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Purpose: Assessing the metastasis status of the sentinel lymph nodes (SLNs) for hematoxylin and eosin-stained frozen tissue sections by pathologists is an essential but tedious and time-consuming task that contributes to accurate breast cancer staging. This study aimed to review a challenge competition (HeLP 2019) for the development of automated solutions for classifying the metastasis status of breast cancer patients., Materials and Methods: A total of 524 digital slides were obtained from frozen SLN sections: 297 (56.7%) from Asan Medical Center (AMC) and 227 (43.4%) from Seoul National University Bundang Hospital (SNUBH), South Korea. The slides were divided into training, development, and validation sets, where the development set comprised slides from both institutions and training and validation set included slides from only AMC and SNUBH, respectively. The algorithms were assessed for area under the receiver operating characteristic curve (AUC) and measurement of the longest metastatic tumor diameter. The final total scores were calculated as the mean of the two metrics, and the three teams with AUC values greater than 0.500 were selected for review and analysis in this study., Results: The top three teams showed AUC values of 0.891, 0.809, and 0.736 and major axis prediction scores of 0.525, 0.459, and 0.387 for the validation set. The major factor that lowered the diagnostic accuracy was micro-metastasis., Conclusion: In this challenge competition, accurate deep learning algorithms were developed that can be helpful for making a diagnosis on intraoperative SLN biopsy. The clinical utility of this approach was evaluated by including an external validation set from SNUBH.

Prognostic implications of therapeutic response of metastatic lymph nodes (LNs) to neoadjuvant chemotherapy (NAC) remain unclear in patients with breast cancer. We aimed to evaluate the prognostic value of axillary LN regression after NAC in locally-advanced breast cancer patients. Therapeutic response of the LNs was evaluated in 563 breast cancer patients and classified into four grades according to the regression pattern. Initial pathologic N stage was estimated from the sum of the metastatic LNs and those with complete regression. In survival analyses, LN regression grade, pathologic N stage after NAC, and presumed initial pathologic N stage stratified clinical outcome of the patients in the whole group, in both ER-positive and ER-negative subgroups, and in those with residual breast disease. On multivariate analysis, LN regression grade and presumed initial pathologic N stage were revealed as independent prognostic factors. The number of completely-responsive LNs and the ratio of non-responsive LNs also revealed a prognostic value. In conclusion, regression grade of axillary LNs and presumed initial pathologic N stage have prognostic values in breast cancer patients who receive NAC. Thus, regression of axillary LNs should be evaluated and included in pathologic reporting of post-NAC resection specimens.

Myeloid-derived suppressor cells (MDSCs) play an important role in tumor progression through both immunologic and non-immunologic mechanisms. This study was conducted to evaluate the expression of S100A8, a well-known MDSC marker, and the significance of its expression in pre-invasive and invasive breast cancers. S100A8 expression in tumor cells (TCs) and immune cells (ICs) was assessed by immunohistochemistry, and its association with clinicopathologic features and infiltration of other IC subsets including CD4+, CD8+, and FOXP3+ tumor-infiltrating lymphocytes (TILs) and PD-L1+ ICs was evaluated. S100A8 expression in TCs and ICs showed a positive correlation in pre-invasive carcinoma and invasive carcinoma. S100A8+ ICs, but not S100A8+ TCs, were significantly higher in number in invasive carcinoma than in pre-invasive carcinoma. Infiltration of S100A8+ ICs was revealed as a poor prognostic indicator in pre-invasive and invasive carcinomas, especially in hormone receptor-positive subgroup. Infiltration of CD4+, CD8+, and FOXP3+ TIL subsets and PD-L1+ ICs was significantly higher in S100A8+ IC (+) group than in S100A8+ IC (-) group. Combined analyses of IC subset infiltration revealed that infiltration of S100A8+ ICs was associated with poor clinical outcome in the PD-L1+ IC (-), CD8+ TIL-low, and FOXP3+ TIL-low subgroups. In conclusion, S100A8+ ICs seem to undergo a dynamic change during breast cancer progression in association with other IC subset infiltration. The prognostic impact of S100A8+ IC infiltration was greater in less immunogenic tumors.

Background: Apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3B (APOBEC3B) is implicated in anti-viral immune response and cancer mutagenesis. Germline APOBEC3B deletion is associated with increased susceptibility to breast cancer. We aimed to evaluate the association between germline APOBEC3B deletion and clinical phenotypes of breast cancer in Korean patients with operable breast cancer., Methods: Mononuclear blood cell DNA of 103 patients with operable breast cancer was collected at Seoul National University Bundang Hospital in 2009. The DNA was sequenced to analyze APOBEC3B deletion status. Further, tumor-infiltrating lymphocytes (TILs) and programmed cell death-ligand 1 (PD-L1) expression in tumor cells were measured using immunohistochemistry., Results: Median age of breast cancer diagnosis was 46 (25-72). In APOBEC3B deletion analysis, 10 (9.7%), 36 (35.0%), and 57 (55.3%) patients were identified as two-copy deletion (A3B del/del ), one-one copy deletion (A3B del/wt ), and no deletion (A3B wt/wt ), respectively. For other cancer susceptibility gene alterations, 9 (8.7%) patients were identified as pathogenic variants: RAD51D (n = 1), GJB2 (n = 1), BRCA1 (n = 1), BRCA2 (n = 2), ATM (n = 1), USH2A (n = 1), RET (n = 1), BARD1 (n = 1). We observed no significant association between germline APOBEC3B deletion with any clinicopathologic features of breast cancer, such as age, family history of cancer, and bilateral breast cancer. Further, according to follow-up observations, APOBEC3B deletion was not predictive of disease-free survival. In ER+ subtype, a trend toward better survival was observed in patients with A3B del/del genotype as compared to patients with A3B del/wt and A3B wt/wt genotype (log-rank, P = 0.25). In patients with sufficient tumor samples for the assessment of TIL (n = 63) and PD-L1 (n = 71), the A3B del/del genotype was significantly associated with high TILs (> 10%) than other tumor genotypes (6/7 patients in A3B del/del vs. 13/24 in A3B del/wt vs. 15/32 in A3B wt/wt : Fisher's exact test, P = 0.029). However, PD-L1 expression was not associated with APOBEC3B deletion status (1/7 patients > 1% PD-L1 in A3B del/del vs. 4/26 in A3B del/wt vs. 8/38 in A3B wt/wt : P = 0.901)., Conclusion: We identified germline APOBEC3B deletion in 9.7% of Korean patients with operable breast cancer. The relationship between A3B del/del genotype and high TILs suggests that patients carrying this genotype could be potential candidates for immunotherapy.

Tumor-infiltrating lymphocyte (TIL) levels have prognostic and predictive values in treatment-naïve breast cancers. However, there have been controversies regarding TIL subset changes and their clinical implications in post-treatment breast cancers. This study aimed to explore change and prognostic significance of TIL subset infiltration after primary systemic therapy (PST) in breast cancer. One-hundred-fifty-five patients who had residual disease after anthracycline- or anthracycline plus taxane-based PST were included. The quantities of intratumoral and stromal TIL subsets (CD8+, CD4+, and FOXP3+ TILs) in pre- and post-PST breast cancer samples, as well as changes between them, were analyzed along with their correlations with clinicopathologic features and outcome of patients. As a whole, intratumoral CD8+ and CD4+ TILs increased after PST while stromal TILs decreased. Both intratumoral and stromal FOXP3+ TILs decreased after PST. The chemo-sensitive group [residual cancer burden (RCB) class I and II] showed the same pattern of change in intratumoral CD8+ TILs as the whole group, whereas the chemo-resistant group (RCB class III) showed no significant change in intratumoral CD8+ TIL infiltration after PST. Survival analyses for each TIL subset as well as their ratios revealed that high levels of intratumoral, stromal, and total CD8+ TIL infiltration after PST were independent predictors of longer patient survival. In subgroup analyses, CD8+ TIL infiltration after PST revealed prognostic significance in the chemo-resistant group but not in the chemo-sensitive group. In conclusion, infiltration of CD8+, CD4+, and FOXP3+ TIL changed after PST in the intratumoral and stromal compartments. Especially, increase of intratumoral CD8+ TILs was associated with chemo-responsiveness. Moreover, CD8+ TIL status in residual tumors after PST may be used as a potential prognostic marker in breast cancer patients who receive PST and provide additional prognostic information to chemo-resistant group., Competing Interests: The authors have declared that no competing interests exist.

The aim of the study was to evaluate the impact of the updated 2018 American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) guideline on human epidermal growth factor receptor 2 (HER2) interpretation in breast cancer compared with that of the previous guidelines and also the significance of in situ hybridization (ISH) groups proposed by the updated guideline. HER2 ISH reports and immunohistochemistry (IHC) data from 1,348 invasive breast cancers diagnosed at a single institution were included in this study. HER2 IHC was reassessed using the 2018 guideline, and HER2 ISH status was determined by the 2007, 2013, and 2018 guidelines. When applying the updated guideline, most of the HER2 ISH-equivocal cases as per the previous guidelines were reclassified as ISH negative, and 0.8% of HER2 ISH-positive tumors as per the 2007 guideline and 2.5% of those as per the 2013 guideline were changed to ISH negative. Accordingly, the negative HER2 ISH results significantly increased in the 2018 guideline compared with the 2013 guideline. HER2 ISH-positive tumors in ISH group 3 (HER2/chromosome enumeration probe 17 [CEP17] ratio <2.0 and average HER2 copy number ≥6.0 per cell) were characterized by equivocal HER2 protein expression, CEP17 copy number gain, and low HER2 copy numbers compared with classic HER2 ISH-positive tumors in ISH group 1 (HER2/CEP17 ratio ≥2.0 and average HER2 copy number ≥4.0 per cell). HER2 ISH-negative tumors in ISH group 4 (HER2/CEP17 ratio <2.0 with average HER2 copy number ≥4.0 and < 6.0 per cell) revealed more aggressive clinicopathologic features and poorer clinical outcomes than those in ISH group 5 (HER2/CEP17 ratio <2.0 and average HER2 copy number <4.0 per cell), especially in the hormone receptor-positive subgroup. In conclusion, implementation of the updated 2018 ASCO/CAP guideline leads to a significant increase in HER2 ISH-negative results compared with the 2013 guideline, mainly via reclassification of the ISH-equivocal cases to ISH-negative ones. ISH groups proposed by the updated guideline provide additional information on the clinicopathologic characteristics of the tumors., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: The immune microenvironment in ductal carcinoma in situ (DCIS) and its significance are not well established. This study was conducted to evaluate the immune microenvironment of DCIS including the composition of tumor-infiltrating lymphocyte (TIL) subsets and PD-L1+ immune cells and to compare it with that of invasive breast cancer., Materials and Methods: A total of 671 cases including three different disease groups of pure DCIS, DCIS with microinvasion (DCIS-M), and invasive carcinoma were included in this study. CD4+, CD8+, and FOXP3+ TIL subsets and PD-L1+ immune cells were detected with immunohistochemistry using tissue microarrays and were analyzed in relation to clinicopathologic characteristics and different disease groups., Results: In pure DCIS, high infiltrations of CD4+, CD8+, and FOXP3+ T cells and the presence of PD-L1+ immune cells were associated with high nuclear grade, comedo-type necrosis, hormone receptor (HR) negativity, and high Ki-67 proliferation index. All immune cell infiltrations were higher in invasive carcinoma than in pure DCIS regardless of the HR status. While CD4+ T cells were more abundant than CD8+ T cells in pure DCIS, CD8+ T cells were dominant in invasive carcinoma, especially in HR-negative tumors. Within individual cases of invasive carcinoma with DCIS component, all immune cell subset infiltration was higher in the invasive component than in the DCIS component; however, CD4+ TIL infiltration did not differ between the two components in HR-negative tumors. Comparing pure DCIS, DCIS-M, and DCIS associated with invasive carcinoma (DCIS-INV), CD4+ TIL infiltration revealed a gradual increase from pure DCIS to DCIS-M and DCIS-INV in the HR-negative group, whereas FOXP3+ TIL infiltration was significantly increased in DCIS-INV than in pure DCIS in the HR-positive group. The high infiltration of FOXP3+ TIL and the presence of PD-L1+ immune cells were associated with tumor recurrence in patients with pure DCIS., Conclusions: Our study showed that the immune microenvironment differs significantly not only between DCIS and invasive carcinoma but also between pure DCIS, DCIS-M, and DCIS-INV depending on the HR status.

Background: Multiplicity in breast cancer is common. Studies on multiple breast cancers have revealed high concordance in biomarker status among individual lesions. However, genomic differences among multiple lesions are not well-established. We aimed to investigate the potential genomic heterogeneity of multiple breast cancer., Methods: Twenty-one patients with radiologically and histologically evident multiple breast cancer with similar histology were included. Two lesions from each of the 21 patients were selected, and biomarker status was evaluated for each lesion. Capture-based targeted next-generation sequencing was performed using a cancer gene panel consisting of 170 genes., Results: We identified discordance in intrinsic subtype in 2 (10%) of the 21 patients. Pathogenic mutations were detected in 13 of the 21 patients, of whom 11 shared oncogenic variants in the two lesions. The remaining two patients yielded different mutation results for TP53, ATM, and PIK3CA. Difference in copy number alteration was observed in 7 (33%) of the 21 patients including ERBB2 (n = 2), FGFR1 (n = 2), and FGFR2 (n = 1) genes., Conclusion: Despite similar histologic features of the individual lesions, inter-lesional genomic difference was identified in more than one-third of the patients. Inter-lesional genomic heterogeneity needs to be considered when performing a genomic test in multiple breast cancers.

Chromosomal instability (CIN) is known to be associated with prognosis and treatment response in breast cancer. This study was conducted to determine whether copy number gain of centromere 17 (CEP17) reflects CIN, and to evaluate the prognostic and predictive value of CIN in breast cancer. CIN status was determined by summing copy number gains of four centromeric probes (CEP1, CEP8, CEP11, and CEP16) based on fluorescence in situ hybridization and CIN scores were calculated using next generation sequencing data. High CIN was associated with adverse clinicopatholgical parameters of breast cancer. Among them, positive HER2 status, high Ki-67 index and CEP17 copy number gain were found to be independent predictors of high CIN. High CIN was associated with poor clinical outcome of the patients in the whole group, as well as in luminal/HER2-negative and HER2-positive subtypes. CEP17 copy number was significantly higher in the high-CIN-score group than in the low-CIN-score group. A positive linear correlation between the mean CEP17 copy number and the CIN score was found. In conclusion, CEP17 copy number was confirmed as a useful predictor for CIN in breast cancer, and high CIN was revealed as an indicator of poor prognosis in breast cancer.

Chemotherapy can alter the makeup of a tumor cell population by exerting selection pressure. We examined the change in Shannon index, a mathematical diversity measure used in ecology, for c-MYC copy number variation (CNV) after neoadjuvant chemotherapy and evaluated its clinical significance in breast cancer. Associations between Shannon indices for c-MYC CNV in pre- and post-neoadjuvant chemotherapy breast cancer samples and clinicopathologic features of tumors as well as patient survival were analyzed in 144 patients. A change in c-MYC amplification and copy number gain status was found in 14.3% and 33.6% with most cases showing positive to negative conversion. The chemo-sensitive group showed a significant decrease in Shannon index after neoadjuvant chemotherapy. However, there was no difference in diversity indices between pre- and post-neoadjuvant chemotherapy specimens in the chemo-resistant group. In survival analyses, high Shannon indices for c-MYC CNV in post-neoadjuvant chemotherapy samples as well as those in pre-neoadjuvant chemotherapy samples were revealed as independent prognostic factors for poor disease-free survival not only in the whole group but also in the chemo-resistant subgroup. These findings suggest that a change in Shannon index for c-MYC CNV after neoadjuvant chemotherapy reflects chemo-responsiveness and that Shannon indices after neoadjuvant chemotherapy have a prognostic value in breast cancer patients who receive neoadjuvant chemotherapy.

Purpose: Alteration of biomarker status after primary systemic therapy (PST) is occasionally found in breast cancer. This study was conducted to clarify the clinical implications of change of biomarker status in breast cancer patients treated with PST., Materials and Methods: The pre-chemotherapeutic biopsy and post-chemotherapeutic resection specimens of 442 breast cancer patients who had residual disease after PST were included in this study. The association between changes of biomarker status after PST and clinicopathologic features of tumors, and survival of the patients, were analyzed., Results: Estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) status changed after PST in 18 (4.1%), 80 (18.1%), and 15 (3.4%) patients,respectively. ER and PR mainly underwent positive to negative conversion,whereas HER2 status underwent negative to positive conversion. Negative conversion of ER and PR status after PST was associated with reduced disease-free survival. Moreover, a decline in the Allred score for PR in post-PST specimens was significantly associated with poor clinical outcome of the patients. HER2 change did not have prognostic significance. In multivariate analyses, negative PR status after PST was found to be an independent adverse prognostic factor in the whole patient group, in the adjuvant endocrine therapy-treated subgroup, and also in pre-PST PR positive subgroup., Conclusion: ER and HER2 status changed little after PST, whereas PR status changed significantly. In particular, negative conversion of PR status was revealed as a poor prognostic indicator, suggesting that re-evaluation of basic biomarkers is mandatory in breast cancer after PST for proper management and prognostication of patients.

Pathology archives with linked clinical data are an invaluable resource for translational research, with the limitation that most cancer samples are formalin-fixed paraffin-embedded (FFPE) tissues. Therefore, FFPE tissues are an important resource for genomic profiling studies but are under-utilised due to the low amount and quality of extracted nucleic acids. We profiled the copy number landscape of 356 breast cancer patients using DNA extracted FFPE tissues by shallow whole genome sequencing. We generated a total of 491 sequencing libraries from 2 kits and obtained data from 98.4% of libraries with 86.4% being of good quality. We generated libraries from as low as 3.8 ng of input DNA and found that the success was independent of input DNA amount and quality, processing site and age of the fixed tissues. Since copy number alterations (CNA) play a major role in breast cancer, it is imperative that we are able to use FFPE archives and we have shown in this study that sWGS is a robust method to do such profiling., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Context: - The Ki-67 index is strongly prognostic and is used as a surrogate marker to distinguish luminal A from luminal B breast cancer types., Objective: - To investigate differences in Ki-67 index between core needle biopsy samples and matched surgical samples in breast cancer., Design: - We included patients with invasive breast cancer who did not receive neoadjuvant therapy. A total of 89 pairs of core needle biopsies and surgical specimens were collected, and the Ki-67 index was assessed in hot spot areas using an image analyzer. We applied a 14% Ki-67 index to define low versus high groups., Results: - The Ki-67 index was significantly higher in core needle biopsies than in surgical specimens ( P < .001), with a median absolute difference of 3.5%. When we applied 14% as a cutoff, 16 of 89 cases (18%) showed discrepancy. Thirteen cases showed a high Ki-67 index in core needle biopsies but a low Ki-67 index in surgical samples. There were 10 cases (11.2%) that showed discordant luminal A/B types between core needle biopsy and the matched surgical specimen. The reasons for the discordance were poor staining of MIB1 accompanied by fixation issues and intratumoral heterogeneity of the Ki-67 index., Conclusions: - A significant difference in the Ki-67 index between core biopsy and surgical specimens was observed. Our findings indicate that it may be better to perform the Ki-67 assay on the core needle biopsy and the surgical specimen than on only one sample.

Purpose: To determine whether apparent diffusion coefficient (ADC) values vary according to tumor-stroma ratio, dominant stroma type, or presence of central fibrosis in estrogen receptor-positive breast cancer., Materials and Methods: Institutional review board approval was obtained, and patient consent was waived. Sixty-one patients with estrogen receptor-positive invasive ductal carcinoma-not otherwise specified who underwent breast magnetic resonance (MR) imaging with diffusion-weighted (DW) imaging were included in this study. The ADC values of the lesions were measured. Two pathologists evaluated the tumor-stroma ratio, dominant stroma type (collagen, fibroblast, lymphocyte), and central fibrosis. Detectability on DW images was compared between the two groups according to the tumor-stroma ratio (stroma rich or stroma poor). Mean ADC values were retrospectively compared with the tumor-stroma ratio, dominant stroma type, and presence of a central fibrosis. Multiple linear regression analysis was performed to determine variables independently associated with ADC., Results: On DW images, detectability was not significantly different between stroma-rich and stroma-poor groups (P = .244). ADC values were significantly lower in the stroma-poor group (P < .001). The mean ADC values in the collagen-dominant type were lower than in fibroblast-dominant or lymphocyte-dominant types (P = .021). In multiple linear regression analysis, tumor-stroma ratio (P = .007), tumor size (P = .007), and dominant stroma type (collagen dominant, P = .029) were independently correlated with ADC., Conclusion: In estrogen receptor-positive breast cancers, ADC values showed significant differences according to the tumor-stroma ratio and dominant stroma type., (RSNA, 2013)

Fibroblasts in the stromal component of a tumor may influence tumor progression in various organs. The prognostic significance of tumor-infiltrating lymphocytes is also frequently reported. However, the prognostic significance of the stromal component in breast cancers, particularly those of high grade, has not been established. In this study, we analyzed surgically resected specimens from 545 patients with breast carcinoma, including 193 high-grade tumors, for tumor-stroma ratio, dominant stroma type [collagen (C), fibroblast (F), or lymphocyte (L) dominant type], and central fibrosis on hematoxylin-eosin-stained histological sections. We correlated these features with clinical prognosis. Among the 533 specimens examined, 127 (23.3 %) were of C type, 292 (53.6 %) of F type, and 114 (20.9 %) of L type. Central fibrosis was found in 99 tumors (18 %). The dominant stroma type was a significant prognostic factor on univariate and multivariate analyses, together with T classification, nodal status, and Bloom-Richardson grade. Tumor-stroma ratio and central fibrosis did not predict survival on multivariate analysis. Even in high-grade tumors, relapse-free intervals differed significantly according to dominant stroma type. Thus, conventional hematoxylin-eosin-stained tumor slides may contain more prognostic information than previously thought; in particular, the dominant stroma type in invasive breast cancer may potentially be used to predict outcome.