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Supplementary Table 1 lists genes comprising the RNA expression signatures measured with the BC360 panel

Supplementary Figure 3 shows association between RNA signatures and overall survival

Purpose:Although optimal treatment in early triple-negative breast cancer (TNBC) remains unclear, de-escalated chemotherapy appears to be an option in selected patients within this aggressive subtype. Previous studies have identified several pro-immune factors as prognostic markers in TNBC, but their predictive impact regarding different chemotherapy strategies is still controversial.Experimental Design:ADAPT-TN is a randomized neoadjuvant multicenter phase II trial in early patients with TNBC (n = 336) who were randomized to 12 weeks of nab-paclitaxel 125 mg/m2 + gemcitabine or carboplatin d 1,8 q3w. Omission of further (neo-) adjuvant chemotherapy was allowed only in patients with pathological complete response [pCR, primary endpoint (ypT0/is, ypN0)]. Secondary invasive/distant disease-free and overall survival (i/dDFS, OS) and translational research objectives included quantification of a predictive impact of markers regarding selection for chemotherapy de-escalation, measured by gene expression of 119 genes (including PAM50 subtype) by nCounter platform and stromal tumor-infiltrating lymphocytes (sTIL).Results:After 60 months of median follow-up, 12-week-pCR was favorably associated (HR, 0.24; P = 0.001) with 5y-iDFS of 90.6% versus 62.8%. No survival advantage of carboplatin use was observed, despite a higher pCR rate [HR, 1.04; 95% confidence interval (CI), 0.68–1.59]. Additional anthracycline-containing chemotherapy was not associated with a significant iDFS advantage in pCR patients (HR, 1.29; 95% CI, 0.41–4.02). Beyond pCR rate, nodal status and high sTILs were independently associated with better iDFS, dDFS, and OS by multivariable analysis.Conclusions:Short de-escalated neoadjuvant taxane/platinum-based combination therapy appears to be a promising strategy in early TNBC for using pCR rate as an early decision point for further therapy (de-) escalation together with node-negative status and high sTILs.See related commentary by Sharma, p. 4840

Supplementary Data from De-escalated Neoadjuvant Chemotherapy in Early Triple-Negative Breast Cancer (TNBC): Impact of Molecular Markers and Final Survival Analysis of the WSG-ADAPT-TN Trial

Supplementary Figure from De-escalated Neoadjuvant Chemotherapy in Early Triple-Negative Breast Cancer (TNBC): Impact of Molecular Markers and Final Survival Analysis of the WSG-ADAPT-TN Trial

Purpose:To identify associations of biological signatures and stromal tumor-infiltrating lymphocytes (sTIL) with pathological complete response (pCR; ypT0 ypN0) and survival in the Phase II WSG-ADAPT HER2+/HR− trial (NCT01817452).Experimental Design:Patients with cT1-cT4c, cN0–3 HER2+/HR− early breast cancer (EBC) were randomized to pertuzumab+trastuzumab (P+T, n = 92) or P+T+paclitaxel (n = 42). Gene expression signatures were analyzed in baseline biopsies using NanoString Breast Cancer 360 panel (n = 117); baseline and on-treatment (week 3) sTIL levels were available in 119 and 76 patients, respectively. Impacts of standardized gene expression signatures on pCR and invasive disease-free survival (iDFS) were estimated by logistic and Cox regression.Results:In all patients, ERBB2 [OR, 1.70; 95% confidence interval (CI), 1.08–2.67] and estrogen receptor (ER) signaling (OR, 1.72; 95% CI, 1.13–2.61) were favorable, whereas PTEN (OR, 0.57; 95% CI, 0.38–0.87) was unfavorable for pCR. After 60 months median follow-up, 13 invasive events occurred (P+T: n = 11, P+T+paclitaxel: n = 2), none following pCR. Gene signatures related to immune response (IR) and ER signaling were favorable for iDFS, all with similar HR about 0.43–0.55. These patterns were even more prominent in the neoadjuvant chemotherapy-free group, where additionally BRCAness signature was unfavorable (HR, 2.00; 95% CI, 1.04–3.84). IR signatures were strongly intercorrelated. sTILs (baseline/week 3/change) were not associated with pCR or iDFS, though baseline sTILs correlated positively with IR signatures.Conclusions:Distinct gene signatures were associated with pCR versus iDFS in HER2+/HR− EBC. The potential role of IR in preventing recurrence suggests that patients with upregulated IR signatures could be candidates for de-escalation concepts in HER2+ EBC.

Supplementary Table from De-escalated Neoadjuvant Chemotherapy in Early Triple-Negative Breast Cancer (TNBC): Impact of Molecular Markers and Final Survival Analysis of the WSG-ADAPT-TN Trial

Background: De-escalating strategies seem promising inHER2-positive early breast cancer (EBC) and chemo-free regimens are thus of keyinterest. Recent data have underlined the role of tumor immunogenicity inresponse to de-escalated neoadjuvant anti-HER2 therapy. Therefore, theprospective single arm hypothesis-generating phase II KEYRICHED-1 trial (NCT03988036)investigates the pCR rate in patients with HER2-enriched EBC receiving fourcycles of the dual anti-HER2 blockade in combination with the checkpointinhibitor pembrolizumab. Initial studies with dual antibody-based HER2 blockadealone were able to achieve pCR-rates of 20-40%, which did not quite match the pCRrates obtained with concurrent chemotherapy. KEYRICHED-1 aims at achieving pCR-ratescomparable to standard chemotherapy-containing regimens by incorporating appropriatemolecular selection and immune oncology. Methods: A total of 48 pre- and postmenopausal patients with newly diagnosed HER22+ or 3+ EBC (stage I-III) and HER2-enriched (HER2-E) subtype by PAM50 wereenrolled in this single-arm study. All patients received four cycles of studytreatment with pembrolizumab (200mg), trastuzumab biosimilar (Trazimera®,loading dose 8mg/kg bodyweight (BW), maintenance dose 6mg/kg BW), and pertuzumab(loading dose 840mg/kg BW, maintenance dose 420mg/kg BW) q21d . Primaryendpoint was centrally confirmed pCR (ypT0/is, ypN0). The trial was planned asa Simon's two-Stage design (null and alternative pCR were 40% and 60%); interimanalysis after 16 patients had to show a pCR rate of at least 50% to continuerecruitment. Results: Between 05/2020 and 03/2021, 98 patients werescreened. N=52 (55%) had HER2-E subtype,of whom 48 patients entered thetreatment phase. Median patient age was 57 years (28-83). 65% had tumors > 2cm and 30% positive lymph node status. Centrally confirmed pCR rate in surgicalspecimens was 46% (95% CI 0.31-0.62) in the 43 patients of the per protocolpopulation, and 52% (95%CI 0.37-0.67) in all 46 evaluable patients (localassessment; two pCRs verified only by core biopsy) (p=0.22 and p=0.06 for nullhypothesis, respectively). Despite HER2-E subtype, no pCR was observed in the 4patients with immunohistochemical (IHC) HER2 2+/ISH-positive status in contrastto 20/39 (51.2%) pCRs in IHC HER2 3+ tumors. Centrally confirmed pCR rate in HR+/HER2+tumors was38.5% compared to 58.5% in HR-/HER2+ tumors. No new safety signals wereobserved. Conclusions: These are the first results of a neoadjuvant chemotherapy-free12-week de-escalation anti-HER2-regimen with trastuzumab and pertuzumab incombination with the PD-1 inhibitor pembrolizumab in patients with a HER2-E EBC.In the context of the WSG ADAPT HER2+ de-escalation trials the observed pCRrates compare favourably in HR+ as well as HR- HER2 EBC. Moreover, KEYRICHED-1demonstrates that with appropriate molecular patient selection clinicallymeaningful pCR rates in the range of those obtained with longer, more toxicchemotherapy-containing regimens can be achieved. Citation Format: Sherko Kuemmel, Oleg Gluz, Mattea Reinisch, Athina Kostara, Iris Scheffen, Monika Graeser, Kerstin Luedtke-Heckenkamp, Andreas Hartkopf, Felix Hilpert, Angela Kentsch, Carsten Ziske, Reinhard Depenbusch, Michael Braun, Jens Blohmer, Christine zu Eulenburg, Matthias Christgen, Stephan Bartels, Hans Kreipe, Enrico Pelz, Peter Schmid, Nadia Harbeck. KEYRICHED-1 - A prospective, multicenter, open label, neoadjuvant phase II single arm study with pembrolizumab in combination with dual anti-HER2 blockade with trastuzumab and pertuzumab in early breast cancer patients with molecular HER2-enriched intrinsic subtype [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-13-03.

In intermediate risk hormone receptor (HR) positive, HER2 negative breast cancer (BC), the decision regarding adjuvant chemotherapy might be facilitated by multigene expression tests. In all, 142 intermediate risk BCs were investigated using the PAM50-based multigene expression test Prosigna® in a prospective multicentric study. In 119/142 cases, Prosigna® molecular subtyping was compared with local and two central (C1 and C6) molecular-like subtypes relying on both immunohistochemistry (IHC; HRs, HER2, Ki-67) and IHC + tumor grade (IHC+G) subtyping. According to local IHC, 35.4% were Luminal A-like and 64.6% Luminal B-like subtypes (local IHC+G subtype: 31.9% Luminal A-like; 68.1% Luminal B-like). In contrast to local and C1 subtyping, C6 classified >2/3 of cases as Luminal A-like. Pairwise agreement between Prosigna® subtyping and molecular-like subtypes was fair to moderate depending on molecular-like subtyping method and center. The best agreement was observed between Prosigna® (53.8% Luminal A; 44.5% Luminal B) and C1 surrogate subtyping (Cohen’s kappa = 0.455). Adjuvant chemotherapy was suggested to 44.2% and 88.6% of Prosigna® Luminal A and Luminal B cases, respectively. Out of all Luminal A-like cases (locally IHC/IHC+G subtyping), adjuvant chemotherapy was recommended if Prosigna® testing classified as Prosigna® Luminal A at high / intermediate risk or upgraded to Prosigna® Luminal B.

Purpose: Although optimal treatment in early triple-negative breast cancer (TNBC) remains unclear, de-escalated chemotherapy appears to be an option in selected patients within this aggressive subtype. Previous studies have identified several pro-immune factors as prognostic markers in TNBC, but their predictive impact regarding different chemotherapy strategies is still controversial. Experimental Design: ADAPT-TN is a randomized neoadjuvant multicenter phase II trial in early patients with TNBC (n = 336) who were randomized to 12 weeks of nab-paclitaxel 125 mg/m2 + gemcitabine or carboplatin d 1,8 q3w. Omission of further (neo-) adjuvant chemotherapy was allowed only in patients with pathological complete response [pCR, primary endpoint (ypT0/is, ypN0)]. Secondary invasive/distant disease-free and overall survival (i/dDFS, OS) and translational research objectives included quantification of a predictive impact of markers regarding selection for chemotherapy de-escalation, measured by gene expression of 119 genes (including PAM50 subtype) by nCounter platform and stromal tumor-infiltrating lymphocytes (sTIL). Results: After 60 months of median follow-up, 12-week-pCR was favorably associated (HR, 0.24; P = 0.001) with 5y-iDFS of 90.6% versus 62.8%. No survival advantage of carboplatin use was observed, despite a higher pCR rate [HR, 1.04; 95% confidence interval (CI), 0.68–1.59]. Additional anthracycline-containing chemotherapy was not associated with a significant iDFS advantage in pCR patients (HR, 1.29; 95% CI, 0.41–4.02). Beyond pCR rate, nodal status and high sTILs were independently associated with better iDFS, dDFS, and OS by multivariable analysis. Conclusions: Short de-escalated neoadjuvant taxane/platinum-based combination therapy appears to be a promising strategy in early TNBC for using pCR rate as an early decision point for further therapy (de-) escalation together with node-negative status and high sTILs. See related commentary by Sharma, p. 4840

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, < 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 < 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 < 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 & 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.

Invasive lobular breast carcinoma (ILC) is the second most common breast carcinoma (BC) subtype and is mainly driven by loss of E-cadherin expression. Correct classification of BC as ILC is important for patient treatment. This study assessed the degree of agreement among pathologists for the diagnosis of ILC. Two sets of hormone receptor (HR)-positive/HER2-negative BCs were independently reviewed by participating pathologists. In set A (61 cases), participants were provided with hematoxylin/eosin (HE)-stained sections. In set B (62 cases), participants were provided with HE-stained sections and E-cadherin immunohistochemistry (IHC). Tumor characteristics were balanced. Participants classified specimens as non-lobular BC versus mixed BC versus ILC. Pairwise inter-observer agreement and agreement with a pre-defined reference diagnosis were determined with Cohen's kappa statistics. Subtype calls were correlated with molecular features, including CDH1/E-cadherin mutation status. Thirty-five pathologists completed both sets, providing 4,305 subtype calls. Pairwise inter-observer agreement was moderate in set A (median κ = 0.58, interquartile range [IQR]: 0.48-0.66) and substantial in set B (median κ = 0.75, IQR: 0.56-0.86, p

Background: De-escalating strategies seem promising in HER2-positive early breast cancer (EBC) and chemo-free regimens are thus of key interest. Recent data have underlined the role of tumor immunogenicity in response to de-escalated neoadjuvant anti-HER2 therapy. Therefore, the prospective single arm hypothesis-generating phase II KEYRICHED-1 trial (NCT03988036) investigates the pCR-rate in patients with HER2-enriched EBC receiving four cycles of the dual anti-HER2 blockade in combination with the checkpoint inhibitor pembrolizumab. Initial studies with dual antibody-based HER2 blockade alone were able to achieve pCR-rates of 20-40%, which did not quite match the pCR-rates obtained with concurrent chemotherapy. KEYRICHED-1 aims at achieving pCR-rates comparable to standard chemotherapy-containing regimens by incorporating appropriate molecular selection and immune oncology.. Methods: A total of 48 pre- and postmenopausal patients with newly diagnosed HER2 2+ or 3+ EBC (stage I-III) and HER2-enriched (HER2-E) subtype by PAM50 were enrolled in this single-arm study. All patients received four cycles of study treatment with pembrolizumab (200mg), trastuzumab biosimilar (Trazimera®, loading dose 8mg/kg bodyweight (BW), maintenance dose 6mg/kg BW), and pertuzumab (loading dose 840mg/kg BW, maintenance dose 420mg/kg BW) q21d. Primary endpoint was centrally confirmed pCR (ypT0/is, ypN0). The trial was planned as a Simon's two-stage design (null and alternative pCR were 40% and 60%); interim analysis after 16 patients had to show a pCR rate of at least 50% to continue recruitment.. Results: Between 05/2020 and 03/2021, 98 patients were screened. N=52 (55%) had HER2-E subtype, of whom 48 patients entered the treatment phase. Median patient age was 57 years (28-83). 65% had tumors > 2 cm and 30% positive lymph node status. Centrally confirmed pCR-rate in surgical specimens was 46% (95% CI 0.31-0.62) in the 43 patients of the per-protocol-population, and 52% (95%CI 0.37-0.67) in all 46 evaluable patients (local assessment; two pCRs verified only by core biopsy) (p=0.22 and p=0.06 for null hypothesis, respectively). Despite HER2-E subtype, no pCR was observed in the four patients with immunohistochemical (IHC) HER2 2+/ISH-positive status in contrast to 20/39 (51.2%) pCR in IHC HER2 3+ tumors. Centrally confirmed pCR-rate in HR+/HER2+ tumors was 38.5% compared to 58.5% in HR-/HER2+ tumors. No new safety signals were observed.. Conclusions: These are the first results of a neoadjuvant chemotherapy-free 12-week de-escalation anti-HER2-regimen with trastuzumab and pertuzumab in combination with the PD-1 inhibitor pembrolizumab in patients with a HER2-E EBC. In the context of the WSG ADAPT HER2+ de-escalation trials the observed pCR-rates compare favorably in HR+ as well as HR- HER2+ EBC. Moreover, KEYRICHED-1 demonstrates that with appropriate molecular patient selection clinically meaningful pCR-rates in the range of those obtained with longer, more toxic chemotherapy-containing regimens can be achieved. Citation Format: Sherko Kuemmel, Oleg Gluz, Mattea Reinisch, Athina Kostara, Iris Scheffen, Monika Graeser, Rachel Wuerstlein, Ulrike Nitz, Kerstin Luedtke-Heckenkamp, Andreas Hartkopf, Felix Hilpert, Angela Kentsch, Carsten Ziske, Reinhard Depenbusch, Michael Braun, Jens Blohmer, Christine zu Eulenburg, Matthias Christgen, Stephan Bartels, Hans Kreipe, Enrico Pelz, Peter Schmid, Nadia Harbeck. Keyriched-1- A prospective, multicenter, open label, neoadjuvant phase ii single arm study with pembrolizumab in combination with dual anti-HER2 blockade with trastuzumab and pertuzumab in early breast cancer patients with molecular HER2-enriched intrinsic subtype [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD10-11.

BRCA1 is an important protein in the repair of DNA double strand breaks (DSBs), which are induced by alkylating chemotherapy. A BRCA1-like DNA copy number signature derived from tumors with a BRCA1 mutation is indicative for impaired BRCA1 function and associated with good outcome after high dose (HD) and tandem HD DSB inducing chemotherapy. We investigated whether BRCA1-like status was a predictive biomarker in the WSG AM 01 trial. WSG AM 01 randomized high-risk breast cancer patients to induction (2× epirubicin-cyclophosphamide) followed by tandem HD chemotherapy with epirubicin, cyclophosphamide and thiotepa versus dose dense chemotherapy (4× epirubicin-cyclophospamide followed by 3× cyclophosphamide-methotrexate-5-fluorouracil). We generated copy number profiles for 143 tumors and classified them as being BRCA1-like or non-BRCA1-like. Twenty-six out of 143 patients were BRCA1-like. BRCA1-like status was associated with high grade and triple negative tumors. With regard to event-free-survival, the primary endpoint of the trial, patients with a BRCA1-like tumor had a hazard rate of 0.2, 95% confidence interval (CI): 0.07-0.63, p = 0.006. In the interaction analysis, the combination of BRCA1-like status and HD chemotherapy had a hazard rate of 0.19, 95% CI: 0.067-0.54, p = 0.003. Similar results were observed for overall survival. These findings suggest that BRCA1-like status is a predictor for benefit of tandem HD chemotherapy with epirubicin-thiotepa-cyclophosphamide.