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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.

Background Lymphovascular invasion (LVI) is regulated through complex molecular mechanisms. Cyclin B1 (CCNB1) was previously determined as being associated with LVI using large cohorts of breast cancer (BC) and artificial neural network (ANN) technique. In this study, we aimed to assess the association between CCNB1 and LVI, other clinicopathological and other LVI-related biomarkers at the molecular (RNA transcriptomic) and proteomic levels in BC. Methods Two transcriptomic BC cohorts (n = 2834) were used to assess the association between the expression of CCNB1 at the mRNA level and clinicopathological characteristics and patient outcome. Tissue microarrays (TMAs) from a well-characterised BC cohort (n = 2480) with long-term outcome were also used to assess the clinical significance of CCNB1 protein expression using immunohistochemistry. Results High CCNB1 mRNA expression was associated with aggressive tumour behaviour, including LVI, larger size, higher tumour grade, high lymph nodal stage, hormonal receptor negativity, HER2 positivity and poor clinical outcome (all p p CCNB1- and LVI-related biomarkers including N-cadherin, P-cadherin and TWIST2 at the transcriptomic and proteomic level. Multivariate analysis revealed that CCNB1 was an independent predictor of shorter BC-specific survival (HR = 1.3; 95% CI 1.2–1.5; p = 0.010). Conclusion CCNB1 is a key gene associated with LVI in BC and has prognostic value. More functional studies are warranted to unravel the mechanistic role of CCNB1 in the development of LVI.

Mitotic count in breast cancer is an important prognostic marker. Unfortunately, substantial inter- and intraobserver variation exists when pathologists manually count mitotic figures. To alleviate this problem, we developed a new technique incorporating both haematoxylin and eosin (HE) and phosphorylated histone H3 (PHH3), a marker highly specific to mitotic figures, and compared it to visual scoring of mitotic figures using HE only.Two full-face sections from 97 cases were cut, one stained with HE only, and the other was stained with PHH3 and counterstained with HE (PHH3-HE). Counting mitoses using PHH3-HE was compared to traditional mitoses scoring using HE in terms of reproducibility, scoring time, and the ability to detect mitosis hotspots. We assessed the agreement between manual and image analysis-assisted scoring of mitotic figures using HE and PHH3-HE-stained cells. The diagnostic performance of PHH3 in detecting mitotic figures in terms of sensitivity and specificity was measured. Finally, PHH3 replaced the mitosis score in a multivariate analysis to assess its significance.Pathologists detected significantly higher mitotic figures using the PHH3-HE (median ± SD, 20 ± 33) compared with HE alone (median ± SD, 16 ± 25), P 0.001. The concordance between pathologists in identifying mitotic figures was highest when using the dual PHH3-HE technique; in addition, it highlighted mitotic figures at low power, allowing better agreement on choosing the hotspot area (k = 0.842) in comparison with standard HE (k = 0.625). A better agreement between image analysis-assisted software and the human eye was observed for PHH3-stained mitotic figures. When the mitosis score was replaced with PHH3 in a Cox regression model with other grade components, PHH3 was an independent predictor of survival (hazard ratio [HR] 5.66, 95% confidence interval [CI] 1.92-16.69; P = 0.002), and even showed a more significant association with breast cancer-specific survival (BCSS) than mitosis (HR 3.63, 95% CI 1.49-8.86; P = 0.005) and Ki67 (P = 0.27).Using PHH3-HE-stained slides can reliably be used in routine scoring of mitotic figures and integrating both techniques will compensate for each other's limitations and improve diagnostic accuracy, quality, and precision.

Chromosome 17 alterations affect the assessment of HER2 gene amplification in breast cancer (BC), but its clinical significance remains unclear. This study aimed to identify the prevalence of centromere enumeration probe 17 (CEP17) alterations, and its correlation with response to neoadjuvant therapy (NAT) in BC patients with human epidermal growth factor receptor 2 (HER2) immunohistochemistry-equivocal score.A large BC cohort (n = 6049) with HER2 immunohistochemistry score 2+ and florescent in-situ hybridisation (FISH) results was included to assess the prevalence of CEP17 alterations. Another cohort (n = 885) with available clinicopathological data was used to evaluate the effect of CEP17 in the setting of NAT. HER2-amplified tumours with monosomy 17 (CEP17 copy number 1.5 per nucleus), normal 17 (CEP17 1.5- 3.0) and polysomy 17 (CEP17 ≥ 3.0) were observed in 16, 59 and 25%, respectively, compared with 3, 74 and 23%, respectively, in HER2-non-amplified tumours. There was no significant relationship between CEP17 alterations and pathological complete response (pCR) rate in both HER2-amplified and HER2-non-amplified tumours. The independent predictors of pCR were oestrogen (ER) negativity in HER2-amplified tumours [ER negative versus positive; odds ratio (OR) = 11.80; 95% confidence interval (CI) = 1.37-102.00; P = 0.02], and histological grade 3 in HER2 non-amplified tumours (3 versus 1, 2; OR = 5.54; 95% CI = 1.61-19.00; P = 0.007).The impacts of CEP17 alterations are not as strong as those of HER2/CEP17 ratio and HER2 copy number. The hormonal receptors status and tumour histological grade are more useful to identify BC patients with a HER2 immunohistochemistry-equivocal score who would benefit from NAT.

The MRE11 nuclease is essential during DNA damage recognition, homologous recombination, and replication. BRCA2 plays important roles during homologous recombination and replication. Here, we show that effecting an MRE11 blockade using a prototypical inhibitor (Mirin) induces synthetic lethality (SL) in BRCA2-deficient ovarian cancer cells, HeLa cells, and 3D spheroids compared to BRCA2-proficient controls. Increased cytotoxicity was associated with double-strand break accumulation, S-phase cell cycle arrest, and increased apoptosis. An in silico analysis revealed Mirin docking onto the active site of MRE11. While Mirin sensitises DT40 MRE11+/− cells to the Top1 poison SN-38, it does not sensitise nuclease-dead MRE11 cells to this compound confirming that Mirin specifically inhibits Mre11 nuclease activity. MRE11 knockdown reduced cell viability in BRCA2-deficient PEO1 cells but not in BRCA2-proficient PEO4 cells. In a Mirin-resistant model, we show the downregulation of 53BP1 and DNA repair upregulation, leading to resistance, including in in vivo xenograft models. In a clinical cohort of human ovarian tumours, low levels of BRCA2 expression with high levels of MRE11 co-expression were linked with worse progression-free survival (PFS) (p = 0.005) and overall survival (OS) (p = 0.001). We conclude that MRE11 is an attractive SL target, and the pharmaceutical development of MRE11 inhibitors for precision oncology therapeutics may be of clinical benefit.

Using digitalized whole slide images (WSI) in routine histopathology practice is a revolutionary technology. This study aims to assess the clinical impacts of WSI quality and representation of the corresponding glass slides. 40,160 breast WSIs were examined and compared with their corresponding glass slides. The presence, frequency, location, tissue type, and the clinical impacts of missing tissue were assessed. Scanning time, type of the specimens, time to WSIs implementation, and quality control (QC) measures were also considered. The frequency of missing tissue ranged from 2% to 19%. The area size of the missed tissue ranged from 1–70%. In most cases (>75%), the missing tissue area size was p < 0.05). None of the WSI with missing tissues resulted in a change in the final diagnosis. Missing tissue on breast WSI is observed but with variable frequency and little diagnostic consequence. Balancing between WSI quality and scanning time/image file size should be considered and pathology laboratories should undertake their own assessments of risk and provide the relevant mitigations with the appropriate level of caution.

Although counting mitoses is part of breast cancer grading, concordance studies showed low agreement. Refining the criteria for mitotic counting can improve concordance, particularly when using whole slide images (WSIs). This study aims to refine the methodology for optimal mitoses counting on WSI. Digital images of 595 hematoxylin and eosin stained sections were evaluated. Several morphological criteria were investigated and applied to define mitotic hotspots. Reproducibility, representativeness, time, and association with outcome were the criteria used to evaluate the best area size for mitoses counting. Three approaches for scoring mitoses on WSIs (single and multiple annotated rectangles and multiple digital high-power (×40) screen fields (HPSFs)) were evaluated. The relative increase in tumor cell density was the most significant and easiest parameter for identifying hotspots. Counting mitoses in 3 mm

Background: Inner centromere protein (INCENP) is a member of the chromosomal passenger complex (CPC) and plays a key role in mitosis and cell proliferation. This study aims to evaluate the clinical and prognostic significance of INCENP in invasive breast cancer (BC). Material and Methods: INCENP protein expression was evaluated on a tissue microarray of a large BC cohort (n=1295) using immunohistochemistry. At the mRNA level, INCENP expression was assessed using the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) (n=1980) and Cancer Genome Atlas (TCGA) BC cohorts (n=854). The correlations between INCENP expression, clinicopathological parameters and patient outcome were investigated. Results: INCENP protein expression was detected in the nucleus and cytoplasm of the tumour cells. Its expression was significantly associated with features characteristic of aggressive BC behaviour including high tumour grade, larger tumour size and high Nottingham Prognostic Index scores. High INCENP nuclear expression was a predictor of shorter BC-specific survival (BCSS) in the whole cohort, as well as in the luminal subtype (p

Supplementary Figure from Hypoxia Drives Centrosome Amplification in Cancer Cells via HIF1α-dependent Induction of Polo-Like Kinase 4

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 Data from Ecdysoneless Overexpression Drives Mammary Tumorigenesis through Upregulation of C-MYC and Glucose Metabolism

Supplementary Figure Legends

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.

Tables and Figures

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

Supplementary Data from Combined Perioperative Lapatinib and Trastuzumab in Early HER2-Positive Breast Cancer Identifies Early Responders: Randomized UK EPHOS-B Trial Long-Term Results

Supplementary Figure from Combined Perioperative Lapatinib and Trastuzumab in Early HER2-Positive Breast Cancer Identifies Early Responders: Randomized UK EPHOS-B Trial Long-Term Results

Western blots, FACS and clonogenic assays

Supplemental Table S1: Clinicopathological characteristics of whole breast cancer cohort 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 PARP and XRCC1 co-expression in breast cancers. Supplementary Table S5: Percentage of cells in various stages of cell cycle. Supplementary Table S6: Patient demographics in DCIS cohort. Supplementary Table S7: XRCC1 and clinic-pathological associations in pure DCIS cohort Supplementary Table 8: List of EMT PCR array genes layout Supplementary Table S9: LD50 for PARP inhibitor sensitivity in XRCC1 deficient/knockout cells.

Supplementary figure 1. BQ323636.1-overexpresion stable cell lines were produced using lentiviral system; Supplementary figure 2. BQ323636.1 overexpression conferred resistance to tamoxifen in vivo using xenografts established with MCF-7; Supplementary figure 3. BQ323636.1 overexpressed did not significantly affect the expressions of NCOR2, GPS2, TBLR1 nor HDAC3; Supplementary figure 4. Chromatin Immunoprecipitation (ChIP) assays show overexpression of BQ323636.1 could reduce the amount of NCOR2 interacting with estrogen responsive element of four genes; Supplementary figure 5. The clinical significance of nuclear expression level of BQ323636.1 in the Hong Kong cohort.; Supplementary figure 6. The clinical significance of nuclear expression level of BQ323636.1 in the United Kingdom cohort; Supplementary figure 7. The significance of nuclear expression level of BQ323636.1 on disease free survival.; Supplementary Table 1. Clinical characteristics of patients with Tamoxifen response information available, N= 358

Purpose:EPHOS-B aimed to determine whether perioperative anti-HER2 therapy inhibited proliferation and/or increased apoptosis in HER2-positive breast cancer.Patients and Methods:This randomized phase II, two-part, multicenter trial included newly diagnosed women with HER2-positive invasive breast cancer due to undergo surgery. Patients were randomized to: part 1 (1:2:2), no treatment (control), trastuzumab or lapatinib; part 2 (1:1:2) control, trastuzumab, or lapatinib and trastuzumab combination. Treatment was given for 11 days presurgery. Coprimary endpoints were change in Ki67 and apoptosis between baseline and surgery tumor samples (biologic response: ≥30% change). Central pathology review scored residual cancer burden (RCB). Relapse-free survival (RFS) explored long-term effects.Results:Between November 2010 and September 2015, 257 patients were randomized (part 1: control 22, trastuzumab 57, lapatinib 51; part 2: control 29, trastuzumab 32, combination 66). Ki67 response was evaluable for 223 patients: in part 1 Ki67 response occurred in 29/44 (66%) lapatinib versus 18/49 (37%) trastuzumab (P = 0.007) and 1/22 (5%) control (P < 0.0001); in part 2 in 36/49 (74%) combination versus 14/31 (45%) trastuzumab (P = 0.02) and 2/28 (7%) control (P < 0.0001). No significant increase in apoptosis after 11 days was seen in treatment groups. Six patients achieved complete pathologic response (pCR, RCB0) and 13 RCB1, all but two in the combination group. After 6 years median follow-up, 28 (11%) had recurrence and 19 (7%) died. No recurrences or deaths were observed among patients who achieved a pCR. Ki67% falls ≥50% associated with fewer recurrences (P = 0.002).Conclusions:Early response after short duration anti-HER2 dual therapy identifies cancers dependent on the HER2 pathway providing a strategy for exploring risk-adapted individualized treatment de-escalation.

Purpose:The purpose of this study is to predict risk of local recurrence (LR) in ductal carcinoma in situ (DCIS) with a new visualization and quantification approach using centrosome amplification (CA), a cancer cell–specific trait widely associated with aggressiveness.Experimental Design:This first-of-its-kind methodology evaluates the severity and frequency of numerical and structural CA present within DCIS and assigns a quantitative centrosomal amplification score (CAS) to each sample. Analyses were performed in a discovery cohort (DC, n = 133) and a validation cohort (VC, n = 119).Results:DCIS cases with LR exhibited significantly higher CAS than recurrence-free cases. Higher CAS was associated with a greater risk of developing LR (HR, 6.3 and 4.8 for DC and VC, respectively; P < 0.001). CAS remained an independent predictor of relapse-free survival (HR, 7.4 and 4.5 for DC and VC, respectively; P < 0.001) even after accounting for potentially confounding factors [grade, age, comedo necrosis, and radiotherapy (RT)]. Patient stratification using CAS (P < 0.0001) was superior to that by Van Nuys Prognostic Index (VNPI; HR for CAS = 6.2 vs. HR for VNPI = 1.1). Among patients treated with breast-conserving surgery alone, CAS identified patients likely to benefit from adjuvant RT.Conclusions:CAS predicted 10-year LR risk for patients who underwent surgical management alone and identified patients who may be at low risk of recurrence, and for whom adjuvant RT may not be required. CAS demonstrated the highest concordance among the known prognostic models such as VNPI and clinicopathologic variables such as grade, age, and comedo necrosis.

Supplementary Figure 1. Expression of CDK7, cyclin H and MAT1 is elevated in breast cancer. Supplementary Figure 2. Relationship between mRNA levels of CDK7, cyclin H and MAT1 in breast cancer. Supplementary Figure 3. Profiling of CDK7, Cyclin H and MAT1 expression in the cell cycle. Supplementary Figure 4. Expression of CDK7, CyclinH and MAT1 is elevated in breast cancer. Supplementary Figure 5. CDK7, CyclinH and MAT1 expression is not estrogen regulated in breast cancer cells. Supplementary Figure 6. Serine 118 phosphorylation is associated with better prognosis.

Legends for Supplementary Figure S1 & Supplementary Figure S2

Supplementary figure legends

Targeting PARP1 for synthetic lethality is a new strategy for breast cancers harboring germline mutations in BRCA. However, these mutations are rare, and reactivation of BRCA-mediated pathways may result in eventual resistance to PARP1 inhibitor therapy. Alternative synthetic lethality approaches targeting more common sporadic breast cancers and preinvasive ductal carcinoma in situ (DCIS) are desirable. Here we show that downregulation of XRCC1, which interacts with PARP1 and coordinates base excision repair, is an early event in human breast cancer pathogenesis. XRCC1-deficient DCIS were aggressive and associated with increased risk of local recurrence. Human invasive breast cancers deficient in XRCC1 and expressing high PARP1 levels also manifested aggressive features and poor outcome. The PARP1 inhibitor olaparib was synthetically lethal in XRCC1-deficient DCIS and invasive breast cancer cells. We conclude that targeting PARP1 is an attractive strategy for synthetic lethality and chemoprevention in XRCC1-deficient breast cancers, including preinvasive DCIS.Significance:These findings show that loss of XRCC1, which is associated with more malignant DCIS, can be exploited by PARP inhibition, suggesting its application as a promising therapeutic and chemoprevention strategy in XRCC1-deficient tumor cells.

Supplementary materials and Methods for Compounds and reagents, Generation of XRCC1 knockouts using CRISPR/Cas-9 system, Clonogenic assays, Cell Proliferation assays, Cell synchronization and functional studies, Confocal microscopy, Invasion and migration assays, qRT-PCR analysis of epithelial-mesenchymal transition (EMT) gene expression, Generation of 3D spheroids, human Invasive breast cancers and DCIS cohorts (demographics and treatment).

Purpose: Adjuvant tamoxifen treatment revolutionized the management of estrogen receptor (ER)–positive breast cancers to prevent cancer recurrence; however, drug resistance compromises its clinical efficacy. The mechanisms underlying tamoxifen resistance are not fully understood, and no robust biomarker is available to reliably predict those who will be resistant. Here, we study BQ323636.1, a novel splice variant of the NCOR2 gene, and evaluate its efficacy in predicting tamoxifen resistance in patients with breast cancer.Experimental Design: A monoclonal anti-BQ323636.1 antibody that specifically recognizes the unique epitope of this splice variant was generated for in vitro mechanistic studies and for in vivo analysis by immunohistochemistry on tissue microarrays of two independent cohorts of 358 patients with more than 10 years clinical follow-up data, who had ER-positive primary breast cancer and received adjuvant tamoxifen treatment. An orthotopic mouse model was also used.Results: Overexpression of BQ323636.1 conferred resistance to tamoxifen in both in vitro and in an orthotopic mouse model. Mechanistically, coimmunoprecipitation showed BQ323636.1 could bind to NCOR2 and inhibit the formation of corepressor complex for the suppression of ER signaling. Nuclear BQ3232636.1 overexpression in patients samples was significantly associated with tamoxifen resistance (P = 1.79 × 10−6, sensitivity 52.9%, specificity 72.0%). In tamoxifen-treated patients, nuclear BQ323636.1 overexpression was significantly correlated with cancer metastasis and disease relapse. Nuclear BQ323636.1 was also significantly associated with poorer overall survival (P = 1.13 × 10−4) and disease-specific survival (P = 4.02 × 10−5).Conclusions: These findings demonstrate that BQ323636.1 can be a reliable biomarker to predict tamoxifen resistance in patients with ER-positive breast cancer. Clin Cancer Res; 24(15); 3681–91. ©2018 AACR.See related commentary by Jordan, p. 3480

Purpose: CDK-activating kinase (CAK) is required for the regulation of the cell cycle and is a trimeric complex consisting of cyclin-dependent kinase 7 (CDK7), Cyclin H, and the accessory protein, MAT1. CDK7 also plays a critical role in regulating transcription, primarily by phosphorylating RNA polymerase II, as well as transcription factors such as estrogen receptor-α (ER). Deregulation of cell cycle and transcriptional control are general features of tumor cells, highlighting the potential for the use of CDK7 inhibitors as novel cancer therapeutics.Experimental Design: mRNA and protein expression of CDK7 and its essential cofactors cyclin H and MAT1 were evaluated in breast cancer samples to determine if their levels are altered in cancer. Immunohistochemical staining of >900 breast cancers was used to determine the association with clinicopathologic features and patient outcome.Results: We show that expressions of CDK7, cyclin H, and MAT1 are all closely linked at the mRNA and protein level, and their expression is elevated in breast cancer compared with the normal breast tissue. Intriguingly, CDK7 expression was inversely proportional to tumor grade and size, and outcome analysis showed an association between CAK levels and better outcome. Moreover, CDK7 expression was positively associated with ER expression and in particular with phosphorylation of ER at serine 118, a site important for ER transcriptional activity.Conclusions: Expressions of components of the CAK complex, CDK7, MAT1, and Cyclin H are elevated in breast cancer and correlate with ER. Like ER, CDK7 expression is inversely proportional to poor prognostic factors and survival. Clin Cancer Res; 22(23); 5929–38. ©2016 AACR.

Supplementary Data

Supplementary Figure Legends 1-9 from Global Histone Modifications in Breast Cancer Correlate with Tumor Phenotypes, Prognostic Factors, and Patient Outcome