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Background: Based on the results of the DESTINY-Breast04 study, the HER2-targeted antibody-drug conjugate trastuzumab deruxtecan (T-DXd) and the Ventana PATHWAY 4B5 companion diagnostic were recently approved by the US Food and Drug Administrated for the treatment and identification of patients with HER2-low (IHC2+/ISH- or IHC1+) (Modi et al. N Engl J Med 2022). The efficacy of T-DXd in HER2-low mBC highlights the need to distinguish lower ranges of HER2 IHC expression, which has been reported to be more challenging than scoring high HER2 expression (Fernandez et al. JAMA Oncol 2022). Here we report on current real-world HER2-low interpretation proficiency and the impact of training for participating pathologists in HER2-low scoring. Methods: Pathologists from laboratories across the US, EU, Japan, Australia, and Brazil were invited to use a digital pathology platform (Pathotrainer) to interpret HER2 digital images using ASCO/CAP 2018 scoring criteria. Two whole-slide imaging sample sets of representative study cases were compiled for Ventana PATHWAY 4B5 or Dako HercepTest (HcT) stained tumors. Another sample set (n = 25) was developed for a 4 hour virtual training session based on the ASCO/CAP 2018 guideline with some practical considerations. A steering committee (SC) of 8 pathology experts was formed to guide the study. Pathologists’ score was compared with a reference score as determined by independent review of 3 experts in HER2 pathology. Paired study cases (n = 14) considered challenging due to difficult-to-interpret staining patterns were reevaluated by the SC members. Concordance and efficacy of training were measured by Cohen’s weighted kappa (κ) coefficient, overall rater agreement (ORA), and receiver operating characteristic curve statistics. The primary endpoint was real-world concordance and ORA. The secondary endpoints were post-training concordance and ORA and correct identification of HER2 IHC 0 and HER2-low. Results: Pre-training baseline or real-world scores were taken by 77 pathologists in 14 countries (n = 49 for 4B5, n = 28 for HcT) and 74 pathologists completed post-training scores (n = 48 for 4B5, n = 26 for HcT). HER2 scoring proficiency of pathologists was high for both assays when assessed on ASCO/CAP binary HER2-negative and -positive status, irrespective of training (4B5: κ = 0.96, ORA =98.9.%; HcT: κ = 0.84, ORA = 94.3%) Concordance per ORA for the new 3-tier classification (HER2 IHC 0 vs HER2-low [IHC 2+/ISH- or IHC 1+] vs HER2-positive [IHC 3+ or IHC 2+/ISH+]) was greater than 80% for both assays at baseline and after training (see Table). In a subgroup analysis assessing training effect for 4B5 assay, concordance rates for HER2 IHC 0 were 74.6% at baseline and 89.2% after training (P < 0.001), and for HER2-low, NPA was 80.6% before and 91.1% after training (P < 0.001); there were no statistically significant changes after training for HcT (data not shown). Conclusions: Results from this real-world global study demonstrate that overall score concordance with a new category of HER2-low was above the 80% ORA benchmark for both 4B5 and HcT and is higher than previously reported (Fernandez et al. JAMA Oncol 2022). These data demonstrate pathologists’ ability to achieve an acceptable level of accuracy for identifying HER2 IHC 0 and HER2-low patients even after short-term training; however, additional training techniques and experience are needed to further improve accuracy. Acknowledgment Judy Yu, a former AstraZeneca employee, provided expertise and technical insights to support the study. Editorial Acknowledgment Under guidance of the authors, assistance in medical writing and editorial support was provided by Toinette Labuschagné, MSc, of ApotheCom, and was funded by Daiichi Sankyo. Funding This study was funded by Daiichi Sankyo and AstraZeneca. Table. Summary of Pathologist Concordance and Interobserver Variability Citation Format: Josef Rüschoff, Alexander Penner, Ian O. Ellis, M. Elizabeth H. Hammond, Annette Lebeau, Robert Y. Osamura, Frédérique Penault-Llorca, Federico Rojo, Neil Atkey, Andreas H. Scheel, Corrado D´Arrigo, Hans-Ulrich Schildhaus, Akira Moh, Chirag Desai, Giuseppe Viale. HER2-13 Proficiency assessment of HER2-low breast cancer scoring with the Ventana PATHWAY 4B5 and Dako HercepTest HER2 assays and the impact of pathologist training [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 HER2-13.

Standardised reporting of breast cancer key pathology data has become the norm in some parts of the world, but are based on national or regional guidelines that differ in certain aspects, resulting in divergent reporting practices and a lack of comparability of data internationally. The International Collaboration on Cancer Reporting (ICCR), a global alliance of major (inter-)national pathology and cancer organizations, have recently produced a new international dataset for the pathology reporting of breast cancer, including resection specimens with invasive cancer and ductal carcinoma in situ (DCIS) of the breast. This initiative aims at providing an international unified approach to reporting cancer. The guidance was prepared by an international expert panel consisting of experienced breast pathologists, a surgeon, and an oncologist. The dataset includes core (essential) and noncore (optional) data items based on a critical review and discussion of current evidence. Commentary is provided for each data item to explain the rationale for selection, its clinical relevance, and to highlight potential areas of disagreement or lack of evidence, in which case a consensus position was formulated. The process concludes with international public consultation, before ratification and publication on the free open access ICCR website, with a synoptic reporting guide. The key aim is to promote high-quality, standardised pathology reporting that can be used worldwide. Histological grade, tumour size, and oestrogen receptor status are used in this article to illustrate this process and the detail provided to support its inclusion.

Supplementary Table S1: CDK12 expression in relation to clinicopathological parameters for the unselected TMA series; Supplementary Table S2: CDK12 expression in relation to clinicopathological parameters for the HER2-positive Herceptin treated series; Supplementary Table S3: CDK12 expression in relation to clinicopathological parameters for the METABRIC TMA series; Supplementary Table S4: Univariate and multivariate analysis of CDK12 in the TMA cohorts; Supplementary Table S5: CDK12 mutations in breast cancer. Taken from cBioportal (42,43); Supplementary Table S6: Correlations of CDK12 mutations, methylation, gene expression and ERBB2 copy number in primary breast cancers from TCGA; Supplementary Table S7: Correlations of CDK12 mutations and gene expression of DNA repair genes in primary tumors from METABRIC. P values from heteroscedastic 2-tailed, t-test; Supplementary Table S8: Correlations of CDK12 protein expression, and miRNA expression in primary tumors from METABRIC. Wilcoxon rank P values are corrected for multiple testing; Supplementary Table S9: Correlations of CDK12 protein expression and gene expression of DNA repair genes in primary tumors from METABRIC. Limma analysis corrected for multiple testing; Supplementary Table S10: Association of CDK12 absent and intermediate (0, 2-6) versus high (7-8) expression with DNA repair proteins in unselected and TNBC. P values from Fishers exact test.

Supplementary Figure S1: Prognostic significance of ATM, BRCA1 and XRCC1 in breast cancers negative for CD8+ TILs. Kaplan-Meier survival curves are shown here. Supplementary Figure S2: Prognostic significance of polbeta (A & B), ERCC1 (C&D), RECQL4 (E&F), RECQL5 (G &H), BLM (I & J), PARP1 (K & L), FEN1 ( M & N), TOPO2A (O & P), Ku70/Ku80 (Q & R), Chk2 (S & T), WRN (U & V) and DNA-PKcs (W & X) in CD8+ TILs positive and CD8+ TILs negative breast cancers respectively. Kaplan-Meier survival curves are shown here. Supplementary Figure S3: Correlation between CD8 counts and ATM (A), BRCA1 (B) and XRCC1 (C). Supplementary Figure S4: Prognostic significance of ATM, BRCA1 and XRCC1 in FOXP3+ or FOXP3 negative breast cancers. Kaplan-Meier survival curves are shown here. Supplementary Figure S5: Prognostic significance of ATM, BRCA1 in PD-L1+ (tumour cells), PD-L1+ (TILs) or PD-1+ (TILs) is shown here. Kaplan-Meier survival curves are shown here. Supplementary Figure S6: Prognostic significance of ATM, BRCA1 and XRCC1 in PD-L1 negative (tumour cells), PD-L1 negative (TILs) and PD-1 negative (TILs) breast cancers. Kaplan-Meier survival curves are shown here. Supplementary Figure S7: Prognostic significance of CD8+ TILs in ER+ breast cancer [whole cohort (A), received no endocrine therapy (B) and received endocrine therapy (C)] is shown here. Supplementary Figure S8: Prognostic significance of BRCA1 (A), XRCC1 (B) or ATM (C) in CD8+ TILs positive ER+ breast cancer. Kaplan-Meier survival curves are shown here. Supplementary Figure S9: Prognostic significance of XRCC1 in CD8+/CD8- (A), PD-1+/PD-1- (B), FOXP3+/FOXP3- (C) and PD-L1+/PD-L1- (D) ER+ breast cancer that received no endocrine therapy. Supplementary Figure S10: A. Prognostic significance of XRCC1 in PD-1+/PD-1- HER2 + breast cancers. B. Prognostic significance of BRCA1 in FOXP3+/FOXP3- HER2 + breast cancers. Kaplan-Meier survival curves are shown here. Supplementary Figure S11: Prognostic significance of CD8+ TILs in ER- breast cancer [whole cohort (A), received no chemotherapy (B) and received chemotherapy (C)] is shown here. Kaplan-Meier survival curves are shown here. Supplementary Figure S12:.Prognostic significance of BRCA1 in PD-L1+/PD-L1- ER- breast cancers that received no chemotherapy. Kaplan-Meier survival curves are shown here.

Bloom syndrome helicase (BLM) has key roles in homologous recombination repair, telomere maintenance, and DNA replication. Germ-line mutations in the BLM gene causes Bloom syndrome, a rare disorder characterized by premature aging and predisposition to multiple cancers, including breast cancer. The clinicopathologic significance of BLM in sporadic breast cancers is unknown. We investigated BLM mRNA expression in the Molecular Taxonomy of Breast Cancer International Consortium cohort (n = 1,950) and validated in an external dataset of 2,413 tumors. BLM protein level was evaluated in the Nottingham Tenovus series comprising 1,650 breast tumors. BLM mRNA overexpression was significantly associated with high histologic grade, larger tumor size, estrogen receptor–negative (ER−), progesterone receptor–negative (PR−), and triple-negative phenotypes (ps < 0.0001). BLM mRNA overexpression was also linked to aggressive molecular phenotypes, including PAM50.Her2 (P < 0.0001), PAM50.Basal (P < 0.0001), and PAM50.LumB (P < 0.0001) and Genufu subtype (ER+/Her2−/high proliferation; P < 0.0001). PAM50.LumA tumors and Genufu subtype (ER+/Her2−/low proliferation) were more likely to express low levels of BLM mRNA (ps < 0.0001). Integrative molecular clusters (intClust) intClust.1 (P < 0.0001), intClust.5 (P < 0.0001), intClust.9 (P < 0.0001), and intClust.10 (P < 0.0001) were also more likely in tumors with high BLM mRNA expression. BLM mRNA overexpression was associated with poor breast cancer–specific survival (BCSS; ps < 0.000001). At the protein level, altered subcellular localization with high cytoplasmic BLM and low nuclear BLM was linked to aggressive phenotypes. In multivariate analysis, BLM mRNA and BLM protein levels independently influenced BCSS. This is the first and the largest study to provide evidence that BLM is a promising biomarker in breast cancer. Mol Cancer Ther; 14(4); 1057–65. ©2015 AACR.

Supplementary Figure S1. RECQL1 and survival in ER+ or ER- breast cancer patients. Supplementary Figure S2. RECQL1-depleted MCF-7 cells exhibit sensitivity to doxorubicin. Supplementary Figure S3. RECQL1 depletion and ERα protein levels.

Supplementary Table S1: Clinicopathological characteristics in the METABRIC cohort Supplementary Table S2: Clinicopathological characteristics of Nottingham Tenovus series Supplemental Table S3: Clinicopathological characteristics of ER- cohort Supplemental Table S4: Clinicopathological characteristics of BRCA germ-line deficient cohort Supplementary Table S5: Antigens, primary antibodies, clone, source, optimal dilution and scoring system used for each immunohistochemical marker Supplementary Table S6. RecQL1 mRNA expression and breast cancer. Supplementary Table S7. RECQL1 protein levels and ER+ breast cancers. Supplementary Table S8. RECQL1 protein levels and ER- breast cancers.

Western blots and immunohistochemistry.

RECQL1 expression and survival in ER+ or ER- breast cancers

Supplementary Figure Legends

Disruption of Cyclin-Dependent Kinase 12 (CDK12) is known to lead to defects in DNA repair and sensitivity to platinum salts and PARP1/2 inhibitors. However, CDK12 has also been proposed as an oncogene in breast cancer. We therefore aimed to assess the frequency and distribution of CDK12 protein expression by IHC in independent cohorts of breast cancer and correlate this with outcome and genomic status. We found that 21% of primary unselected breast cancers were CDK12 high, and 10.5% were absent, by IHC. CDK12 positivity correlated with HER2 positivity but was not an independent predictor of breast cancer–specific survival taking HER2 status into account; however, absent CDK12 protein expression significantly correlated with a triple-negative phenotype. Interestingly, CDK12 protein absence was associated with reduced expression of a number of DDR proteins including ATR, Ku70/Ku80, PARP1, DNA-PK, and γH2AX, suggesting a novel mechanism of CDK12-associated DDR dysregulation in breast cancer. Our data suggest that diagnostic IHC quantification of CDK12 in breast cancer is feasible, with CDK12 absence possibly signifying defective DDR function. This may have important therapeutic implications, particularly for triple-negative breast cancers. Mol Cancer Ther; 17(1); 306–15. ©2017 AACR.

Supplementary table descriptions

Supplementary Tables S1-S9: Supplementary Table S1: Clinicopathological characteristics in the METABRIC cohort Supplementary Table S2: External validation cohorts (pooled n = 2413). Supplementary Table S3: Clinicopathological characteristics of Nottingham cohort Supplementary Table S4: Antigens, primary antibodies, clone, source, optimal dilution and scoring system used for each immunohistochemical marker Supplementary Table S5: BLM (nuclear protein expression) in breast cancer Supplementary Table S6: BLM (cytoplasmic protein expression) in breast cancer Supplementary Table S7: BLM (nuclear and cytoplasmic protein co-expression) in breast cancer Supplementary Table S8. BLM - Rad51 nuclear co-expression and breast cancer Supplementary Table S9: Multivariate analysis in Nottingham cohort.

RECQL1, a key member of the RecQ family of DNA helicases, is required for DNA replication and DNA repair. Two recent studies have shown that germline RECQL1 mutations are associated with increased breast cancer susceptibility. Whether altered RECQL1 expression has clinicopathologic significance in sporadic breast cancers is unknown. We evaluated RECQL1 at the transcriptomic level (METABRIC cohort, n = 1,977) and at the protein level [cohort 1, n = 897; cohort 2, n = 252; cohort 3 (BRCA germline deficient), n = 74]. In RECQL1-depleted breast cancer cells, we investigated anthracycline sensitivity. High RECQL1 mRNA was associated with intClust.3 (P = 0.026), which is characterized by low genomic instability. On the other hand, low RECQL1 mRNA was linked to intClust.8 [luminal A estrogen receptor–positive (ER+) subgroup; P = 0.0455] and intClust.9 (luminal B ER+ subgroup; P = 0.0346) molecular phenotypes. Low RECQL1 expression was associated with shorter breast cancer–specific survival (P = 0.001). At the protein level, low nuclear RECQL1 level was associated with larger tumor size, lymph node positivity, high tumor grade, high mitotic index, pleomorphism, dedifferentiation, ER negativity, and HER-2 overexpression (P < 0.05). In ER+ tumors that received endocrine therapy, low RECQL1 was associated with poor survival (P = 0.008). However, in ER− tumors that received anthracycline-based chemotherapy, high RECQL1 was associated with poor survival (P = 0.048). In RECQL1-depleted breast cancer cell lines, we confirmed doxorubicin sensitivity, which was associated with DNA double-strand breaks accumulation, S-phase cell-cycle arrest, and apoptosis. We conclude that RECQL1 has prognostic and predictive significance in breast cancers. Mol Cancer Ther; 16(1); 239–50. ©2016 AACR.

Supplementary Table S1: Clinicopathological characteristics of Nottingham Tenovus series Supplemental Table S2: Clinicopathological characteristics of ER- cohort Supplementary Table S3: Antigens, primary antibodies, clone, source, optimal dilution and scoring system used for each immunohistochemical marker Supplementary Table S4: Clinicopathological significance of ATM expression in CD8+ TILs positive and CD8+ TILs negative breast cancer Supplementary Table S5: Clinicopathological significance of BRCA1 expression in CD8 positive and CD8 negative breast cancer Supplementary Table S6: Clinicopathological significance of XRCC1 expression in CD8 positive and CD8 negative breast cancers Supplementary Table S7: Clinicopathological significance of ATM expression in FOXP3 positive and FOXP3negative breast cancer Supplementary Table S8: Clinicopathological significance of BRCA1 expression in FOXP3 positive and FOXP3negative breast cancer Supplementary Table S9: Clinicopathological significance of XRCC1 expression in FOXP3 positive and FOXP3negative breast cancer

Supplementary Figure S1: BLM m RNA and breast cancer (external validation cohort). Supplementary Figure S2: Western blots and immunohistochemistry. Supplementary Figure S3: BLM protein expression and survival. Supplementary Figure S4: BLM-Rad51 co-expression and survival. Supplementary Figure S5: BLM-Rad51 co-expression and survival in ER+ breast cancers.

Supplementary Figure from Clinical Validation of EndoPredict in Pre-Menopausal Women with ER-Positive, HER2-Negative Primary Breast Cancer

Supplementary Data from Clinical Validation of EndoPredict in Pre-Menopausal Women with ER-Positive, HER2-Negative Primary Breast Cancer

Purpose:The EndoPredict prognostic assay is validated to predict distant recurrence and response to chemotherapy primarily in post-menopausal women with estrogen receptor–positive (ER+), HER2− breast cancer. This study evaluated the performance of EndoPredict in pre-menopausal women.Experimental Design:Tumor samples from 385 pre-menopausal women with ER+, HER2− primary breast cancer (pT1-3, pN0-1) who did not receive chemotherapy in addition to endocrine therapy were tested with EndoPredict to produce a 12-gene EP molecular score and an integrated EPclin score that includes pathologic tumor size and nodal status. Associations of molecular and EPclin scores with 10-year distant recurrence–free survival (DRFS) were evaluated by Cox proportional hazards models and Kaplan–Meier analysis.Results:After a median follow-up of 9.7 years, both the EP molecular score and the molecular-clinicopathologic EPclin score were associated with increased risk of distant recurrence [HR, 1.33; 95% confidence interval (CI), 1.18–1.50; P = 7.2 × 10–6; HR, 3.58; 95% CI, 2.26–5.66; P = 9.8 × 10–8, respectively]. Both scores remained significant after adjusting for clinical factors in multivariate analysis. Patients with low-risk EPclin scores (64.7%) had significantly improved DRFS compared with high-risk patients (HR, 4.61; 95% CI, 1.40–15.17; P = 4.2 × 10–3). At 10 years, patients with low-risk and high-risk EPclin scores had a DRFS of 97% (95% CI, 93%–99%) and 76% (95% CI, 67%–82%), respectively.Conclusions:The EPclin score is strongly associated with DRFS in pre-menopausal women who received adjuvant endocrine therapy alone. On the basis of these data, pre-menopausal women with EPclin low-risk breast cancer may be treated with endocrine therapy only and safely forgo adjuvant chemotherapy.

Supplementary Figure 1: Examples of staining and coring system for cyclin E; Supplementary Figure 2: Examples of LMW-E cyclin E staining; Supplementary Figure 3: REMARK diagram of biospecimen availability for the MDA Lab00-222, NCI TMA, MDA TMA, and UK TMA cohorts.

S1: Image of full size blot for figure 1A; S2: Image of full size blot for figure 1B; S3: SRC3-pS543 and SRC3 chromatin binding patterns in MCF7 cells; S4: Venn diagram of SRC1, SRC2 and SRC3-pS543 enriched binding sites; S5: A. ChIP-QPCR validation of differential enriched chromatin binding sites for total SRC3 and SRC3-pS543; S6: Hormone dependence and phospho-specificity of SRC3-pS543 at sites either or not shared with total SRC3; S7: Overlap of ERïƒ¡ï€ and SRC3 with other publically available datasets; S8: Fulvestrant decreases SRC3-pS543/chromatin interactions at sites shared with ER; S9: Venn diagram, showing the overlap of GO pathways enriched for genes proximal to chromatin binding sites for SRC3-pS543 unique, SRC3 unique or shared regions; S10: 2D graph of normalized ChIP-seq data, illustrating signal for ER, total SRC3, H3K4me1, H3K4me2, H3K4me3 and H3K27Ac binding events at genomic regions occupied by SRC3-pS543; S11: Histological analysis of SRC3 and SRC3-pS543 expression in representative tumour sections; S12: Scatterplot analysis for total SRC3 versus SRC3-pS543 signal in the Nottingham Tenovus Primary Breast Carcinoma Series; S13: Disease Free Survival and breast cancer specific survival in breast cancer patients based on SRC3 protein expression; S14: Disease Free Survival and breast cancer specific survival in patients with HER2 positive and SRC3 high breast cancers compared with all other patients.

supplementary figure 7-8

R File codes pertaining to NCI cohort

Supplementary Figure 4. Multivariable Cox proportional hazards regression analyses for disease free survival (DFS; left panel) and corresponding forest plots (Right panel).

Supplementary Table (S3). Clinico-pathological characteristics of primary locally advanced triple negative breast cancer cohort (PLA-TNBC; n=110)

Supplementary Table 1. Multivariate Cox Regression Analysis for Time to Distant Metastasis (TTDM)

Interrogation of Oncomine{trade mark, serif} (https://www.oncomine.org/) showed higher expression of eIF4E and eIF5 in breast (top panel) and lung cancer (bottom panel) compared to normal tissue. When these biomarkers were compared for gender, eIF4E and eIF5 expression was proportionately higher in male breast cancer (A, C) but not in lung cancer (B, D), respectively .

Materials and Methods

Supplementary Figure 2. Receiver operating characteristic (ROC) analysis of pathological complete response (pCR), revised pathological TNM stage (yp-TNM stage), Clinical-Pathologic Scoring System (CPS) score and CPS-Oestrogen receptor-histological grade (CPS-EG) were performed for predicting breast cancer specific survival in the training (A), internal validation (B), external validation (C) and combined three cohorts (D).

R File codes pertaining to UK cohort

QPCR primer sequences for ChIP validations.

supplemental figure legend.

A. Mib-1 is required for trastuzumab or lapatinib-induced increase of HES-1 protein expression in MDA-MB-453 cells. B. Notch-1, JAG-1, and HES-1 proteins are increases in trastuzumab resistant BT474 cells compared to sensitive cells.

R File codes pertaining to the MDACC-TMA cohort