Genomic Complexity Profiling Reveals That HORMAD1 Overexpression Contributes to Homologous Recombination Deficiency in Triple-Negative Breast Cancers

UnlabelledTriple-negative breast cancers (TNBC) are characterized by a wide spectrum of genomic alterations, some of which might be caused by defects in DNA repair processes such as homologous recombination (HR). Despite this understanding, associating particular patterns of genomic instability with response to therapy has been challenging. Here, we show that allelic-imbalanced copy-number aberrations (AiCNA) are more prevalent in TNBCs that respond to platinum-based chemotherapy, thus providing a candidate predictive biomarker for this disease. Furthermore, we show that a high level of AiCNA is linked with elevated expression of a meiosis-associated gene, HORMAD1. Elevated HORMAD1 expression suppresses RAD51-dependent HR and drives the use of alternative forms of DNA repair, the generation of AiCNAs, as well as sensitizing cancer cells to HR-targeting therapies. Our data therefore provide a mechanistic association between HORMAD1 expression, a specific pattern of genomic instability, and an association with response to platinum-based chemotherapy in TNBC.SignificancePrevious studies have shown correlation between mutational "scars" and sensitivity to platinums extending beyond associations with BRCA1/2 mutation, but do not elucidate the mechanism. Here, a novel allele-specific copy-number characterization of genome instability identifies and functionally validates the inappropriate expression of the meiotic gene HORMAD1 as a driver of HR deficiency in TNBC, acting to induce allelic imbalance and moderate platinum and PARP inhibitor sensitivity with implications for the use of such "scars" and expression of meiotic genes as predictive biomarkers

HER2-enriched subtype and novel molecular subgroups drive aromatase inhibitor resistance and an increased risk of relapse in early ER+/HER2+ breast cancer

BACKGROUND: Oestrogen receptor positive/ human epidermal growth factor receptor positive (ER+/HER2+) breast cancers (BCs) are less responsive to endocrine therapy than ER+/HER2- tumours. Mechanisms underpinning the differential behaviour of ER+HER2+ tumours are poorly characterised. Our aim was to identify biomarkers of response to 2 weeks’ presurgical AI treatment in ER+/HER2+ BCs. METHODS: All available ER+/HER2+ BC baseline tumours (n=342) in the POETIC trial were gene expression profiled using BC360™ (NanoString) covering intrinsic subtypes and 46 key biological signatures. Early response to AI was assessed by changes in Ki67 expression and residual Ki67 at 2 weeks (Ki672wk). Time-To-Recurrence (TTR) was estimated using Kaplan-Meier methods and Cox models adjusted for standard clinicopathological variables. New molecular subgroups (MS) were identified using consensus clustering. FINDINGS: HER2-enriched (HER2-E) subtype BCs (44.7% of the total) showed poorer Ki67 response and higher Ki672wk (p<0.0001) than non-HER2-E BCs. High expression of ERBB2 expression, homologous recombination deficiency (HRD) and TP53 mutational score were associated with poor response and immune-related signatures with High Ki672wk. Five new MS that were associated with differential response to AI were identified. HER2-E had significantly poorer TTR compared to Luminal BCs (HR 2.55, 95% CI 1.14–5.69; p=0.0222). The new MS were independent predictors of TTR, adding significant value beyond intrinsic subtypes. INTERPRETATION: Our results show HER2-E as a standardised biomarker associated with poor response to AI and worse outcome in ER+/HER2+. HRD, TP53 mutational score and immune-tumour tolerance are predictive biomarkers for poor response to AI. Lastly, novel MS identify additional non-HER2-E tumours not responding to AI with an increased risk of relapse

Abstract PD15-06: Estrogen receptor expression thresholds by IHC and mRNA for Ki67 response to aromatase inhibition: A POETIC study

Abstract Background. Estrogen receptor (ER) expression is the key determinant for endocrine treatment of breast cancer (BC). In clinical practise ER analysis is currently almost entirely by immunohistochemistry (IHC). ASCO/CAP recommends cut-offs of &amp;lt;1% (negative) and 1-10% (low) to minimise the risk of false negatives. There is uncertainty in the level of responsiveness of those with low ER. mRNA assessment could be acceptable or preferable if a similar or better threshold was shown to identify response to endocrine therapies. Change of Ki67 after 2 weeks’ presurgical treatment with aromatase inhibitors (AIs) relates to reduction in risk of recurrence. We aimed to define IHC and mRNA cut-points for predicting change in Ki67 in response to AI. Method. POETIC is a phase III trial in postmenopausal patients with ER+ (by local assessment) BC randomized to receive 2-weeks’ pre-surgical AI vs no presurgical treatment. Cases were selected from the AI treatment group. All HER2+ cases were analysed. To enrich the HER2- study set for lower ER values, we selected all of the 15% poorest Ki67 suppression (ie PR, &amp;lt;40% suppression) and 30% of the remainder categorised as intermediate (IR, 40-79%) and good-responders (GR, &amp;gt;79%). 770 baseline FFPE samples were analysed (582 HER2-, 188 HER2+). IHC used the NCL-L-ER6F11 (Novocastra, Leica) antibody with the DAKO Flex kit and were scored centrally. mRNA was assessed by rtPCR. Values were expressed according to an arbitrary scale (“A units”). Results. ER IHC was available from 517 HER2- and 188 HER2+ tumors and ER mRNA from 376 HER2- and 173 HER2+ tumors. The correlation between ER levels by IHC and rtPCR was moderately high (Rho=0.616, p&amp;lt;0.00001). In IHC &amp;lt;1%, 1-10% and &amp;gt;10% tumors median (range) mRNA levels were 1.290 (0.188-11.346), 2.368 (0.575-13.863) and 111.150 (0.826-1001.423), respectively. The tables show the %age of PR, IR and GR for each category of ER by IHC or mRNA. The number of cases was adjusted to cater for the analysis of only 30% of the HER2- IR and GR , by multiplying the HER2- IR and GR number of cases by 3.3, resulting in this not being an integer for some categories. IHC: 87% of cases had &amp;gt;60% of cells +ve and only 11-12% of these were PRs. For cases with &amp;lt;10% cells +ve (4.9% of the population) over 90% of cases were PRs and there were no GRs. Cases between 1 and 10% +ve were not less responsive than those &amp;lt;1% +ve. For cases with &amp;gt;10% &amp;lt;20% cells +ve there was a substantial number of GRs and IRs (14.6% and 19.0%, respectively). mRNA: 82% of cases had ≥ 40 A units and only 10-14% of these showed PR. For cases with 5 A units (4.2% of the population) there were 9.5% % IR and no GR. In the next category, ≥5 &amp;lt;10 A units, 34.4% of cases were IR or GR. The levels of ER that distinguished PRs from IR/GRs was no different between HER2+ and HER2- cases. Conclusions. Ki67 responsiveness increases with increasing levels of ER by both IHC and mRNA. There was little responsiveness at IHC&amp;lt;10% and no distinction in responsiveness between &amp;lt;1% and 1-10% cells positive. Above 10% positive cells substantial numbers of patients showed IR or GR. Similar separation of PRs from IR/GRs was achieved by IHC and mRNA. IHC (%)0 &amp;lt; 1≥1 &amp;lt; 10≥10 &amp;lt; 20≥20 &amp;lt; 40≥40 &amp;lt; 60≥60 &amp;lt; 80≥80No. of cases42.32922.625.671.7239.81010.6PR87.596.566.462.541.811.710.8IR12.53.419.020.741.640.933.1GR0.00.014.616.816.647.456.1 mRNA (A units)&amp;lt;5≥5 &amp;lt; 10≥10 &amp;lt; 20≥20 &amp;lt; 40≥40 &amp;lt; 80≥80 &amp;lt; 120≥120No. of cases45.318.336.589.2168.1177.6550.4PR90.565.638.426.913.710.710.7IR9.416.458.947.946.835.226.8GR0.018.02.725.239.554.162.5 Citation Format: Elena Lopez Knowles, Simone Detre, Margaret Hills, Gene F Schuster, Maggie CU Cheang, Holly Tovey, Lucy Kilburn, Judith Bliss, John Robertson, Ian Smith, Mitch Dowsett, POETIC investigators. Estrogen receptor expression thresholds by IHC and mRNA for Ki67 response to aromatase inhibition: A POETIC study [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 PD15-06.</jats:p

Abstract PD15-02: HER2-enriched subtype and novel molecular subgroups drive aromatase inhibitor resistance and an increased risk of relapse in early ER+/HER2+ breast cancer

Abstract Background: Approximately 10% of estrogen receptor positive (ER+) early breast cancers (EBC) are human epidermal growth factor positive/overexpressed (HER2+). Resistance to aromatase inhibitors (AIs) is poorly understood among ER+HER2+ BC. We aimed to identify biomarkers of response and resistance using the HER2+ tumours in the POETIC trial. Methods: POETIC was a phase III trial of post-menopausal women with ER+ EBC randomized 2:1 to 2-weeks of peri-surgical AI vs control, followed by standard-of-care. Baseline formalin-fixed paraffin-embedded samples from 342 ER+HER2+ BC (237 treated/105 controls) were gene expression profiled using Breast cancer 360™ panel (NanoString) covering intrinsic subtypes and 40 key biological signatures. New molecular subgroups were identified using consensus clustering (CC).Proliferation rate was estimated as percentage of cancer-cells staining for Ki67. Ki67 response was defined by percentage-changes from baseline to surgery: poor (PR; reduction &amp;lt;50%), intermediate (IR; 50-75%) and good (GR; &amp;gt;75%). Residual Ki67 at 2 weeks timepoint (Ki672w) was defined as High (H) (≥ 10%) vs Low (L) (&amp;lt;10%). Association of molecular features with Ki67 response was assessed using logistic regression models and with time to recurrence (TTR) using multivariable Cox regression models adjusted for post-surgery clinicopathological variables: grade, tumour size, nodal status and age. Results: Ki672w was measured for all treated samples (n = 227, 48% H, 52% L) and a fraction of controls (n = 50, 96% H, 4% L). At baseline, 45% of tumours were HER2-enriched (HER2-E), 53% Luminal A/B and 2% Basal-Like. HER2-E associated with high Ki672W (84% H, 16% L) compared to non-HER2-E (29% H, 71% L, p&amp;lt;0.001). Amongst the 40 BC360 signatures, 9 were significantly associated with Ki67 response (Table). High expression of endocrine-related and apoptosis signatures associated with GR while HRD and TP53 with PR. These features were also related to Ki672W, with additional signatures involved in immune-checkpoint component and immune-enrichment (IM) (IDO1, PD-L1, IFN-gamma and T-reg) associated with high Ki672W.. Using CC, 4 molecular subgroups predicting differential response to AI were identified. Cluster (C) 1 was characterized by overexpression of IM features and low ER signaling (42%); C2 low IM but highest ERBB2 expression (15%); C3 ESR1 high and PgR low (4%) and C4 high endocrine signaling and lowest ERBB2 expression (39%). Distribution of Intrinsic subtypes varied across these molecular subgroups with the majority of C1 being HER2-E (62%) and Luminal B (27%); C2 HER2-E (74%); C3 Luminal B (64%) and C4 Luminal B (55%) and Luminal A (30%). HER2-E had significantly poorer TTR (HR 2.14; 95%CI1.11-4.17; p=0.0224) compared to Luminal tumours, remaining significant (HR 2.55, 95%CI1.14-5.69; p=0.0222) in the multivariable analysis. C2 (HR 4.67, 95%CI 1.90-11.51; p&amp;lt;0.001) and C4 (HR 2.42, 95%CI1.05-4.5.58; p=0.0385) were independent predictors of shorter TTR compared to C1, adding significant value beyond intrinsic subtypes (Likelihood ratio test, p=0.00429). Conclusions: Our results confirm that HER2-E, TP53 mutation, HRD and IM features are the main components driving poor early response to AI in ER+/HER2+ EBC. Novel molecular subgroups identify additional non-HER-E tumours not responding to AI with an increased risk of relapse. The appropriate additional treatment warrants further investigation. List of significant gene expression signatures associated with response as defined by reduction of Ki67. Odds ratio (OR) were determined by ordinal logistic regression analyses. OR: &amp;lt;1 indicating associated with good response and &amp;gt; 1.0 associated with worse response.Odds Ratiop-valuep-adjusted (False Discovery Rate)ESR10.65&amp;lt; 0.001&amp;lt; 0.001Estrogen Receptor Signaling0.44&amp;lt; 0.001&amp;lt; 0.001ERBB21.52&amp;lt; 0.001&amp;lt; 0.001TP53 mutant signature2.65&amp;lt; 0.001&amp;lt; 0.001FOXA10.42&amp;lt; 0.001&amp;lt; 0.001Apoptosis0.26&amp;lt; 0.0010.00228Homologous Recombination Deficiency (HRD)2.33&amp;lt; 0.0010.00349PgR0.820.001480.00737Hypoxia2.180.001660.00737 Citation Format: Milana A Bergamino Sirvén, Elena López-Knowles, Gabriele Morani, Holly Tovey, Lucy Kilburn, Chris Holcombe, Anthony Skene, Ian Smith, John Robertson, Gene Schuster, Judith M Bliss, Mitch Dowsett, Maggie CU Cheang, POETIC investigators. HER2-enriched subtype and novel molecular subgroups drive aromatase inhibitor resistance and an increased risk of relapse in early ER+/HER2+ breast cancer [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 PD15-02.</jats:p