HER2-enriched Research Articles

Multiomic factor analysis for pathologic complete response (pCR) after 12 weeks of pembrolizumab + trastuzumab + pertuzumab in HER2-enriched (HER2-E) early breast cancer (eBC) in the Keyriched-1 trial.

590 Background: pCR successfully identified candidates for (chemotherapy) de-escalation in the ADAPT HER2+ trial program. Here, we report biomarker analysis for pCR in the single arm, hypothesis-generating Keyriched-1 trial (NCT03988036) in HER2-E eBC treated with pembrolizumab and dual HER2 blockade. The trial rationale is based on high efficacy of anti-HER2 agents and potentially high immunogenicity and immune checkpoint protein expression in HER2-E eBC. Methods: 48 patients with HER2 2+ (ISH+) or 3+ eBC (stage I-III) and HER2-E subtype received pembrolizumab (200 mg), trastuzumab biosimilar (loading dose, LD 8 mg/kg, maintenance dose, MD 6 mg/kg), and pertuzumab (LD 840 mg/kg, MD 420 mg/kg) q3w for 12 weeks. Primary outcome was pCR (ypT0/is ypN0). Multiomic factor analysis was performed using different combinations of biomarker datasets: baseline BC360 single genes and gene signatures, multiplexed immunohistochemistry (mIHC), stromal tumor infiltrating lymphocytes (sTILs), somatic mutations and tumor mutational burden, and changes in circulating tumor DNA status under therapy. Factors representing combined markers developed from these pooled datasets were identified based on eigenvalue >1 criteria. Logistic regression with factors dichotomized by median score was conducted to analyze their association with pCR. Furthermore, logistic regression with elastic net regularization was performed using all single markers, factors from multiomic factor analysis, and clinical characteristics. Results: Factor involving gene expression (mainly related to immune response and tumorigenesis), mIHC markers (immune and HER2) in stroma, and sTILs datasets was significantly associated with pCR. Using the median as cutoff, higher scores increased the odds of pCR (unadj. OR 5.00, 95%CI 1.35, 18.56; p=.016). pCR rates were 66.7% and 28.6% in the high and low score groups, respectively. Effect of the factor (high vs low; OR 2.19) was also important in a model that additionally included clinical markers: grade (3 vs 2; OR 2.57), ER status (ER+ vs ER-; OR 0.89), cT (cT 2/3 vs 1; OR 0.69), cN (cN 1/2 vs 0; OR 0.51), and PR status (PR+ vs PR-; OR 0.34). When standardized values for all individual markers from gene expression, mIHC, and sTIL datasets were analyzed, markers with greatest impact on pCR vs. non-pCR were genes FNBP1, CD36, MYCN, and SIX1 (OR 1.43-1.62) and PR status (OR 0.65), with stronger impact than grade (OR 1.22) or nodal status (OR 0.90). Conclusions: Our multiomic analysis uncovered potential biomarkers for pCR in HER2-E eBC treated with immune therapy and dual-HER2 blockade alone. These results together with the results from the ADAPT translational research program support validation in larger trials investigating chemotherapy-free regimens in selected patients with HER2+ eBC. Clinical trial information: NCT03988036 .

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 .

Association of tumor-infiltrating lymphocytes (TILs) and immune signatures with PAM50 intrinsic subtyping hormone receptor-positive (HR+)/HER2-negative (HER2-) early breast cancer (BC): A translational analysis of two multicentric neoadjuvant trials.

558 Background: HR+/HER2− BCs are generally described as immunologically cold, with low levels of TILs and PD-L1. Nevertheless, the addition of immunotherapy (ICI) to chemo has been reported to increase pCR rates in high-risk early HR+/HER2– BCs. Assessing the interaction between immune features and tumor biology may identify of a subset of patients (pts) with HR+/HER2− BC that would be the ideal candidates for the combination of chemo and ICI. Methods: Gene-expression data from two multicentric neoadjuvant phase II trials (GIADA, LETLOB) enrolling stage II-IIIA HR+HER2- BC pts was retrieved (Dieci, CCR 2022; Guarneri, JCO 2014). In the GIADA trial, 758 genes were assessed on baseline tumor samples by nCounter (N=43). In the LETLOB trial, gene-expression data (Affymetrix platform) from baseline core-biopsies was available for 66 pts. Intrinsic subtype was assigned using a research-based PAM50 predictor. TILs were evaluated following guidelines. Association of TILs and immune signatures with intrinsic subtyping was assessed by Kruskal-Wallis test. Results: PAM50 subtype distribution (N=109) was 44% Luminal B (LumB), 33% Luminal A (LumA), 18% Basal-like, 5% HER2-enriched (HER2-E). TILs were assessed on 101 samples: median 2 (range 0-100). Basal-like BCs showed higher TILs than other subtypes (p=0.008). No significant difference was observed between LumA and LumB BCs (Table). Basal-like tumors showed higher levels of previously published immune signatures associated with CD8 T-cells (p<0.001), Cytotoxic cells (p<0.001), IFN-gamma (p<0.001), and response to ICI in the GeparNuevo trial (p=0.003) (N=109). PD-1 (p=0.002), PD-L1 (p=0.001), and PD-L2 (p=0.001) genes were also more expressed in Basal-like BCs (evaluable in 43 samples). No significant difference was observed between LumA and LumB tumors. A significant weak to moderate positive correlation was observed between continuous TILs and immune signatures and basal-like PAM50 signature (p<0.001, Spearman coefficient=0.372 for TILs, from 0.354 to 0.648 for immune signatures). Conclusions: PAM50 intrinsic subtyping and TILs/immune signatures capture only partially overlapping information on the biology of HR+/HER2- BCs. Pts with HR+/HER2- BCs simultaneously showing features of biological aggressiveness and enhanced immunogenicity may represent the ideal candidates for the combination of ICI and chemo. [Table: see text]

Abstract PS09-01: Individual patient data meta-analysis of clinical and translational biomarkers for prediction of pathological complete response (pCR) after de-escalated therapy in HER2+ breast cancer in four trials of the West German Study Group

Abstract Background Introduction of anti-HER2 therapies substantially improved outcomes for HER2-positive early breast cancer (HER2+ eBC). However, a considerable proportion of patients (pts) may still be overtreated with systemic chemotherapy (CTx) combinations. Establishing safe de-escalation strategies to avoid toxicities requires precise patient selection. Therefore, we set out to determine predictors for efficacy and survival in the unique setting of four de-escalation trials investigating short (12-week) neoadjuvant treatments (NAT) in HER2+ eBC. We investigated the prognostic ability of clinical and translational biomarkers for pCR to identify pts with the best prognosis after de-escalated systemic CTx-free NAT. Methods 756 pts who received de-escalated NAT were analyzed: trastuzumab + pertuzumab (T + P, n=92) and T + P + paclitaxel (pac, n=42) in ADAPT-HR-/HER2+ (NCT01817452); trastuzumab emtansine (T-DM1, n=118), T-DM1 + endocrine therapy (ET, n=125), and T + ET (n=129) in ADAPT-HR+/HER2+ (NCT01779206), T + P + pac (n=107) and T + P + ET (n=100) in TP-II (NCT03272477); and T + P + pembrolizumab (n=43) in Keyriched-1 (NCT03988036) in HER2-enriched (HER2-E) subtype by PAM50. The primary endpoint of each trial was pCR (ypT0/is ypN0). Survival was a secondary endpoint in all trials but Keyriched-1; survival data for TP-II are pending. Baseline gene expression was analyzed by BC360 assay; intrinsic molecular subtypes were determined using the PAM50 predictor. Stromal tumor infiltrating lymphocytes (sTILs) were evaluated at baseline and at week 3 of NAT. sTILs were categorized using a 30% threshold and the low cellularity category ( < 500 invasive tumor cells) at week 3. Prognostic markers for pCR were identified with univariate and multivariable logistic regression models with backstep algorithm excluding variables with p≥0.1; considered variables included age, sTILs (baseline, 3-weeks), cT, cN, grade, hormone receptor and HER2 status, intrinsic subtype, and standardized ERBB2 and ESR1 gene expression. These analyses were performed separately for NAT involving systemic CTx (i.e. containing pac, n=149) and systemic CTx-free NAT (n=607). Results Overall pCR rate was 39.1% (CTx: 66.4%; CTx-free: 32.5%). Multivariable analysis identified predictors of pCR after CTx-free NAT: low cellularity at week 3 (vs < 30% sTILs: OR 3.21, 95%CI 1.85-5.57), ERBB2 (OR 1.90, 95%CI 1.41-2.55), HER2 3+ (vs 1+/2+ and ISH+: OR 8.01, 95%CI 1.65-38.99), LumA/LumB/Basal subtype (vs HER2-E: OR 0.57, 95%CI 0.34-0.97), cT2 (vs 1: OR 0.54, 95%CI 0.33-0.89), and cN1-3 (vs 0, OR 0.46, 95%CI 0.27-0.78). In CTx-containing NAT, only ERBB2 (OR 2.08, 95%CI 1.28-3.40) and additionally ESR1 (OR 0.38, 95%CI 0.23-0.61) were prognostic for pCR. Updated analysis including expanded gene expression data from ADAPT-HR+/HER2+ and a prognostic score for pCR after CTx-free NAT developed using machine learning methods will be presented at the meeting. Conclusions This pooled analysis demonstrated a 66% pCR rate after a short (12-week) de-escalated NAT in HER2+ eBC. In one-third of patients (with mainly stage I cancer, higher HER2 expression by immunohistochemistry and gene expression analysis, and low cellularity at 3 weeks), pCR can be achieved without systemic CTx. Identified biomarkers could pave the way for developing new patient selection strategies to spare CTx-associated acute and late toxicities in a clinically meaningful number of patients. Citation Format: Monika Graeser, Oleg Gluz, Christine zu Eulenburg, Sherko Küemmel, Raquel von Schumann, Matthias Christgen, Rachel Wuerstlein, Enrico Pelz, Hans-Heinrich Kreipe, Peter Schmid, Marc Thill, Michael Braun, Jochem Potenberg, Claudia Schumacher, Joke Tio, Andreas Hartkopf, Marianne Just, Christian Schem, Kerstin Lüdtke-Heckenkamp, Eva-Maria Grischke, Felix Hilpert, Angela Kentsch, Ronald Kates, Ulrike Nitz, Nadia Harbeck. Individual patient data meta-analysis of clinical and translational biomarkers for prediction of pathological complete response (pCR) after de-escalated therapy in HER2+ breast cancer in four trials of the West German Study Group [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 PS09-01.

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 PO4-23-09: Dual inhibition of FGFR4 and HER2/EGFR with Lapatinib improves targeting of Erk and Akt signaling in HER2+ breast cancer cells

Abstract In HER2-enriched (HER2E) breast cancer (BC), the dual HER2/EGFR inhibitor Lapatinib is a standard of care for advanced staged disease. Anti-HER2 therapies disrupt tumor progression through inhibition of the PI3K/AKT pathway. Some treated tumors will override inhibition by shifting to MAPK/ERK signaling to fuel cell growth, thus fostering resistance to anti-HER2 therapy. The tyrosine receptor kinase FGFR4 is included in the profile for a HER2E subtype. FGFR4 is overexpressed in ~30% of BC and can signal through PI3K/AKT and MAPK/ERK. Inhibitors for FGFR4 such as BLU554 are being investigated in phase I/II clinical trials for hepatocellular carcinoma, showing success in tumors that are positive for FGFR4’s preferred ligand FGF19. According to cBioPortal patient data, FGF19 is amplified in ~20% of BC, and has been shown to trigger an autocrine loop in triple negative BC through FGFR4. We examined BLU554 as a dual therapeutic with Lapatinib and dissected intracellular signaling stemming from the combination in HER2E BC. MDA-MB-453 BC cells (HER2+FGFR4+) were used for a dual strategy with BLU554 and Lapatinib. Cell viability assays were performed at matched doses, then as a dose response matrix, ranging from 0.1-20 µM. Findings were used for synergy analysis using synergyfinder.fimm.fi. Western blots determined protein activity in response to treatments. Ridaforolimus (mTOR inhibitor) was used in combination with either BLU554 or Lapatinib to assess MAPK/ERK and PI3K/AKT pathway crosstalk in MB-453 cells. Additionally, FGF19 was added to cells after a 2-hour pre-treatment to challenge BLU554 + Lapatinib and compare changes in signaling with FGF19 present. MB-453 cells treated with BLU554 decreased cell viability similarly to Lapatinib at 10 µM (65% [SD ± 3.3] and 63% [SD ± 2.1], respectively). Dual treatment decreased viability compared to single agents at all concentrations but the greatest difference was observed at 20 µM (38.5% in dual compared to Lapatinib [p-value = 0.00006]; 45.1% in dual compared to BLU554 [p-value = 0.00016]). Dose-response matrix of dual treatment yielded a ZIP synergy score of 16.852 ± 2.97 (>10 is synergistic), with the greatest synergy and inhibition at 10 µM and 20 µM of both drugs. BLU554 caused a therapeutic potency shift of Lapatinib, such that lower doses of Lapatinib (0.1 µM, 1 µM) are effective at higher doses of BLU554 (10 µM, 20 µM). Westerns of MB-453 with one dose of BLU554 increased phospho-ERK1/2 (pERK1/2) at 16- or 48-hr time points versus control, but when BLU554 was re-dosed then pERK1/2 was successfully inhibited. Both pAKT and pS6RP were increased by re-dosing BLU554. Cells treated for 2 hrs with Lapatinib decreased pAKT and pS6RP and did not change pERK, whereas a 2-hr treatment of BLU554 increased pAKT and decreased pERK. Dual treatment exhibited inhibition of ERK, AKT, 4EBP1, and S6RP phosphorylation all to a greater extent than single agents in combination with Ridaforolimus. Challenge by ectopic FGF19 of BLU554 + Lapatinib was unable to restore downstream signaling of AKT and ERK. BLU554 synergistically interacts with Lapatinib to decrease cell viability while increasing its therapeutic potency. Lapatinib primarily inhibits AKT signaling, whereas BLU554 targets ERK. Dual treatment simultaneously blocks MAPK/ERK and PI3K/AKT signaling and yields decreases of mTOR downstream effectors pS6RP and p4EBP1. These effects are maintained even when challenged with FGF19, suggesting that dual inhibition of FGFR4 and HER2 could prevent pathway activation from external stimuli. Citation Format: Jordan Beardsley, Joseph Goode, Deborah Altomare. Dual inhibition of FGFR4 and HER2/EGFR with Lapatinib improves targeting of Erk and Akt signaling in HER2+ breast cancer cells [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 PO4-23-09.

Abstract PS09-08: Genomic characterization of endocrine resistance in ER+HER2+ breast cancers in the POETIC Trial

Abstract Background: Mechanisms of resistance to endocrine therapy are not well understood within ER+HER2+ breast cancer (BC). Our prior work suggested that intrinsic HER2-Enriched (HER2E) molecular subtype predicts early resistance to aromatase inhibitors (AI) (Bergamino eBioMedicine 2022) and high on-treatment (on-Txt) Ki67 levels predict poor survival (Smith Lancet Oncol 2020). Improved early detection of persistent proliferating tumor cells with endocrine resistance pathways could be targeted by pre-emptive personalized therapy and reduction in recurrence. In this study, we proposed to further identify additional alterations/features from genomic and spatial data to provide unprecedented new insight into intrinsic and adaptive resistant pathways in tumor cells that may assist to identify molecular targets for treatment. Materials: POETIC was a phase III trial of post-menopausal patients with ER/PR+ invasive BC (n = 4480) randomized 2:1 to 2 weeks of peri-operative AI (POAI) vs control, followed by standard-of-care treatment. Ki67 was assessed by IHC and intra-tumor heterogeneity was evaluated (5-15 regions) for all the POETIC POAI samples (N = 2487). ER+HER2+ samples were classified as good responders (GR) or poor responders (PR) based on a reduction in Ki67 between pre-treatment (pre-Txt) and 2-week on-Txt samples. Tumor-infiltrating lymphocytes were assessed; multiplex Immunofluorescence (mIF) was performed to measure immune cell densities in tumor and stroma compartments (CD3, CD20, CD68, FOXP3, and CD3 FOXP3 co-expression). Gene expression profiles by BC360™ (Nanostring) on all 210 pairs of POAI treated ER+/HER2+; whole exome sequencing (WES, 100X) were performed on pre-Txt tumor and blood samples from 13 GR, 17 PR, and 9 HER2E GR. We performed GeoMx Whole Transcriptome on 4 pairs (pre-Txt and on-Txt) of GR and GeoMx Proteins (77 including IO proteins) on 6 pairs of GRs and 6 pairs of PRs. Results: The most frequently mutated genes were TP53, PIK3CA, GATA3, and CHD4. Only TP53 was associated with PR (Fisher’s exact p=0.01). TP53 mutated cases had higher expression of TP53 mutant-like gene expression signature compared to wild-type cases (Wilcoxon test p=0.001), mIF FOXP3 (Wilcoxon test p = 0.0005), and CD68 (Wilcoxon test p = 0.019) density score. However, within the HER2-E subset, we found that TP53 mutations were associated with GR (Fisher’s exact p=0.02). We found spatial heterogeneity of Ki67 IHC levels across POAI samples. Examining IHC, while there was higher heterogeneity of Ki67 in the ER+HER2- samples (n = 2264) with 3% of pre-Txt and 9% on-Txt, 6% of ER+HER2+ samples (13/223, 6 LumA, 5 LumB, and 2 HER2E) showed heterogeneity of Ki67 exclusively on-Txt. Even in GR tumors with Ki67 < 10% on-Txt, we identified hotspots with retained proliferating Ki67+ cells after 2 weeks of POAI. The lobular tumors were GR and had characteristic CDH1 mutations. Importantly, cases with persistent areas of Ki67+ cells, regardless of Her2 status, were associated with late relapse. To further explore intratumoral heterogeneity, we performed spatial whole transcriptomics profiling on 95 regions from 4 pairs of GR samples (Ki67 > 10% at baseline and Ki67 < 10% on-Txt) and found low intratumoral heterogeneity in the pre-Txt samples that increased at 2 weeks on-Txt. In a larger set of samples including both GR and PR with the GeoMx protein method, we found increased intratumoral heterogeneity in the PR vs GR. Conclusion: While TP53 mutation was generally a predictor of poor response; in HER2-E it paradoxically was associated with a good early response to aromatase inhibitor which warrants further investigation. Ki67 levels in ER+HER2+ showed higher intratumoral heterogeneity in a subset of patients on treatment suggesting the potential of persistent, proliferating cells leading to later recurrence. Our spatial RNA and protein data further observe the intratumoral heterogeneity that identifies pathways for use as potential spatial biomarkers. Citation Format: Maggie Chon U Cheang, Xixuan Zhu, Orsolya Sipos, Anastasia Alataki, Mikayla Feldbauer, Elena López-Knowles, Holly Tovey, Lucy Kilburn, Milana Bergamino Sirvén, Dhrusti Patel, Hui Xiao, Perry Maxwell, Anthony Skene, Chris Holcombe, Manuel Salto-Tellez, Nicholas Turner, Andrew Dodson, Ian Smith, John Robertson, Judith Bliss, Gene Schuster, Roberto Salgado, Mitch Dowsett, Katherine A Hoadley. Genomic characterization of endocrine resistance in ER+HER2+ breast cancers in the POETIC 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 PS09-08.

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 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 PO4-06-04: Understanding metastasis mixed-treatment responses through genomic analyses

Abstract Background Early-stage and metastatic breast cancer (MBC) exhibit genomic heterogeneity, with inter-patient variability between primary tumors, intra-patient variability of primary tumors versus metastasis, and even between different metastatic sites within the same patient. Response to therapy in MBC can also be heterogeneous, termed "mixed response", with different degrees of responsiveness to the same drug(s) across metastatic sites within the same patient. Whether this treatment response variability is influenced by factors such as intrinsic characteristics of metastatic lesions and/or the microenvironment is unknown. Through genomic analysis, we aim to identify genomic features that may explain mixed clinical responses across metastatic sites, which will also provide valuable insights into treatment sensitivity and resistance. Methods Eligible patients with MBC were identified from the UNC Rapid Autopsy Program (RAP) with primary tumors and multiple metastases (≥ 2 different sites) with RNA sequencing data. An independent radiologist retrospectively reviewed imaging studies to identify patients with mixed radiographic responses to a defined line of therapy. The response to treatment was categorized for each metastasis, defined as a complete or partial response, as stabilization ≥ 6 months. Tumor measurements were performed for each patient at different time points. We performed supervised learning with linear regression incorporating "RNA sequencing-based gene expression signatures" as a fixed effect and "patient" as a random effect. We performed two different analyses: (1) Using all tumors and (2) using Basal-like tumors only. We finally selected significant signatures with a p-value >0.05. Results Ten patients with mixed response were identified, and 33 metastases were used for the analysis; 3 had de novo stage IV disease, 5 had triple-negative (TNBC), 4 HER2+, and 4 hormone receptor+ (HR+/HER2-) disease. Of 33 metastases, 6 were brain, 8 liver, 7 lung, and 12 “other”. Regarding intrinsic subtype, 15 metastatic tumors were Basal-like, 3 HER2-Enriched (HER2E), 6 Luminal A, and 9 Luminal B. In this dataset, 6 metastatic lesions showed response and 27 non-response. When analyzing all 33 metastases, KRAS high expression, KRAS amplicon, 16q23 amplicon, and apocrine features correlated with response independently of metastatic site. Higher T reg and CDKN2A gene expression levels were associated with non-response in metastasis. Within Basal-like subtype metastases only (N=15), CD8 T cells, PI3K pathway, KRAS gene and amplicon, 16q23 amplicon, and Basal-like-associated 5q11 loss correlated with response. However, T reg cells, higher levels of CDKN2A gene, and stem-cell-like features were associated with treatment non-response. Conclusions By examining matched metastases with discordant responsiveness to treatment within individual patients, this genomic analysis provides valuable insight into treatment sensitivity and resistance. Higher T reg cells and CDKN2A gene expression values correlate with non-response, while the KRAS gene, KRAS amplicon, and the 16q23 amplicon were associated with response. These results suggest that the molecular tumor and microenvironment features of metastases may contribute to sensitivity and resistance to therapy in MBC. Further validation and exploration are needed in larger multi-metastatic cohorts to fully understand the clinical implications of these findings. Citation Format: Susana Garcia-Recio, Paola Zagami, Amy Wheless, Kerry Thomas, Lisa Carey, Charles M Perou. Understanding metastasis mixed-treatment responses through genomic analyses [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 PO4-06-04.

IVIM and DCE-MRI in Predicting Phenotypic Subtypes and Nottingham Prognostic Index of Breast Cancer.

To explore the value of intravoxel incoherent motion (IVIM) and dynamic contrast enhanced MRI (DCE-MRI) for predicting phenotypic subtypes and Nottingham prognostic index (NPI) of breast cancer. Descriptive study. Place and Duration of the Study: Department of Radiology, Affiliated Hospital of Jining Medical University, Jining, Shandong, China, from March 2020 to January 2022. One hundred and forty-one breast cancer patients with preoperative IVIM and DCE imaging were collected. IVIM parameters of D, D*, f, and DCE-MRI parameters of Ktrans, Kep, and Ve were measured. Receiver operating characteristic curves were conducted to assess the diagnostic efficacies. Additionally, 40 patients collected from February 2022 to July 2022 were enrolled as validation cohort. The D value in HER2-enriched (HER2-E) was lower than that in non-HER-E, while D*, Ktrans, and Ve values were higher than that in non-HER-E (p < 0.001, 0.046, < 0.001, and < 0.001, respectively). D + Ktrans + Ve showed an optimal diagnostic efficiency (AUC = 0.868). Meanwhile, D* and f values of triple-negative breast cancer (TNBC) were higher than those of non-TNBC, and Ve value of TNBC was lower than that of non-TNBC (p = 0.013, 0.006, and < 0.001, respectively). D* + f + Ve showed the best prediction performance (AUC = 0.849). Additionally, D and Kep were independent predictors of NPI (p < 0.001, and 0.002, respectively). D + Kep showed a good diagnostic efficiency (AUC = 0.818). The combined IVIM and DCE-MRI model showed enhanced diagnostic efficiency in predicting phenotypic subtypes and NPI of breast cancer, and might thus be considered efficient in therapy decision-making for patients. Breast neoplasms, Intravoxel incoherent motion, Dynamic contrast enhanced magnetic resonance imaging, Phenotypic subtypes, Nottingham prognostic index.

Comparison of the Response to Neoadjuvant Therapy Between Immunohistochemistry HER2 (3+) and HER2 (2+)/ISH+ Early-Stage Breast Cancer: A Retrospective Multicenter Cohort Study.

According to the American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) criteria, both immunohistochemical HER2 (3+) and HER2 (2+)/in situ hybridization (ISH) amplified [HER2 (2+)/ISH+] breast cancers (BCs) fall under the HER2-positive BC category. However, there is a lack of studies exploring the difference of neoadjuvant therapeutic response between patients with HER2 (3+) and HER2 (2+)/ISH+ early BC. We aimed to evaluate the neoadjuvant therapeutic response, long-term outcome, and intrinsic subtype heterogeneity between HER2 (3+) and HER2 (2+)/ISH+ BC. We examined 2 distinct cohorts. Cohort 1 (C1) encompassed 2648 patients with HER2-positive early BC diagnoses, and they received neoadjuvant therapy (NT) and surgery between January 1, 2009 and December 31, 2022, from the Shanghai Jiao Tong University Breast Cancer Data Base. Cohort 2 (C2) comprised 135 patients with early-stage HER2-positive BC who underwent NT and surgery at Henan Cancer Hospital from January 1, 2021, to December 31, 2022. These patients had available genomic and transcriptomic data at their disposal. C1 and C2 were further categorized into 2 patient cohorts as follows: (1) patients with IHC HER2 (3+) early BC [HER2 (3+) group], (2) patients with HER2 (2+)/ISH+ early BC [HER2 (2+)/ISH+ group]. Among those excluded from the analysis were patients < 18 years or >80 years of age. Clinicopathological parameters, long-term outcomes, and intrinsic subtypes were analyzed. In the C1 population, 83.7% had HER2 (3+) BC, while 16.3% had HER2 (2+)/ISH+ BC. Patients with HER2 (3+) had a significantly higher pathological complete response (PCR) rate (38.9%) than patients with HER2 (2+)/ISH+ (18.1%; P < .001), but the disease-free survival (DFS) was comparable after a median follow-up of 29 months (P = .556). The addition of trastuzumab or trastuzumab plus pertuzumab to neoadjuvant chemotherapy (NAC) improved PCR rates and DFS in HER2 (3+) BC but not in HER2 (2+)/ISH+ BC. In the C2 population, 97.75% HER2 (3+) and 52.17% HER2 (2+)/ISH+ were HER2 enriched (HER2E) subtype (P < .001). HER2E showed increased PCR rates compared to non-HER2E (P = .004). Compared to HER2 (3+) BC, the limited effectiveness of neoadjuvant trastuzumab and pertuzumab therapy for HER2 (2+)/ISH+ BC is due to subtype heterogeneity. Reassessment of targeted therapy efficacy in patients with HER2 (2+)/ISH+ BC is essential.

DNAJC9 expression in basal-like and luminal A breast cancer subtypes predicts worse survival.

This study aimed to analyze the role of genetic/epigenetic alterations and the prognostic value of the DNAJC9 gene in breast cancer. RT-PCR and Q-RT-PCR methods are used to examine DNAJC9 expression in breast cell lines. Survival ratios of breast cancer patients were evaluated by using bc-GenExMiner. Combined bisulfite restriction analysis and UALCAN in-silico tool were used to assess the methylation level of the DNAJC9 promoter. Mutations were searched with the help of Sanger Cosmic database and direct sequencing. DNAJC9 mRNA expression is significantly higher in basal-like, HER2-Enriched (HER2-E), luminal A and luminal B breast cancer subtypes compared to normal breast-like samples based on DNA microarray datasets (P < 0.001). Similar results were obtained in RNA-seq datasets, except for the luminal A breast cancer subtype (P > 0.1). We did not find any mutation at the core promoter region of DNAJC9 in breast cancer and normal cell lines. Mutations of DNAJC9 are infrequent in clinical samples (<%1). DNAJC9 promoter region is hypomethylated in tumor and normal samples. DNAJC9 expression is unfavorable for survival in basal-like and luminal A breast cancer subtypes. Mutations or promoter hypomethylation do not appear to have a role in high DNAJC9 gene expression in breast cancer. DNAJC9 expression could be suggested as a novel biomarker in basal-like and luminal A breast cancer subtypes.

Genomic characteristics of triple negative apocrine carcinoma: a comparison to triple negative breast cancer.

Apocrine carcinoma is a rare breast cancer subtype. As such, the genomic characteristics of apocrine carcinoma with triple negative immunohistochemical results (TNAC), which has been treated as triple negative breast cancer (TNBC), have not been revealed. In this study, we evaluated the genomic characteristics of TNAC compared to TNBC with low Ki-67 (LK-TNBC). In the genetic analysis of 73 TNACs and 32 LK-TNBCs, the most frequently mutated driver gene in TNAC was TP53 (16/56, 28.6%), followed by PIK3CA (9/56, 16.1%), ZNF717 (8/56, 14.3%), and PIK3R1 (6/56, 10.71%). Mutational signature analysis showed enrichment of defective DNA mismatch repair (MMR)-related signatures (SBS6 and SBS21) and the SBS5 signature in TNAC, whereas an APOBEC activity-associated mutational signature (SBS13) was more prominent in LK-TNBC (Student's t test, p < 0.05). In intrinsic subtyping, 38.4% of TNACs were classified as luminal A, 27.4% as luminal B, 26.0% as HER2-enriched (HER2-E), 2.7% as basal, and 5.5% as normal-like. The basal subtype was the most dominant subtype (43.8%) in LK-TNBC (p < 0.001), followed by luminal B (21.9%), HER2-E (21.9%), and luminal A (12.5%). In the survival analysis, TNAC had a five-year disease-free survival (DFS) rate of 92.2% compared to 59.1% for LK-TNBC (P = 0.001) and a five-year overall survival (OS) rate of 95.3% compared to 74.6% for LK-TNBC (P = 0.0099). TNAC has different genetic characteristics and better survival outcomes than LK-TNBC. In particular, normal-like and luminal A subtypes in TNAC have much better DFS and OS than other intrinsic subtypes. Our findings are expected to impact medical practice for patients diagnosed with TNAC.

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]

A Multiparameter Molecular Classifier to Predict Response to Neoadjuvant Lapatinib plus Trastuzumab without Chemotherapy in HER2+ Breast Cancer.

Clinical trials reported 25% to 30% pathologic complete response (pCR) rates in HER2+ patients with breast cancer treated with anti-HER2 therapies without chemotherapy. We hypothesize that a multiparameter classifier can identify patients with HER2-"addicted" tumors who may benefit from a chemotherapy-sparing strategy. Baseline HER2+ breast cancer specimens from the TBCRC023 and PAMELA trials, which included neoadjuvant treatment with lapatinib and trastuzumab, were used. In the case of estrogen receptor-positive (ER+) tumors, endocrine therapy was also administered. HER2 protein and gene amplification (ratio), HER2-enriched (HER2-E), and PIK3CA mutation status were assessed by dual gene protein assay (GPA), research-based PAM50, and targeted DNA-sequencing. GPA cutoffs and classifier of response were constructed in TBCRC023 using a decision tree algorithm, then validated in PAMELA. In TBCRC023, 72 breast cancer specimens had GPA, PAM50, and sequencing data, of which 15 had pCR. Recursive partitioning identified cutoffs of HER2 ratio ≥ 4.6 and %3+ IHC staining ≥ 97.5%. With PAM50 and sequencing data, the model added HER2-E and PIK3CA wild-type (WT). For clinical implementation, the classifier was locked as HER2 ratio ≥ 4.5, %3+ IHC staining ≥ 90%, and PIK3CA-WT and HER2-E, yielding 55% and 94% positive (PPV) and negative (NPV) predictive values, respectively. Independent validation using 44 PAMELA cases with all three biomarkers yielded 47% PPV and 82% NPV. Importantly, our classifier's high NPV signifies its strength in accurately identifying patients who may not be good candidates for treatment deescalation. Our multiparameter classifier differentially identifies patients who may benefit from HER2-targeted therapy alone from those who need chemotherapy and predicts pCR to anti-HER2 therapy alone comparable with chemotherapy plus dual anti-HER2 therapy in unselected patients.

Prognostic and predictive impact of gene expression in node-positive early breast cancer patients receiving dose-dense versus standard-dose adjuvant chemotherapy.

The utility of multigene expression assays in advanced (≥ 4 positive lymph nodes) early breast cancer (EBC) is limited. We conducted exploratory transcriptomic analysis of 758 genes (Breast Cancer 360 panel, nCounter® platform; NanoString) in primary tumor samples collected during a phase 3 trial comparing adjuvant taxane-containing dose-dense chemotherapy (ddCTX) versus standard-dosed chemotherapy (stCTX) in resected EBC with ≥ 4 positive lymph nodes. Prognostic and predictive associations with disease-free survival (DFS) and overall survival (OS) were evaluated by Cox regression with false discovery rate (FDR) adjustment. Data were available from tumor samples of 141/226 patients (median follow-up: 14 years). Several genes/signatures, including immune markers, showed prognostic relevance in unadjusted analyses. Of these, two remained significant after multiplicity adjustment: a positive effect on DFS of programmed cell death 1 ligand-2 (PD-L2) in the ddCTX arm (univariate HR: 0.53, FDR-adjusted P = 0.036) and a negative effect on OS of HER2-enriched (HER2-E) signature in the stCTX arm (univariate HR: 5.40, FDR-adjusted P = 0.036). Predictive analyses showed greater DFS benefit of ddCTX in tumors with high antigen processing machinery (APM) expression (multivariate interaction P = 0.024). Multigene expression assays have a prognostic and predictive potential in advanced EBC, and further investigation is warranted in order to identify candidates for de-escalated treatment. In addition, intrinsic subtype and immune gene expression have predictive potential.

Prognostic value of intrinsic subtypes in hormone-receptor-positive metastatic breast cancer: systematic review and meta-analysis

In hormone receptor-positive (HoR+) breast cancer (BC), gene expression analysis identifies luminal A (LumA), luminal B (LumB), human epidermal growth factor receptor 2 (HER2)-enriched (HER2-E), basal-like (BL) intrinsic subtypes and a normal-like group. This classification has an established prognostic value in early-stage HoR+ BC. Here, we carried out a trial-level meta-analysis to determine the prognostic ability of subtypes in metastatic BC (MBC). We systematically reviewed all the available prospective phase II/III trials in HoR+ MBC where subtype was assessed. The primary endpoint was progression-free survival (PFS)/time to progression (TTP) of the LumA subtype compared to non-LumA. Secondary endpoints were PFS/TTP of each individual subtype, according to treatment, menopausal and HER2 status and overall survival (OS). The random-effect model was applied, and heterogeneity assessed through Cochran's Q and I2. Threshold for significance was set at P < 0.05. The study was registered in PROSPERO (ID: CRD42021255769). Seven studies were included (2536 patients). Non-LumA represented 55.2% and was associated with worse PFS/TTP than LumA [hazard ratio (HR) 1.77, P < 0.001, I2= 61%], independently of clinical HER2 status [Psubgroup difference (Psub)= 0.16], systemic treatment (Psub= 0.96) and menopausal status (Psub= 0.12). Non-LumA tumors also showed worse OS (HR 2.00, P < 0.001, I2= 65%), with significantly different outcomes for LumB (PFS/TTP HR 1.46; OS HR 1.41), HER2-E (PFS/TTP HR 2.39; OS HR 2.08) and BL (PFS/TTP HR 2.67; OS HR 3.26), separately (PFS/TTP Psub= 0.01; OS Psub= 0.005). Sensitivity analyses supported the main result. No publication bias was observed. In HoR+ MBC, non-LumA disease is associated with poorer PFS/TTP and OS than LumA, independently of HER2, treatment and menopausal status. Future trials in HoR+ MBC should consider this clinically relevant biological classification.

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 P5-02-03: Combined biomarker analysis for prediction of pathological complete response (pCR) after 12 weeks of pembrolizumab + trastuzumab + pertuzumab in HER2-enriched early breast cancer: Keyriched-1 trial

Abstract Background In unselected HER2+ early breast cancer (EBC), de-escalated chemotherapy-free neoadjuvant therapy (NAT) with dual HER2-blockade induces pCR rates of only 20%-40%. In order to achieve pCR rates by de-escalated therapy comparable to those achieved by chemotherapy-based regimens, patient selection and more effective chemotherapy-free regimens are thus key. KEYRICHED-1 (NCT03988036), a single-arm phase 2 study, is the first trial to investigate chemotherapy-free NAT with dual HER2 blockade and pembrolizumab in HER2-enriched HER2+ EBC. In a translational subproject, we analyzed gene signatures together with tumor cell proliferation and spatiotemporal immune cell profiling to identify predictive factors for pCR. Methods 48 pre- and postmenopausal patients with newly diagnosed HER2 2+ (ISH positive) or 3+ EBC (stage I-III) and HER2-enriched (HER2-E) subtype by PAM50 were included in the study. All patients received 4 cycles of pembrolizumab (200 mg), trastuzumab biosimilar ABP 980 (loading dose (LD) 8 mg/kg bodyweight (BW), maintenance dose (MD) 6 mg/kg BW), and pertuzumab (LD 840 mg/kg BW, MD 420 mg/kg BW) q21d. Primary objective was pCR (centrally confirmed absence of invasive tumor in breast and lymph nodes: ypT0/is, ypN0). NanoString Breast Cancer 360 panel was performed in baseline biopsies (n=42). ≥30% Ki67 decrease, &amp;lt; 500 invasive tumor cells or no evidence of tumor in week 3 biopsies (on treatment) were classified as early response. sTILs were analyzed at baseline (n=42) and week 3 (n=28). Ongoing analyses include whole exome sequencing and multiplexed immunohistochemistry for expression of PD1, PDL1, CD4, CD8, CD68, and CD20 levels in tumor and stroma at baseline and at week 3. Impact of standardized expression of single genes, signatures, and sTILs on pCR was evaluated with univariable and multivariate logistic regression analyses and summarized with odds ratios (OR) and 95% confidence intervals (95%CI). Results 42 patients with BC360 and sTILs data at baseline were included in the analysis. Median age was 55 years (range: 22-83), 11 patients (31%) had node-positive EBC. At baseline, 28 patients had sTIL levels ≥30% and 14 had sTILs &amp;lt; 30%; the corresponding pCR rates were 57.1% (n=16) and 28.6% (n=4, p=0.108). At week 3 (on treatment), 16 patients had sTIL levels ≥30%, 50% (n=8) had a pCR vs 8.3% in those with &amp;lt; 30% sTILs (one patient out of 12, p=0.039). 37 patients had early response, 54.1% of them (n=20) had a pCR vs 0% in early non-responders (n=5, p=0.049). In univariate analysis, IDO1, ERBB2, IFNγ, cytotoxic cells, cytotoxicity, CD8 T-cells, TIGIT, and tumor inflammation signatures were statistically significantly associated with pCR (OR 2.3-3.6); ERBB2, IDO1, IFNγ and CD8 T-cells remained significant after adjusting for hormone receptor (HR) and central HER2 status (OR 2.2-4.3). 70 single genes were predictive for pCR; none of them remained significant after false discovery rate adjustment (25%). In multivariable analysis for baseline markers including signatures, sTILs, HR and central HER2 status, only ERBB2 (OR 8.7, 95%CI 1.9-39.0, p=0.0046) and cytotoxic cells signatures (OR 4.6, 95%CI 1.6-13.5, p=0.0059) were predictive for pCR. Results of whole exome sequencing, and multiplexed immunohistochemistry analysis of immune cell markers will be presented at the Symposium. Conclusions Biomarker analysis in the unique KEYRICHED-1 cohort revealed that early response at week 3, ERBB2 and immune related signatures as well as on-therapy sTIL levels predict pCR after a chemotherapy-free combination of immunotherapy and dual HER2 blockade in HER2-enriched EBC. These results pave the way for validation in larger de-escalation trials investigating short, chemotherapy-free regimens in selected patients with HER2+ EBC. Funding for this research was provided by MSD Sharp &amp; Dohme GmbH. Citation Format: Monika Graeser, Sherko Kuemmel, Oleg Gluz, Friedrich Feuerhake, Valery Volk, Daniel Ulbrich-Gebauer, Claudia Biehl, Mattea Reinisch, Athina Kostara9, Iris Scheffen, Kerstin Luedtke-Heckenkamp, Andreas Hartkopf, Felix Hilpert, Angela Kentsch, Carsten Ziske, Reinhard Depenbusch, Michael Braun, Jens-Uwe Blohmer, Christine zu Eulenburg, Matthias Christgen, Ronald Kates, Stephan Bartels, Hans-Heinrich Kreipe, Enrico Pelz, Peter Schmid, Nadia Harbeck. Combined biomarker analysis for prediction of pathological complete response (pCR) after 12 weeks of pembrolizumab + trastuzumab + pertuzumab in HER2-enriched early breast cancer: Keyriched-1 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 P5-02-03.