PURPOSE:Patients with breast cancer in Ethiopia are generally diagnosed with late-stage disease, and there is a high proportion of young female and male patients. The role of genetic predisposition is yet unknown in this setting. To increase the knowledge about hereditary breast cancer in Ethiopia, this study investigated germline pathogenic variants (PVs) in breast cancer-predisposing genes in a high-risk cohort of young women and men with breast cancer. MATERIALS AND METHODS:One hundred women (age 18-39) and men (all ages) diagnosed with breast cancer at the Tikur Anbessa Specialized Hospital, Addis Ababa, were included. Basic patient and tumor characteristics, and family history were collected, and blood samples drawn. DNA was extracted locally before transport to Lund University, Sweden, for DNA analysis using next-generation sequencing. Type and frequency of PVs in breast cancer-related genes (ATM, BARD1, BRCA1, BRCA2, CDH1, CHEK2, PALB2, PTEN, RAD51C, RAD51D, STK11, and TP53) were analyzed. RESULTS:A high proportion of the 89 study participants with successful genetic analysis carried PVs in breast cancer susceptibility genes (23.6%; 95% CI 15.2 to 33.8). In total, 22 PVs were detected in BRCA1 (n = 7), BRCA2 (n = 8), PALB2 (n = 4), BARD1 (n = 1), PTEN (n = 1), and ATM (n = 1). Potential founder variants and two novel PVs were found (BRCA1 c.5278-864_5332+621del and PALB2 c.1169_1170del). CONCLUSION:Genetic predisposition plays an important role among young women and men in the study setting, with nearly one in four patients carrying a PV. The results call for further research and interventions targeting individuals at high risk, with the long-term potential of reduced morbidity and mortality.

There are two text errors in the original publication [...]

ABSTRACT Background Population-scale molecular profiling integrated into routine healthcare could accelerate biomarker discovery, validation, and implementation, but the feasibility and sustainability of such an approach have rarely been demonstrated prospectively. The Sweden Cancerome Analysis Network - Breast (SCAN-B) Initiative was established to integrate prospective molecular profiling with population-based breast cancer care and create an infrastructure for translating molecular discoveries into clinical practice ( ClinicalTrials.gov identifier NCT02306096 ). Methods We evaluated the first 10 full calendar years of SCAN-B, encompassing patients with primary invasive breast cancer enrolled between August 30, 2010 and December 31, 2020. Enrollment and biospecimen collection were compared with all eligible breast cancer diagnoses in participating hospitals to assess population coverage and representativeness. Clinicopathological characteristics, treatments, recurrence-free survival, overall survival, RNA-sequencing-based molecular subtypes and risk-of-recurrence, and somatic mutations were evaluated. We additionally report the translation of SCAN-B molecular profiling from the research setting into routine clinical diagnostics. Results Among 16,381 estimated eligible breast cancer diagnoses, 13,940 patients (85.1%) were prospectively enrolled across participating Swedish hospitals. Baseline blood samples were obtained from 98.4% of enrolled patients and tumor specimens from 71.1%; 9,323 tumors (94.0% of submitted tumor specimens) underwent RNA-sequencing. The enrolled cohort was broadly representative of the underlying breast cancer population across major clinicopathological characteristics. Integration of longitudinal clinical data with molecular profiling enabled characterization of real-world treatment patterns, long-term outcomes, molecular subtypes, risk-of-recurrence, and the somatic mutational landscape in this population-based cohort. Building on prospective real-time RNA-sequencing and subsequent development and validation of single-sample molecular subtype and risk-of-recurrence predictors, the SCAN-B workflow was transferred into routine clinical molecular diagnostics in Skåne and Blekinge in 2021. Through January 2026, more than 3,000 patients had received clinical RNA-sequencing-based molecular subtype and risk-of-recurrence reports, while prospective SCAN-B enrollment and transfer of samples and molecular data into the research infrastructure continued. Patient enrollment continues prospectively, with over 23,000 patients accrued as of January 2026. Conclusions A prospective, population-based molecular profiling program can be integrated into routine breast cancer care at scale while maintaining high population coverage and representativeness. Over more than a decade, SCAN-B progressed from prospective biosampling and molecular profiling through biomarker development and validation to implementation of RNA sequencing-based testing in routine healthcare. This model establishes a continuous framework linking population-based molecular research, biomarker discovery and validation, and clinical implementation, and provides a strategy for integrating precision oncology research with routine cancer care. Trial registration ClinicalTrials.gov identifier NCT02306096

ABSTRACT Thyroid function has been suggested to be associated with breast cancer risk, but not much is known about thyroid hormones as a potential prognostic factor for breast cancer. This study investigated the relation between thyroid hormones in breast cancer patients and mortality. A total of 2000 breast cancer patients from the Swedish SCAN‐B cohort were followed for 9 years. Blood samples were collected at the time of diagnosis, before surgery, and analysed for free and total T3 and T4. All‐cause mortality and recurrent disease were compared between quartiles (Q) using Cox proportional hazard analysis adjusted for prognostic factors, yielding hazard ratios (HR) with 95% confidence intervals (CIs). During follow‐up, 310 deaths and 167 recurrences occurred. Free T3 was associated with a lower mortality; the combined group Q2 to Q4 had an adjusted HR of 0.73 (0.58–0.93) as compared to Q1. Moreover, a high ratio of free T3/T4 was associated with a low mortality in Q2, Q3 and Q4, respectively. High levels of free and total T4 were associated with a high risk of all‐cause mortality; the crude HR for free T4 Q4 vs. Q1 was 1.53 (1.14–2.07) and the corresponding HR for total T4 was 1.63 (1.20–2.22). These results did not remain in the adjusted analysis. No clear associations were found regarding recurrent disease. We conclude that high levels of free T3, and a high free T3/T4 ratio were associated with a lower risk of all‐cause mortality and may be a marker of a favourable prognosis.

Persistent xcirculating tumor DNA (ctDNA) during neoadjuvant treatment (NAT) of early breast cancer (EBC) indicates high-risk disease. Similarly, detection of ctDNA post-resection indicates molecular residual disease (MRD) and impending relapse. For ctDNA to be integrated into EBC management, accessible and scalable diagnostics are required. Here we apply an ultrasensitive, personalized tumor-informed approach to ctDNA evaluation predicated on analyses of structural variants (SVs) using a novel digital PCR (dPCR) multiplex SV technology. 136 patients eligible for NAT (29.4 For 136 patients with early breast cancer, 1497 plasma samples were collected during neoadjuvant therapy and post-operative adjuvant therapy follow-up (median 6.5 years) and analyzed for ctDNA using an ultrasensitive tumor-informed structural variant-based personalized multiplex dPCR approach. For 136 patients with early breast cancer, 1497 plasma samples were collected during neoadjuvant therapy and post-operative adjuvant therapy follow-up (median 6.5 years) and analyzed for ctDNA using an ultrasensitive tumor-informed structural variant-based personalized multiplex dPCR approach.

BACKGROUND:Homologous recombination deficiency (HRD) originating from inactivation of genes like BRCA1/BRCA2 is a targetable abnormality common in triple-negative breast cancer (TNBC). In estrogen-receptor (ER)-positive HER2-negative (ERpHER2n) breast cancer (BC), HRD prevalence and clinical impact are unclear. METHODS:We analyzed 502 ERpHER2n tumors from patients recruited via the population-representative Swedish SCAN-B study by whole genome sequencing (WGS), defining mutational signatures-based HRD, as well as matched transcriptional, DNA methylation, clinicopathological, adjuvant treatment, and outcome data. RESULTS:We show that HRD is much less frequent in ERpHER2n BC (8.4%) compared to TNBC, though induced by similar genetic/epigenetic mechanisms acting on mainly BRCA1/BRCA2/RAD51C/PALB2 together, providing a plausible HR-inactivation mechanism for 71.4% of HRD tumors. Our modelled estimate of HRD in Western European/Nordic BC is ~10-13%. HRD tumors were observed across all PAM50 gene expression subtypes with the exception of Luminal A tumors ( < 1%) and did not exhibit a unique, defining transcriptional or DNA methylation profile. While HRD status was not statistically associated with differences in patient outcome for patients treated with combined chemotherapy and endocrine therapy, a nonsignificant trend of poorer outcome for patients with HRD tumors was observed for patients treated with adjuvant endocrine therapy only. CONCLUSIONS:ERpHER2n HRD tumors show features of aggressive disease, but do not display a distinct transcriptional or DNA methylation profile that clearly differentiates them from HR-proficient tumors. Though numbers are limited, we present early evidence that HRD stratification by WGS could impact therapeutic strategies, as HRD BCs trended to poorer outcomes when not treated with chemotherapy.

10567 Background: Breast cancer incidence is rapidly increasing in low-and-middle-income countries (LMICs), where access to care is limited and survival outcome is poor. Young women and men are overrepresented among breast cancer (BC) patients diagnosed in LMICs in Sub-Saharan Africa (SSA), for reasons not yet fully understood. As hereditary cancer is more common in young women and men with BC, genetic factors may play a significant role. Even though carriers of germline pathogenic variants (PV) in the genes BRCA1 , BRCA2 , and PALB2 have a very high risk of BC, studies of PV in these genes are very limited in SSA. In order to increase knowledge, this study investigated the prevalence of PV in high-risk BC susceptibility genes in young women and men diagnosed with BC in Ethiopia. Methods: This is a descriptive cross-sectional study. One-hundred young women (age 18-39) and men (all ages) diagnosed with invasive BC were included from Departments of Oncology and Surgery at Tikur Anbessa Specialized Hospital, Addis Ababa, Ethiopia. Potential participants were given oral and written information by a trained physician, and those consenting were included in the study. Basic patient- and tumor characteristic as well as information about family history was collected. DNA was extracted from blood samples, before shipment to BRCAlab, Lund University, Sweden for genetic analysis of genes BRCA1 , BRCA2 and PALB2 , using a gene panel and next generation sequencing on an Illumina platform. Results: Genetic analysis results were available for 89 study patients. There was a high proportion (21.3%) of PV in tested genes. In total, 19 PV were found in BRCA1 (n = 7), BRCA2 (n = 8) and PALB2 (n = 4). One of the PV was in a male. There were five individuals with an identical PV in BRCA1 (c.4524G > A, NM_007294.3), three individuals with identical PV in BRCA2 (c.5159C > A, NM_000059.3), and two individuals with identical PV in PALB2 (c.1216delG, NM_024675.4). Two novel PV not previously reported in literature were found, BRCA1 c.5278-864_5332+621del, NM_007294.3 and PALB2 c.1169_1170del, NM_024675.4. Conclusions: This study demonstrates that germline PV in BRCA1 , BRCA2 and PALB2 are common among young women and men diagnosed with BC in Ethiopia, with over 1 out of 5 patients carrying a PV. Genetic predisposition appears to play an important role in the tumor genesis in the studied group. Multiple patients carried identical PV, which could indicate that the detected PV are founder variants. Since the majority of the patients in Ethiopia are young, and male BC seem more prevalent compared to in western countries, efforts directed to these groups and development of services for genetic testing and follow-up programs for carriers of PV should be further emphasized. This approach has the potential to reduce BC incidence, morbidity and mortality through increased awareness, risk-reducing procedures and earlier cancer detection.

Background: The immune cell component of the tumor microenvironment is an important modulator of tumor progression and suppression. In patients with breast cancer, tumor-infiltrating lymphocytes (TILs) represent a large part of antitumor immunity along with tertiary lymphoid structure (TLS), as they predict favorable prognosis and good treatment response. In this study, we scored for immune-related histology features using whole slide H&E images of the TCGA-BRCA dataset and analyzed these distinct features relative to gene expression patterns and molecular intrinsic subtypes. Method: A total of 1035 cases from TCGA-BRCA dataset were evaluated for TILs, plasma cells, high-endothelial venule associated lymphoid aggregate (HALA) and TLS. For HALA and TLS, location relative to tumor (non-tumor, peritumor, and intratumoral area) and number was determined. For each pathologically defined immune feature (i.e., no TLS vs TLS+), we then searched for associated gene expression features and used the top 100 significantly upregulated genes to make signatures for tumor group-based analyses. Upregulated immune modules in high-TIL tumors using a 10% TIL level cutoff were compared based on PAM50 molecular subtype (luminal A/B, HER2-enriched, basal-like). The clinical significance of histology-based gene signatures and high-TIL related immune modules was validated using bulk RNAseq and survival data from the SCAN-B cohort (N=6329). Results: HER2-enriched (HER2E) and basal-like breast tumors exhibited the highest mean TILs, with plasma cells more frequently observed in these subtypes. HALAs were present in 36.4% of cases and TLS were found in 6.5% of cases, also predominantly in HER2E and basal-like tumors. Gene signatures related to intratumoral HALA and intratumoral TLS correlated with better survival outcomes, while peritumoral HALA correlated with worse outcomes on the overall TCGA data set. In addition to TILs- and plasma cell-associated gene signatures, intratumoral HALA and intratumoral TLS correlated with HER2E and basal like tumors. High-TIL tumors revealed 164 enriched immune modules, with unique upregulated modules in each molecular subtype. For example, in luminal tumors, immune modules had little relation to favorable prognosis. High-TIL HER2E and basal-like tumors had distinct immune gene signatures linked to improved survival, with HER2E being more associated with B-cell prognostic features and basal-like subtype more associated with T-cell prognostic features. Conclusion: Different subtypes of breast cancer exhibit distinct tumor immune microenvironments, both histologically and molecularly. These differences in immune properties and biological characteristics should be considered when developing precise treatment strategies to achieve the best therapeutic efficacy for patients. Citation Format: Yoon Jin, Constandina E. O’Connell, Benjamin C. Calhoun, Christian Brueffer, Christer Larsson, Åke Borg, Lao H. Saal, and Charles M. Perou. Genomic characteristics related to histology-based immune features in breast cancer [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr PS1-05.

Polygenic risk scores (PRS) are not yet standard in clinical risk assessments for familial breast cancer in Sweden. This study evaluated the distribution and impact of an established PRS (PRS313) in women undergoing clinical sequencing for hereditary breast cancer. We integrated PRS313 into a hereditary breast cancer gene panel used in clinical practice and calculated scores for 262 women. Comparisons were made between women with unilateral and contralateral breast cancer, as well as those with and without pathogenic variants in breast cancer susceptibility genes. PRS313 was significantly higher in women with contralateral breast cancer (median + 1.3 SD, n = 33, P = 8e-9) compared to those with unilateral disease (median + 0.66 SD, n = 197, P = 5e-10). Elevated PRS313 was also observed in women with pathogenic variants, including those in high-penetrance genes (+ 0.65 SD) and moderate-penetrance genes (+ 0.93 SD), compared to population controls. Incorporating PRS313 into a clinical risk model (BOADICEA), shifted 20

3057 Background: Persistent circulating tumor DNA (ctDNA) detection during neoadjuvant treatment (NAT) of early-breast cancer (EBC) indicates high-risk disease. Following surgical resection, ctDNA-positivity indicates molecular residual disease (MRD) and heralds occult metastatic disease relapse. To incorporate ctDNA into EBC management, scalable and widely accessible diagnostic methods are necessary. Here we apply an ultrasensitive, personalized tumor-informed approach to ctDNA analysis leveraging structural variant (SV) detection using a novel multiplex digital PCR (dPCR) technology. Methods: 116 patients with stage I-III EBC (31.0% TNBC, 43.1% HR+/HER2- and 24.1% HER2+) and eligible for NAT were recruited through the prospective SCAN-B study (NCT02306096, substudy NeoCircle) between December 2014 and March 2019 and have been analyzed for ctDNA. Whole genome sequencing was performed on tumor material and personalized multiplex dPCR assays tracking up to 16 SVs were used for ctDNA monitoring. Plasma samples were collected at baseline, during NAT, pre- and post-surgery and at 6-monthly intervals during follow up. Results: High baseline detection was observed across all stages and subtypes (90.5% overall), and ctDNA-positivity at end-of-NAT (end-NAT) was a significant predictor of eventual disease relapse and death (relapse-free interval, RFI, hazard ratio, HR, 3.7, 95% CI 1.4-9.7; overall survival HR 7.7, 95% CI 2.2-26.6). A significant association was observed between end-NAT ctDNA clearance and pathological complete response (pCR), whereas non-pCR by itself was not a significant predictor of relapse or death in this cohort. At one or more post-operative timepoints, MRD+ was detected in 10 patients who experienced distant recurrence, with lead times up to 4 years (median 13.9 months, range 1.8-47.7 months). Similarly, ctDNA was detected in 3 of 4 patients with local recurrences and 1 of 2 patients with CNS-only recurrences. For 2 patients without presentation of clinical recurrence to date, ctDNA was detected post-operatively, with subsequent clearance during follow-up. Post-operative MRD associated with poor RFI (HR 45.5, 95% CI 13.0-159.8) and OS (HR 15.3, 95% CI 4.5-52.9). Conclusions: In this analysis of 116 patients from a prospective study in patients with EBC receiving NAT, we monitored ctDNA using an ultrasensitive tumor-informed dPCR assay tracking patient-specific SVs. ctDNA detection post-NAT and prior to surgery was associated with high-risk of disease relapse and death, outperforming pCR. Moreover, post-operative ctDNA detection was also significantly associated with disease relapse and death, with long lead-times over standard-of-care clinical assessments. These findings further validate the feasibility of SVs as an MRD analyte and support the clinical use of this approach in EBC.

BACKGROUND:Breast cancer is the most frequently diagnosed cancer in women. Survival is generally considered favourable, yet some patients remain at risk of early death. We aimed to assess whether comprehensive whole-genome sequencing (WGS) linked to mortality data could add prognostic value to existing clinical measures and identify patients who might respond to targeted therapeutics. METHODS:In this integrative, retrospective analysis, we analysed 2445 breast cancer tumours (any stage and molecular subtype) collected from 2403 patients recruited through 13 National Health Service Genomic Medicine Centres or hospitals in England affiliated to the 100 000 Genomes Project (100kGP) between 2012 and 2018. We linked 2208 (90%) cases with clinical data; mortality data were obtained for 1188 patients. Following high-depth WGS of tumour and matched normal DNA, we performed comprehensive WGS profiling seeking driver mutations, mutational signatures, and compound algorithmic scores for homologous recombination repair deficiency (HRD), mismatch repair deficiency, and tumour mutational burden. Data from 1803 additional patients with breast cancer from three independent cohorts were used to validate various findings. To evaluate the prognostic value of WGS features, we performed univariable and multivariable Cox regression on data from patients with stage I-III, ER-positive, HER2-negative breast cancer with a cancer-specific mortality endpoint (around 5-year follow-up). FINDINGS:Among 2445 tumours in the 100kGP breast cancer cohort, we observed genomic characteristics with immediate personalised medicine potential in 656 (26·8%), including features reporting HRD (298 [12·2%] total cases and 76 [6·3%] ER-positive, HER2-negative cases), highly individualised driver events, mutations underpinning resistance to endocrine therapy, and mutational signatures indicating therapeutic vulnerabilities. 373 (15·2%) cases had WGS features with potential for translational research, including compromised base excision repair and non-homologous end-joining dependency. Structural variation burden (hazard ratio 3·9 [95 CI% 2·4-6·2]; p<0·0001), high levels of APOBEC signatures (2·5 [1·6-4·1]; p<0·0001), and TP53 drivers (3·9 [2·4-6·2]; p<0·0001) were independently prognostic of customary clinical measures (age at diagnosis, stage, and grade) in patients with ER-positive, HER2-negative breast cancer. We developed a prognosticator for ER-positive, HER2-negative breast cancer capable of identifying patients who require either increased intervention or therapy de-escalation, validating the framework in the independent Swedish Cancerome Analysis Network-Breast (SCAN-B) dataset. INTERPRETATION:We show that breast cancer genomes are rich in predictive and prognostic value. We propose a two-step model for effective clinical application. First, the identification of candidates for targeted therapies or clinical trials using highly individualised genomic markers. Second, for patients without such features, the implementation of enhanced prognostication using genomic features alongside existing clinical decision-making factors. FUNDING:National Institute of Health Research, Breast Cancer Research Foundation, Dr Josef Steiner Cancer Research Award 2019, Basser Gray Prime Award 2020, Cancer Research UK, Sir Jeffrey Cheah Early Career Fellowship, the Mats Paulsson Foundation, the Fru Berta Kamprads Foundation, and the Swedish Research Council.

ER-positive/HER2-negative (ERpHER2n) breast cancer classified as PAM50 HER2-enriched (ERpHER2n-HER2E) represents a small high-risk patient subgroup. In this study, we investigate genomic, transcriptomic, and clinical features of ERpHER2n-HER2E breast tumors using two primary ERpHER2n cohorts comprising a total of 5640 patients. We show that ERpHER2n-HER2E tumors exhibit aggressive clinical features and poorer clinical outcomes compared to Luminal A and Luminal B tumors. Furthermore, ERpHER2n-HER2E breast cancer does not consist of misclassified or HER2-low cases, has little impact of ERBB2, is highly proliferative and less ER dependent than other luminal subtypes. It is not an obvious biological entity but is nevertheless associated with potentially targetable molecular features, notably a high immune response and high FGFR4 expression. Strikingly, molecular features that define the HER2E subtype in luminal disease are also consistent in HER2-positive disease, including an epigenetic mechanism for high FGFR4 expression in breast cancer.

The immune cell component of the tumor microenvironment is an important modulator of tumor progression. In patients with breast cancer, tumor-infiltrating lymphocytes (TILs) and tertiary lymphoid structures (TLS) represent core aspects of antitumor immunity, both increasingly recognized for clinical relevance. In this study, we evaluated immune-related histology features using whole-slide hematoxylin and eosin (H&E) images of The Cancer Genome Atlas Breast Invasive Carcinoma (TCGA-BRCA) data set (n = 1035) and analyzed these distinct features relative to gene expression, PAM50 subtypes, and patient survival. H&E images were evaluated for TILs, plasma cells (PCs), high-endothelial venule-associated lymphoid aggregates (HALA), and mature TLS. For HALA and TLS, location relative to the tumor (nontumor, peritumor, and intratumor) was determined. HER2-enriched (HER2E) and basal-like breast tumors exhibited the highest mean TILs and the presence of PCs. HALA were present in 35.1% of cases and TLS in 6.5% of cases, also predominantly in HER2E and basal-like tumors. We derived gene expression signatures for 10 histologically defined immune features and tested their clinical significance using transcriptomic and survival data from the Sweden Cancerome Analysis Network - Breast (SCAN-B) cohort. Signatures related to TILs, PCs, HALA/TLS, TLS, and specifically intratumor HALA and TLS were associated with better survival in HER2E and basal-like tumors. Peritumor HALA/TLS and nontumor signatures were nonsignificant or associated with worse outcomes. Furthermore, we compared the immune microenvironment of high-TIL (TILs > 10%) tumors from TCGA-BRCA by PAM50 subtype through supervised analyses of 200+ immune gene expression signatures, and unique immune features were identified for each subtype. In high-TIL luminal tumors, enriched immune signatures had little relation to prognosis. High-TIL HER2E and basal-like tumors had distinct immune signatures linked to improved survival, related to B and T cells, respectively. Overall, PAM50 subtypes of breast cancer exhibit distinct immune microenvironments, both histologically and molecularly. These differences in immune properties should be considered when developing precise treatment strategies to achieve optimal therapeutic efficacy for patients.

Triple-negative breast cancer (TNBC) is a clinically challenging and molecularly heterogenous breast cancer subgroup. Here, we investigate the DNA methylation landscape of TNBC. By analyzing tumor methylome profiles and accounting for the genomic context of CpG methylation, we divide TNBC into two epigenetic subtypes corresponding to a Basal and a non-Basal group, in which characteristic transcriptional patterns are correlated with DNA methylation of distal regulatory elements and epigenetic regulation of key steroid response genes and developmental transcription factors. Further subdivision of the Basal and non-Basal subtypes identifies subgroups transcending genetic and proposed TNBC mRNA subtypes, demonstrating widely differing immunological microenvironments, putative epigenetically-mediated immune evasion strategies, and a specific metabolic gene network in older patients that may be epigenetically regulated. Our study attempts to target the epigenetic backbone of TNBC, an approach that may inform future studies regarding tumor origins and the role of the microenvironment in shaping the cancer epigenome.

The clinical management of ER-positive/HER2-negative (ERpHER2n) breast cancer is complicated by a heterogeneous patient population, with some patients exhibiting endocrine resistance and an increased risk of recurrence. Among these high-risk subgroups, ERpHER2n Basal-like (ERpHER2n-Basal) breast cancer, as defined by PAM50 gene expression subtyping, remains poorly characterized due to limited available material. However, understanding the somatic molecular features driving treatment resistance and progression is critical for optimizing therapy. To address these challenges, we comprehensively characterized the patient subgroup by comparing it to both ERpHER2n and triple-negative breast cancer (TNBC) patients. We investigated 4474 Swedish patients with primary ERpHER2n tumors (Basal-like = 76, Luminal A = 3049, Luminal B = 1349) with clinical and RNA-sequencing data available, including 16 Basal-like tumors with whole-genome sequencing and matched global DNA methylation data. For TNBC comparisons, we used an additional 228 cases with available WGS, RNA-sequencing, and DNA methylation data. ER-positivity was defined as ≥ 10

568 Background: Persistent circulating cell-free tumor DNA (ctDNA) detection during neoadjuvant treatment of early-breast cancer (EBC) indicates high-risk disease. Detection of ctDNA post-resection of EBC (molecular residual disease, MRD) indicates occult metastatic disease and impending disease relapse. For ctDNA to be integrated into EBC management, accessible and scalable diagnostic tools are required. Here, we apply a highly sensitive, personalized tumor-informed approach to ctDNA evaluation predicated on analyses of structural variants (SVs) using a novel digital PCR (dPCR) SV technology. Methods: 170 patients with EBC and eligible for neoadjuvant therapy (NAT) were recruited through the prospective SCAN-B study (NCT02306096) between Dec 2014 and Mar 2019 (25.8% TNBC, 47.1% HR+/HER2- and 24.1% HER2+). Interim results are presented for the first 46 consecutive patients (comprising 567 plasma samples) where minimum QC criteria were met (10% tumor content and 10x sequencing depth). Whole genome sequencing (WGS) was performed on tumor material and personalized multiplex dPCR assays designed tracking up to 8 SVs for use in ctDNA analyses. Plasma samples were collected at baseline, during NAT, pre- and post-surgery and at 6-monthly intervals during follow up. Clinical characteristics and recurrence outcomes were recorded. Results: ctDNA was detected at one or more timepoints prior to surgery in 43/46 (93%) patients across all breast cancer subtypes, at a median variant allele frequency (VAF) of 0.14% (range 0.0002% - 27.6%). ctDNA levels remained detectable at the end of NAT in 24% patients (11/46); 5/11 (45%) ctDNA positive patients experienced disease relapse versus 1/35 (3%) ctDNA negative patients (P=0.002, Fisher’s exact test). At one or more post-operative timepoints, ctDNA was detected in 6/6 (100%) patients who experienced clinical recurrence, with lead times up to 52 months (median 11.8 months, range 3.5 to 52 months). In 40 patients without presentation of clinical recurrence to date, ctDNA was undetectable across 285/287 (99.3%) plasma timepoints collected post-surgery. Post-operative detection of ctDNA associated with poor overall survival (OS) compared to absence of ctDNA (log-rank P<0.0001). Conclusions: In this interim analysis of an ongoing prospective study in patients with EBC receiving NAT, we analyzed plasma for ctDNA using a novel tumor-informed dPCR assay tracking patient-specific SVs. ctDNA detection post-neoadjuvant therapy, prior to surgery associated with high-risk of disease relapse. Postoperative ctDNA detection was observed in 100% patients with clinical recurrence, and associated with poorer OS and long lead-times. Our data demonstrate the feasibility of SVs as an MRD analyte and provide evidence for high levels of clinical sensitivity achievable with this approach in EBC. Clinical trial information: NCT02306096 .

DPD (dihydropyrimidine dehydrogenase, encoded by DPYD) is the rate-limiting enzyme for fluoropyrimidine catabolism and a key enzyme for the metabolism of chemotherapy drugs such as fluorouracil and capecitabin. Testing for certain DPYD variants is recommended by international clinical guidelines to predict toxicity in patients and adjust treatment accordingly. A consensus has been reached to screen for four DPYD variants, namely c.1905+1G>A, c.1679T>G, c.2846A>T and c.1236G>A. Considering all four variants combined, around 7% of Europeans carry at least one decreased-function DPYD variant. Testing for DPYD variants is done using germline DNA, however, considering the high frequency in which capecitabine is used in breast cancer treatment and the high number of primary tumors for which RNAseq is performed for diagnostic purposes, we hypothesised that DPYD status could be read from tumor RNAseq and evaluated sensitivity and specificity compared to blood-DNA genotyping. We studied a SCAN-B cohort of over 6000 breast tumors with RNAseq data, for which over 1700 patients also had available OncoArray germline genotyping. Since c.1905+1G>A is located in an intronic splice-site, an RNAseq model was developed to infer this SNP using exon-skipping events and ratios of supporting reads. In preliminary results, there was no case with c.1679T>G variant in either RNAseq data or OncoArray. Variant c.2846A>T was not included in OncoArray but 20 cases were identified by RNAseq. For variant c.1236G>A, RNAseq data analysis showed 67 true positive (TP) cases, 1660 true negative (TN) cases, 1 false positive case (FP), and 5 false negative (FN) cases. For the variant call c.1905+1G>A, RNAseq data analysis showed 28 TP, 1692 TN, 0 FP and 7 FN cases. Overall, using an estimated DPYD SNP prevalence of 7%, the positive predictive value (PPV) and negative predictive value (NPV) were both preliminarily >99%. DPYD variant calling is feasible using RNAseq reads. If RNAseq from breast tumor tissue is available, it may be possible to accurately predict toxicity to 5-FU-containing regimes and thus forgo the need for further specific genomic assays, thus optimizing the diagnostic techniques.

Supplementary Material 1, 2 and 5 from Identification of New MicroRNAs in Paired Normal and Tumor Breast Tissue Suggests a Dual Role for the ERBB2/Her2 Gene

Breast cancer that is estrogen receptor positive (ER+) and human epidermal growth factor receptor 2 negative (HER2-) can be further stratified into intrinsic molecular subtypes by employing PAM50-based gene expression profiling. Despite its association with poor outcome, the HER2-enriched (HER2E) subtype currently holds no implications for clinical treatment decisions due to a lack of insight into its distinguishing features. Therefore, we aimed to evaluate the response of patients to conventionally administered therapies and identify molecular features to inform therapy optimization. Two primary breast cancer cohorts provided clinical, transcriptomic, and genomic data on ER+/HER2- cases: SCAN-B 4413 (HER2E: 89, Luminal A (LumA): 3049, Luminal B (LumB): 1349) and METABRIC 1227 (HER2E: 58, LumA: 601, LumB: 340), including whole genome sequencing data for 32 HER2E cases. Focal points were analyses based on overall survival and invasive disease-free survival (IDFS), differential gene expression, pathway enrichment, mutational frequencies and signatures, and copy number alterations. Clinicopathological analyses highlighted HER2E breast cancer to be a small subgroup associated with worse prognoses than other luminal PAM50 subtypes, regardless of whether patients were treated with endocrine therapy (E) or both endocrine and chemotherapy (EC), independent of tumor size, grade, age, and lymph node status (IDFS; E: HR 4.0, CI95 2.0-7.8; EC: HR 2.8; CI95 1.16-6.9; LumA as reference). Furthermore, HER2E tumors were larger and higher graded than LumA tumors. Next to highly proliferative characteristics akin to LumB, HER2E tumors were distinguished by their low ESR1 expression, high FGFR4 expression, high immune response, and high frequency of TP53 mutations. The HER2E subtype within ER+/HER2- disease is a small but clinically relevant patient subgroup that is not constituted by misclassified cases and is less ER dependent than other luminal subtypes. It does not represent a distinct biological entity, but it is nevertheless associated with potentially targetable molecular features, for instance in form of a high immune response and high FGFR4 expression.