Lorenza Mittempergher

PURPOSE: Neoadjuvant immunotherapy (IO) has become the standard of care for early-stage triple-negative breast cancer (TNBC), but not yet for other subtypes. We previously developed a clinical-grade mRNA-based immune classifier (ImPrint) predicting response to IO that is now being used in I-SPY2.2 as part of the response predictive subtypes. We report the performance of ImPrint in hormone receptor–positive and human epidermal growth factor receptor 2–negative (HR+HER2–) patients from five IO arms. METHODS: A total of 204 HR+HER2– (MammaPrint high-risk) patients from five IO arms (anti–PD-1, anti–PD-L1/poly [ADP-ribose] polymerase inhibitor combination, anti–PD-1/toll-like receptor 9 dual-IO combination, and anti–PD-1 ± lymphocyte activation gene 3 dual-IO combination) and 191 patients from the chemotherapy-only control arm were included in this analysis. Patients were classified as ImPrint+ (likely sensitive) versus ImPrint– (likely resistant), using pretreatment mRNA. Performance of ImPrint for predicting pathologic complete response (pCR) to IO-containing arms was characterized and compared with tumor grade (III), MammaPrint (ultra) High2 risk (MP2), and estrogen receptor (ER)-low (ER ≤ 10%). RESULTS: Overall, the pCR rate across the five IO arms was 33%. 26% of HR+HER2– patients were ImPrint+, and pCR rates with IO were 75% in ImPrint+ versus 17% in ImPrint–, with the highest pCR rate >90% in a dual-IO arm. In the control arm, pCR rates were 33% in ImPrint+ and 8% in ImPrint–. Tumor grade (III), MP2, and ER-low showed pCR rates in IO of 45%, 56%, and 63%, respectively, with lower pCR odds ratios (OR < 7.5) compared with ImPrint (OR = 14.5). CONCLUSION: Using an accurate selection strategy, HR+HER2– patients could achieve pCR rates similar to what is seen with best neoadjuvant therapies in TNBC and HER2+ (ie, pCR rate >65%-70%). ImPrint, an Food and Drug Administration IDE-enabled assay, may represent a way to identify HR+HER2– patients for IO that best balances likely benefit versus risk of serious immune-related adverse events.

Tumor-infiltrating lymphocytes (TILs) have been associated with outcomes in HER2-positive breast cancer patients treated with neoadjuvant chemotherapy and trastuzumab. However, it remains unclear if TILs could be a prognostic and/or predictive biomarker in the context of dual HER2-targeting treatment. In this study, we evaluated the association between TILs and pathological response (pCR) and invasive-disease free survival (IDFS) in 389 patients with stage II-III HER2 positive breast cancer who received neoadjuvant anthracycline-containing or anthracycline-free chemotherapy combined with trastuzumab and pertuzumab in the TRAIN-2 trial. Although no significant association was seen between TILs and pCR, patients with TIL scores ≥60% demonstrated an excellent 3-year IDFS of 100% (95% CI 100–100), regardless of hormone receptor status, nodal stage and attainment of pCR. Additionally, in patients with hormone receptor positive disease, TILs as a continuous variable showed a trend to a positive association with pCR (adjusted Odds Ratio per 10% increase in TILs 1.15, 95% CI 0.99–1.34, p = 0.070) and IDFS (adjusted Hazard Ratio per 10% increase in TILs 0.71, 95% CI 0.50–1.01, p = 0.058). We found no interactions between TILs and anthracycline treatment. Our results suggest that high TIL scores might be able to identify stage II-III HER2-positive breast cancer patients with a favorable prognosis.

PURPOSE: At the primary analysis, the APHINITY trial reported a statistically significant but modest benefit of adding pertuzumab to standard adjuvant chemotherapy plus trastuzumab in patients with histologically confirmed human epidermal growth factor receptor 2 (HER2)–positive early-stage breast cancer. This study evaluated whether the 80-gene molecular subtyping signature (80-GS) could identify patients within the APHINITY population who derive the most benefit from dual anti-HER2 therapy. METHODS: In a nested case-control study design of 1,023 patients (matched event to control ratio of 3:1), the 80-GS classified breast tumors into functional luminal type, HER2 type, or basal type. Additionally, 80-GS distinguished tumor subtypes that exhibited a single-dominant functional pathway versus tumors with multiple activated pathways. The primary end point was invasive disease-free survival (IDFS). Hazard ratios (HRs) were evaluated by Cox regression. After excluding patients without appropriate consent and those with missing data, 964 patients were included. RESULTS: The 80-GS classified 50% (n = 479) of tumors as luminal type, 28% (n = 275) as HER2 type, and 22% (n = 209) as basal type. Most luminal-type tumors (86%) displayed a single-activated pathway, whereas 49% of HER2-type and 42% of basal-type tumors were dual activated. There was no significant difference in IDFS among different conventional 80-GS subtypes (single- and dual-activated subtypes combined). However, basal single-subtype tumors were significantly more likely to have an IDFS event (hazard ratio, 1.69 [95% CI, 1.12 to 2.54]) compared with other subtypes. HER2 single-subtype tumors displayed a trend toward greater beneficial effect on the addition of pertuzumab (hazard ratio, 0.56 [95% CI, 0.27 to 1.16]) compared with all other subtypes. CONCLUSION: The 80-GS identified subgroups of histologically confirmed HER2-positive tumors with distinct biological characteristics. Basal single-subtype tumors exhibit an inferior prognosis compared with other subgroups and may be candidates for additional therapeutic strategies. Preliminary results suggest patients with HER2-positive, genomically HER2 single-subtype tumors may particularly benefit from added pertuzumab, which warrants further investigation.

Extracellular-matrix features distinguish between long-term versus shorter-term survivors with OligoMBC.

Purpose: BluePrint (BP) is an 80-gene molecular subtyping test that classifies early-stage breast cancer (EBC) into Basal, Luminal, and HER2 subtypes. In most cases, breast tumors have one dominant subtype, representative of a single activated pathway. However, some tumors show a statistically equal representation of more than one subtype, referred to as dual subtype. This study aims to identify and examine dual subtype tumors by BP to understand their biology and possible implications for treatment guidance. Methods: The BP scores of over 15,000 tumor samples from EBC patients were analyzed, and the differences between the highest and the lowest scoring subtypes were calculated. Based upon the distribution of the differences between BP scores, a threshold was determined for each subtype to identify dual versus single subtypes. Results: Approximately 97% of samples had one single activated BluePrint molecular subtype, whereas ~ 3% of samples were classified as BP dual subtype. The most frequently occurring dual subtypes were the Luminal-Basal-type and Luminal-HER2-type. Luminal-Basal-type displays a distinct biology from the Luminal single type and Basal single type. Burstein’s classification of the single and dual Basal samples showed that the Luminal-Basal-type is mostly classified as ‘luminal androgen receptor’ and ‘mesenchymal’ subtypes, supporting molecular evidence of AR activation in the Luminal-Basal-type tumors. Tumors classified as Luminal-HER2-type resemble features of both Luminal-single-type and HER2-single-type. However, patients with dual Luminal-HER2-type have a lower pathological complete response after receiving HER2-targeted therapies in addition to chemotherapy in comparison with patients with a HER2-single-type. Conclusion: This study demonstrates that BP identifies tumors with two active functional pathways (dual subtype) with specific transcriptional characteristics and highlights the added value of distinguishing BP dual from single subtypes as evidenced by distinct treatment response rates.

Background: The addition of pertuzumab to neoadjuvant trastuzumab-containing chemotherapy significantly improves pathological complete response (pCR) rates in patients with HER2-positive breast cancer. However, effects on long-term survival outcomes have been modest. While prior studies suggest that only a subset of patients benefits most from the addition of pertuzumab, a reliable biomarker for response prediction has not been established. BluePrint is an 80-gene molecular subtyping test that classifies breast tumors as Basal-, Luminal- or HER2-subtype. Recent data shows that some tumors have more than one activated pathway and can be classified as dual subtype. Secondary analyses from the Aphinity trial (NCT01358877) suggest that pertuzumab benefit is largely restricted to patients with single-activated HER2-type tumors and less pronounced in patients with other single- or dual-activated subtypes. In this study, we evaluated BluePrint subtype as a biomarker for predicting response to trastuzumab-containing neoadjuvant chemotherapy with or without pertuzumab in a large nationwide cohort of patients. Methods: We collected data of all patients with stage II-III HER2-positive breast cancer who received neoadjuvant chemotherapy and trastuzumab between January 2013 and January 2016 from the Netherlands Cancer Registry. Pertuzumab was not reimbursed in the Netherlands during this period but was available to patients participating in the TRAIN2 clinical trial (NCT01996267). Pre-treatment biopsies were collected and tumor cell percentage was adequate in 404 patients who received pertuzumab and in 432 matched control patients who did not receive pertuzumab. Matching factors included treatment with anthracyclines (yes vs. no), age and hormone receptor status. BluePrint molecular subtypes were determined sucessfully through microarray in 680 patients. Pathological complete response (pCR) and overall survival (OS) were analyzed by treatment arm and BluePrint subtypes, using logistic and Cox regression analyses. Results: Microarray data was available for 334 patients who received pertuzumab and 346 patients who did not. Tumors were classified as single HER2 (77.6%), Luminal-HER2 (11.3%), single Luminal (8.4%), or other (2.6%). Overall, pCR rate was 65.0% in patients who received pertuzumab and 38.9% in patients who did not. In patients with single HER2-type tumors, pCR rate was significantly higher with pertuzumab (77.0% vs. 46.0%, multivariate adjusted odds ratio [aOR] 3.92, 95% confidence interval [CI] 2.60 - 5.98). In patients with other subtypes, pCR rate was lower overall and not significantly different with or without pertuzumab (23.5% vs. 13.5%, aOR 2.50, 95% CI 0.98 - 6.89). In addition, multivariate Cox regression analyses showed a significant OS benefit with pertuzumab in patients with single HER2-type tumors (adjusted hazard ratio [aHR] 0.33, 95% CI 0.16 - 0.67), but not in other subtypes (aHR 0.94, 95% CI 0.31 - 2.83). However, tests for interactions between pertuzumab and BluePrint subtypes were not significant. Conclusion: We confirmed the results of a prior secondary analysis of the Aphinity trial that the benefit of adding pertuzumab to (neo)adjuvant trastuzumab-based chemotherapy seems most pronounced in patients with a molecularly defined single-activated HER2-subtype. In other subtypes, pathological complete response rates and long-term outcomes are worse overall and no clear benefit of pertuzumab was seen, although tests for interaction between pertuzumab treatment and BluePrint subtype were not significant. Citation Format: Marte C Liefaard, Anna van der Voort, Joyce Sanders, Shiva Vonk, Hugo M Horlings, Sabine Siesling, Linda de Munck, Elise van Leeuwen-Stok, Miranda Kleijn, Lorenza Mittempergher, Midas M Kuilman, Esther H Lips, Gabe S Sonke. Effect of pertuzumab plus neoadjuvant trastuzumab-based chemotherapy in early-stage HER2-positive breast cancer according to BluePrint molecularly defined breast cancer subtypes [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 PD15-07.

MammaPrint® (MP) is a 70‐gene signature that stratifies early‐stage breast cancer patients into low‐ and high risk of distant relapse. Further stratification of MP risk results identifies four risk subgroups, ultra‐low (UL), low, high 1, and high 2, with specific prognostic and predictive outcomes. BluePrint® (BP) is an 80‐gene signature that classifies breast tumors as basal, luminal, or HER2 molecular subtype. To gain insight into their biological significance, we annotated the MP 70‐ and BP 80‐genes with respect to the 10 hallmarks of cancer (HoC). Furthermore, we related gene expression profiles of the extreme ends of the MP low‐ and high‐risk patients (here called, ultra‐low (UL) and ultra‐high (UH) or High2, respectively), to the 10 HoC per BP subtype by differential gene expression and pathway analysis. MP and BP gene functions reflected all 10 HoCs. Most MP and BP genes were associated with sustaining proliferative signaling, followed by genome instability and mutation categories. Based on the gene expression profiles, UL and UH subgroup pathways were down ‐or upregulated, respectively, reflecting proliferative and metastatic features, such as G2M checkpoint, DNA repair, oxidative phosphorylation, immune invasion, PI3K/AKT/mTOR signaling, and hypoxia pathways. Notably, the UH HER2‐type was enriched in several immune signaling pathways, such as IL2/STAT5 signaling and TNFα signaling via NFκB. Our results show that MP and BP gene signatures represent and capture all 10 HoCs and highlight underlying biological processes of MP extreme samples, which might guide treatment decisions as the signature captures the full spectrum of early breast cancers.

PIK3CA mutations are the most common in breast cancer, particularly in the estrogen receptor positive cohort, but the benefit of PI3K inhibitors has had limited success compared with approaches targeting other less common mutations. We found allelic imbalances in the expression of PIK3CA in normal breast tissue and mapped a germline candidate regulatory variant. An imbalance was also frequently observed in the expression of the missense mutant and wild-type PIK3CA alleles in breast tumors from METABRIC and TCGA projects. Moreover, although 60% of tumors preferentially expressed the mutant allele, 10% did preferentially express the wild-type allele. Mechanistically, we show that these imbalances are more frequently due to regulatory variants in cis than altered copy-number and predict that somatic variants have a more significant role than germline ones. We further found that imbalanced allelic expression between mutant and wild-type alleles due to cis- regulatory variants associated with poor prognosis ( p =0.0081). Interestingly, ER ⁺ , PR ⁺ , and Her2 ⁻ tumors expressing very low levels of the mutant allele had the poorest prognosis (DSS + and PR ⁺ tumors and − tumors). Hence, our work provides compelling evidence to support the clinical utility of PIK3CA allelic expression in breast cancer in identifying this cohort of low mutant allele expressing patients of poorer prognosis, who will unlikely benefit from PI3K inhibitors. Furthermore, our work establishes a new model of differential regulation of critical cancer-promoting genes.