tumor subtypes. The lack of inequities for triple negative tumors by SIMD and survival, and IBR for ER+ tumors may be due to standardized treatment protocols and healthcare access. The association with socioeconomic deprivation and survival for HER2+ and recurrence among ER- tumors requires further study on potential other factors such as co-morbidities and adherence to treatment. Citation Format: Ines Mesa-Eguiagaray, Hayley Dunlop, Sarah Wild, Sheila M. Bird, Linda Williams, Peter S. Hall, Jonine D. Figueroa. Association of socioeconomic deprivation with breast cancer incidence, survival and ipsilateral recurrence by molecular subtype in Scotland, United Kingdom [abstract]. In: Proceedings of the 16th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2023 Sep 29-Oct 2;Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(12 Suppl):Abstract nr B083.

Metastatic site patterns by intrinsic subtype and HER2DX in early HER2-positive breast cancer.

Even with contemporary treatment strategies, more than 10% of HER2-positive early stage breast cancer patients may experience distant metastasis as first event during follow-up. Tools for predicting unique patterns of metastatic spread are needed to plan personalized surveillance. We evaluated how molecular heterogeneity affects the pattern of distant relapse in HER2-positive breast cancer. A total of 677 HER2-positive stage I-III breast cancer patients from ShortHER trial, Cher-LOB trial, and 2 institutional cohorts were included. PAM50 molecular subtypes and research-based HER2DX scores were evaluated. The cumulative incidence of distant relapse as the first event (any site and site specific) was evaluated using competing risk analysis. Median follow-up was 8.4 years. Tests of statistical significance are 2-sided. Stage III and high HER2DX risk score identified patients at the highest risk of distant relapse as first event (10-year incidence 24.5% and 19.7%, respectively). Intrinsic molecular subtypes were associated with specific patterns of metastatic spread: compared with other subtypes, HER2-enriched tumors were more prone to develop brain metastases (10-year incidence 3.8% vs 0.6%, P = .005), basal-like tumors were associated with an increased risk of lung metastases (10-year incidence 11.1% vs 2.6%, P = .001), and luminal tumors developed more frequently bone-only metastases (10-year incidence 5.1% vs 2.0%, P = .042). When added to stage or HER2DX risk score in competing risk regression models, intrinsic subtype maintained an independent association with site-specific metastases. The integration of intrinsic molecular subtypes with stage or HER2DX risk score predicts site-specific metastatic risk in HER2-positive breast cancer, with potential implications for personalized surveillance and clinical trials aimed at preventing site-specific recurrence.

Pathological response of different molecular subtypes of locally advanced breast carcinoma to neoadjuvant chemotherapy

Background: This study was done on modified radical mastectomy (MRM) specimens of patients with locally advanced breast carcinoma (LABC) who were categorized into molecular subgroups based on Immunohistochemistry and underwent neoadjuvant chemotherapy (NACT). The purpose of the study was to look for the pathological response of the tumor to the NACT. The molecular subtypes include Luminal A, Luminal B, Human epidermal growth factor receptor 2 (HER2) enriched, and triple negative. For HER2 enriched subtype, trastuzumab was also given along with the chemotherapy regimen. Aims and Objectives: The primary objective of this study is to find out the pathological complete response (pCR) in various molecular subgroups of breast carcinoma following NACT. The secondary objective is to describe the cellular changes in tumor tissue following NACT. Materials and Methods: This is a descriptive study done on MRM specimens of patients with LABC received in histopathology lab of Government Medical College, Kottayam to look for the pCR of the tumor following NACT. Gross examination and histopathology study of the specimen including axillary lymph nodes were done. The trucut biopsies of these cases with immunohistochemistry study were reviewed for confirming the grade and molecular subtype. pCR was assessed based on Chevallier system. Results: Of the 131 MRM specimens studied, overall pathologic complete response rate was 24.4% and the pathologic complete response rate in Luminal A subtype, Luminal B subtype, HER2 enriched tumors, triple negative tumors were 23.6%, 24%, 33%, and 20%, respectively. Conclusion: 33% of HER2 enriched subtype showed a pCR, i.e., no residual foci of neoplasm seen in the MRM specimens following NACT. Following NACT, change in tumor cellularity, changes in the morphology of neoplastic cells, changes in the surrounding breast tissue, and a change in grade of tumor were also noticed.

Pattern Analysis of Serum Galectins-1, -3, and -9 in Breast Cancer.

Galectins have been shown to have roles in cancer progression via their contributions to angiogenesis, metastasis, cell division, and the evasion of immune destruction. This study analyzes galectin-1, -3, and -9 serum concentrations in breast cancer patients through enzyme-linked immunosorbent assay (ELISA) against the characteristics of the patient and the tumor such as stage, molecular subtype, and receptor expression. Galectin-9 was found to be statistically significantly increased in HER2-enriched tumors and reduced in patients with hormone-receptor-positive tumors. Galectin-1 was found to be statistically significantly increased in the serum of patients who had undergone hormonal, immunotherapy, or chemotherapy. These findings provide insight into the changes in galectin levels during the progress of cancer, the response to treatment, and the molecular phenotype. These findings are valuable in the further understanding of the relationships between galectin and tumor biology and can inform future research on therapeutic targets for galectin inhibitors and the utility of galectin biomarkers.

The disparities in prognostic prediction and annualized hazard function in different molecular subtypes between young Chinese and White American women with breast cancer.

The prognostic disparities in different molecular subtypes between young Chinese and White American breast cancer patients remain unclear. The goal of this study was to explore the prognostic differences in different molecular subtypes between Chinese and White American patients aged ≤ 40 years. We included Chinese and White female breast cancer patients at or under the age of 40 from the Surveillance, Epidemiology, and End Results database (SEER) and the West China Hospital of Sichuan University. The chi-square test, log-rank test, and Cox proportional hazards model were employed to evaluate the distribution and survival disparities in the two racial/ethnic cohorts and different molecular subtypes. An annualized hazard function was used to calculate the annual failure rate among different molecular subtypes. This study included 20,859 female breast cancer patients at or under the age of 40, of whom 18,400 were White women and 2,459 were Chinese women. With a median follow-up time of 47 months, the 5-year breast cancer-specific survival (BCSS) rates for young Chinese and White women were 93.9% and 90.0%, respectively (P< 0.001). Molecular subtype was found to be a significant predictor in both young Chinese and White patients (P< 0.001), but different trends were observed in the two racial/ethnic cohorts when exploring the association between BCSS and molecular subtypes. Among young White patients, the hormone receptor (HoR) (+)/epidermal growth factor receptor 2 (HER2) (+) subtype had the best 5-year BCSS rate, while in young Chinese patients, the HoR (+)/HER2 (+) and HoR (+)/HER2 (-) showed comparable survival curves and both showed superior 5-year BCSS than other subtypes. Stratification by molecular subtypes, young Chinese patients demonstrated a superior 5-year BCSS in HoR (+)/HER2 (-) (96.3% vs 92.9%, P< 0.001) and triple-negative subtypes (88% vs 81.7%, P= 0.006) compared to young White American patients, while no significant differences were found in HoR (+)/HER2 (+) and HER2 enriched tumors. The annual hazard function for BCSS showed that there were significantly different trends in the HoR (+)/HER2 (-) and HoR (+)/HER2 (+) subtypes between young Chinese and White patients. There are disparities in prognosis and annualized hazard function between young Chinese and White females with breast cancer in different molecular subtypes.

Gene expression profiling of paired primary breast cancers and brain metastases to identify brain metastases-specific biological changes.

1032 Background: Despite the clinical impact of breast cancer (BC) brain metastases (BM), their biological complexity still remains poorly understood. We here evaluate the genomic profile of paired primary and metastatic samples to characterize biological changes acquired by BC during metastatization to the brain. Methods: The expression of 758 BC–related genes was evaluated using the BC360 Panel (nCounter platform) in matched primary BC and their associated BM. Intrinsic molecular subtyping was determined using the PAM50 subtype predictor (Parker et al. JCO 2009). Hormone receptor (HR) and HER2 status were evaluated on the BCBM. A False Discovery Rate (FDR) corrected paired two-class SAM was used to identify changes in single genes expression between paired BC and BMs samples. Results: Twenty-one matched primary BC and BMs samples were analyzed. BC subtype evaluated on the BMs was distributed as follows: 24% (N=5) HR-/HER2-, 24% (N=5) HR+/HER2- and 52% (N=11) HER2+. A considerable shift in PAM50 subtype was observed between primary BC and paired BM. While subtype concordance was high for HER2-enriched (HER2-E) tumors (100%), all Luminal A (LumA) primary BCs switched to either HER2-E (75%) or Luminal B (LumB, 25%) on the BM sample, as did 45% of Basal-like BCs (Basal to HER2-E). A clinical switch from HER2- primary BC to HER2+ BM (defined by IHC and ISH) was observed in only one pair (5%). Consistently, HER2-E (p&lt;0.001) and LumB (p=0.001) PAM50 signatures were significantly more expressed, while the LumA (p&lt;0.001) and Normal-like (p=0.001) signatures were significantly less expressed in BMs as compared to paired primary BCs (FDR-corrected Wilcoxon paired signed rank test). No significant change was observed in the expression of the Basal-like signature (p=0.562). Among the 758 evaluated genes, 60 and 318 genes, respectively, were significantly up- and downregulated in BMs as compared to primary BC (FDR&lt;5%). Upregulated genes were enriched, among others, in genes involved in survival and migration (e.g. FGFR4), cell proliferation (e.g. CCNB1, CDK1) and HER2-amplicon (e.g. ERBB2, GRB7). Downregulated genes were enriched in genes involved in immune response (e.g. CD8A, CCL5, PDCD1LG2, TNF), angiogenesis (e.g. PDGFRB) and response to hormonal stimuli (e.g. ESR1, BCL2). Conclusions: Gene-expression profiling of matched primary BCs and BMs shows recurrent gene expression modifications in metastatic samples which might have potential therapeutic implications (e.g. acquisition of HER2-E subtyping and increase in ERBB2 mRNA levels). [Table: see text]

Assessing the Correlation of Rate of Pathological Complete Response and Outcome in Post Neoadjuvant Chemotherapy Setting and Molecular Subtypes of Breast Cancer.

Background Neoadjuvant chemotherapy (NAC) is being widely used in treating breast cancer (BC). This study aimed to analyze the correlation between clinicopathological features, immunohistochemistry (IHC)-based molecular subtypes, and the pathological response to NAC and its relationship with disease-free survival (DFS) and overall survival (OS). Materials and methods A retrospective analysis of 211 breast cancer patients who received NAC between 2008 and 2018 was performed. Tumors were classified by IHC into luminal A, luminal B, human epidermal growth factor receptor 2 (HER2)-enriched, and triple-negative subtypes. The chi-square test was used to assess the association between pathological response and clinicopathological parameters. Cox regression analysis was used to assess factors related to DFS and OS. Results Post NAC, 19.4% of patients achieved a pathologic complete response (pCR). Estrogen receptor (ER), progesterone receptor (PR), HER2 (p<0.001, 0.005, and 0.02), Ki67 (p=0.03), molecular subtypes (p<0.001), T stage (p=0.04), and N stage (p=0.01) were significantly associated with pathological response. The rate of pCR was highest among HER2-enriched and triple-negative tumors (45.2% and 28%, respectively) with OR=0.13 and p<0.001 for the HER2-enriched subtype. Patients with pCR were 61% less likely to develop metastasis (adjusted hazard ratio [aHR]=0.39, p=0.06, 95% CI=0.14-1.06) and were significantly associated with better OS (aHR=0.07, p=0.02, 95% CI=0.01-0.61). Patients who were ≤40 years old (aHR=2.1, p=0.01), with T4 (aHR=3.4, p=0.02), grade 3 (aHR=2.5, p=0.01), and node-positive disease (HR=2.24, p=0.02) were at an increased risk of developing metastasis. High Ki67 was found to be significantly associated with better DFS (p=0.006). Conclusion HER2-enriched and triple-negative BC were associated with a higher rate of pCR. Patients with pCR had significantly better DFS and OS. Younger age, advanced stage, higher grade, and lymph node involvement were risk factors for metastasis.

Nuclear and Cytoplasmic hTERT, Tumor-Infiltrating Lymphocytes, and Telomere Elongation Leukocytes Are Independent Factors in the Response to Neoadjuvant Treatment in HER2-Enriched Breast Cancer

HER2-enriched tumors are responsible for 20% of breast tumors and have high rates of immune infiltrates in the tumor stroma that respond favorably to neoadjuvant chemotherapy. In the context of tumors, telomeres control cell death and prevent tumor cells from replicating discontinuously, leading to their immortalization. This study aimed to evaluate the presence of tumor-infiltrating lymphocytes, hTERT expression, hTERT promoter mutation, and leukocyte telomere length in HER2-enriched breast tumors. A total of 103 cases were evaluated, 19 with pathologic complete response. The TILs percentage was above ≥10 in 44 cases (43%) and significantly present in patients ≥50 years of age. hTERT staining positivity was mostly nuclear, significantly present in the non-pCR group, and associated with a lower survival rate. Leukocyte telomeres were elongated for HER2-enriched tumors, and in multivariate analysis, shortening was associated with an increased risk of death. Overall, our results show that the nuclear and cytoplasmic presence of hTERT may indicate a worse prognosis and that leukocyte telomere elongation is a protective factor.

Abstract P2-23-16: Clinico-Pathological and Molecular Characterization of HER2-Enriched Breast Tumors Independently of HER2 Status

Abstract INTRODUCTION: Breast cancer (BC) is a highly prevalent and heterogeneous disease, entailing different so-called intrinsic subtypes (IS) according to gene expression, namely Luminal A, Luminal B, HER2-Enriched (HER2E) and Basal-like, as well as a normal breast-like group. The HER2E is still a poorly understood entity, which needs further biologic characterization to improve therapeutic management. MATERIAL AND METHODS: Patients (pts) treated at Hospital Clinic (Barcelona, Spain) over 18 years with a diagnosis of metastatic BC, with available matched primary and metastatic tumor samples for gene expression analyses were retrospectively recruited. Using the nCounter® Breast Cancer 360 panel, we studied the expression of 776 genes pertaining to different tumor and immune pathway-related signatures, with a focus on HER2E vs. non-HER2E tumors. Significant changes were considered at a false discovery rate (FDR) &amp;lt; 5%. Main clinicopathological features were also compared. IS changes from primary to metastatic disease were assessed. RESULTS: Ninety-one pts with paired tumor samples were included. Briefly, primary tumors were 67.0% hormone receptor-positive (HR+)/HER2-negative (HER2-), 14.8% were HER2-positive (HER2+) and 18.2% were triple-negative (TNBC). IS distribution in primary tumors was the following: 25 Luminal A (28%), 22 Luminal B (24%), 24 HER2E (26%), 12 Basal-like (13%), and 8 Normal-like (9%). In contrast, IS distribution in metastatic tumors was the following: 13 Luminal A (14%), 22 Luminal B (24%), 34 HER2-E (37%), 16 Basal-like (18%), and 6 Normal-like (7%). The HER2E disease proved to be a relatively stable subtype, with 16 (66.7%) tumors not changing IS in the transition to metastatic disease (p=0.078). Particularly, within HR+/HER2-, HER2+ and TNBC, 8/12 (66.7%), 8/10 (80.0%) and 1/2 (50.0%) tumors remained HER2E. Conversely, an overall significant switch from Luminal to non-Luminal tumors at the metastatic progression was observed (p=0.031), with 14/19 (73.7%) new non-Luminal BC being HER2E. When considering all primary and metastatic tumors, HER2E were observed to be less frequently estrogen receptor (ER) positive than non-HER2E tumors (55.8% vs. 78.7%; p=0.002), with lower mean progesterone receptor (PR) levels (15.3% vs. 25.8%; p=0.027). Compared to the other subtypes (as a group), the PAM50 risk of recurrence score (ROR-P) was higher for HER2E tumors (59.1 vs. 41.7; p &amp;lt; 0.001), which were also more frequently HER2+ (36.5% vs. 5.8%) and less likely HR+/HER2- (50.0% vs. 73.6%) and TNBC (13.5% vs. 20.7%), compared to non-HER2E (p &amp;lt; 0.001). No significant differences in grade, TILs, Ki67 and histotype were observed. Overall, 140/776 genes were significantly downregulated in HER2E vs. non-HER2E tumors, including genes related to cell adhesion, migration, DNA damage repair, apoptosis, estrogen signalling pathway, senescence. Conversely, 178/776 genes were significantly upregulated, including the tyrosine-kinase receptor FGFR4, genes involved in nuclear activity, DNA transcription regulation, MAP-kinase signaling pathways, proliferation, cytokine response, epithelial-to-mesenchymal transition (EMT), cell cycle progression, and immune-related genes such as CD274 (PD-L1 gene). DISCUSSION: A switch towards more aggressive IS from primary to advanced disease was observed, with an increase in HER2E prevalence. HER2E tumors, which tend to maintain their IS at the metastatic disease, showed upregulation of genes related to proliferation, survival and EMT, coherently with their well-known worse prognosis. FGFR4 and CD274 upregulation, along with downregulation of genes involved in DNA damage repair suggest these tumors might benefit from targeted therapeutic approaches. Genomic higher risk was not convoyed by consistent clinicopathological features, except for lower PR levels and HER2 overexpression at IHC. Further characterization according to primary/metastatic and HR status is ongoing. Intrinsic subtype distribution across primary and metastatic breast tumors Citation Format: Francisco Javier Muñoz-Carrillo, Laia Paré, Benedetta Conte, Adela Rodríguez, Patricia Galván, Esther Sanfeliu, Blanca González-Farré, Claudette Falato, Isabel Garcia-Fructuoso, Barbara Adamo, Nuria Chic, Olga Martínez-Sáez, Tomás Pascual, Reinaldo Moreno, Montserrat Muñoz, Fara Brasó-Maristany, Aleix Prat, Francesco Schettini, Maria Vidal. Clinico-Pathological and Molecular Characterization of HER2-Enriched Breast Tumors Independently of HER2 Status [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 P2-23-16.

Abstract P5-02-20: Correlative and longitudinal transcriptomic profiling predicts patient outcomes and the efficacy of neoadjuvant HER2-targeted treatments in the randomized PREDIX HER2 trial

Abstract Background: The PREDIX HER2 trial compared standard neoadjuvant therapy with 6 cycles of docetaxel, trastuzumab, and pertuzumab (DTP), versus 6 cycles of trastuzumab emtansine (T-DM1) in 197 patients with HER2-positive breast cancer. There was no difference in pathologic complete response (pCR) rate and event-free survival (EFS) between the two treatments (Hatschek, JAMA Oncology 2021). Here we systematically evaluate the prognostic and predictive molecular biomarkers during neoadjuvant HER2-targeting therapy. Methods: Longitudinal fresh-frozen tissue biopsies (pretreatment (n=194) and after 2 cycles (n=167)) and surgical specimens (n=126) were collected and sequenced by RNA-sequencing (RNA-seq). Differential gene expression (DGE) analyses were conducted using zero-inflated negative binomial mixed model, and P-values were adjusted by the Benjamini-Hochberg method. Potential prognostic and predictive markers including PAM50 intrinsic subtype, cancer hallmark signature (n=50) score, tumor infiltrating lymphocyte fraction (n=9), and immune/stromal score were calculated based on normalized count data. The correlations between above biomarkers and pCR and EFS were analyzed using multivariate logistic and Cox regressions, respectively. We integrated the least absolute shrinkage and selection operator (LASSO) regression and bootstrapping algorithm (iteration=10,000, nfold=5) to choose best predictive features. Results: Downregulation of proliferation-related and extracellular matrix pathways (DGEs with false discovery rate (FDR) &amp;lt; 0.05, |log fold change|&amp;gt;1) was observed throughout treatment in both arms. DTP resulted in early (on-treatment vs baseline) inflammatory (IL-17, TGF-β, TNF signaling pathways) and immune (B cell, NK cell and cytokine signaling) responses, whereas late responses occurred in the T-DM1 arm (post-treatment vs on-treatment). Interestingly, a rebound of HER2 and PI3K-AKT signaling was observed within residual disease after T-DM1. Immune and stromal scores showed similar kinetics between the two treatment arms, with increase after first two cycles and later decrease to baseline levels. PAM50 intrinsic subtype at baseline was independently associated with pCR and EFS after adjusting for treatment arm, tumor size, lymph node status, hormone receptor status, and Ki-67. Luminal A (pCR rate, 11.1%, odds ratio (OR), 0.64, 95% confidence interval (CI): 0.49 to 0.84, P=0.001), Luminal B (pCR rate, 20.0%, OR, 0.63, 95% CI: 0.53 to 0.76, P&amp;lt; 0.001) and basal-like (pCR rate, 33.3%, OR, 0.70, 95% CI: 0.53 to 0.93, P=0.02) subtypes had lower pCR rates compared to HER2-enriched subtype (, 68.9%). Patients with basal-like tumors at baseline had worse EFS than those with HER2-enriched tumors (hazard ratio (HR), 4.66, 95% CI, 1.28 to 16.90, P= 0.02). Moreover, pair-wise analyses revealed that patients with HER2-enriched tumors at baseline switching to other PAM50 subtypes after two cycles, had improved pCR (OR, 1.54, 95% CI: 1.31 to 1.80, P&amp;lt; 0.001) and EFS (HR), 0.26, 95% CI, 0.08 to 0.83, P= 0.02) than the remaining patients. Machine learning based analyses identified best biomarkers for pCR (hallmark estrogen response early, hallmark androgen response, hallmark PI3K AKT mTOR signaling) and EFS (NK and Treg cells fraction, hallmark apical surface). Patients with higher hallmark apical surface score could benefit from T-DM1 (HRT-DM1 vs. DTP = 0.13, 95% CI, 0.02 to 0.79, P=0.03) and vice versa (HRT-DM1 vs. DTP = 5.02, 95% CI, 1.06 to 23.76, P=0.04). Conclusion: This study sheds light on how the tumor transcriptome evolves under anti-HER2 therapy and potentially provides prognostic and predictive biomarkers for standard chemotherapy with dual HER2 blockade versus monotherapy with an antibody-drug conjugate. Further investigations evaluating the spatial and single-cell heterogeneity of HER2-positive BC are ongoing. Citation Format: Kang Wang, Yajing Zhu, Ioannis Zerdes, Alexios Matikas, Emmanouil Sifakis, Dimitrios Salgkamis, Judith Bjöhle, Ellinor Elinder, Sara Margolin, Ana Bosch Campos, Gyula Pekar, Henrik Lindman, Aglaia Schiza, Zakaria Einbeigi, Jamila Adra, Anne Andersson, Lena Carlsson, Ann Charlotte Dreifaldt, Erika Isaksson-Friman, Susanne Agartz, Hemming Johansson, Mats Hellström, Edward Azavedo, Johan Hartman, Jonas Bergh, Thomas Hatschek, Theodoros Foukakis. Correlative and longitudinal transcriptomic profiling predicts patient outcomes and the efficacy of neoadjuvant HER2-targeted treatments in the randomized PREDIX HER2 trial [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 P5-02-20.