Pitfalls in machine learning-based assessment of tumor-infiltrating lymphocytes in breast cancer: A report of the International Immuno-Oncology Biomarker Working Group on Breast Cancer

The clinical significance of the tumor-immune interaction in breast cancer is now established, and tumor-infiltrating lymphocytes (TILs) have emerged as predictive and prognostic biomarkers for patients with triple-negative (estrogen receptor, progesterone receptor, and HER2-negative) breast cancer and HER2-positive breast cancer. How computational assessments of TILs might complement manual TIL assessment in trial and daily practices is currently debated. Recent efforts to use machine learning (ML) to automatically evaluate TILs have shown promising results. We review state-of-the-art approaches and identify pitfalls and challenges of automated TIL evaluation by studying the root cause of ML discordances in comparison to manual TIL quantification. We categorize our findings into four main topics: (1) technical slide issues, (2) ML and image analysis aspects, (3) data challenges, and (4) validation issues. The main reason for discordant assessments is the inclusion of false-positive areas or cells identified by performance on certain tissue patterns or design choices in the computational implementation. To aid the adoption of ML for TIL assessment, we provide an in-depth discussion of ML and image analysis, including validation issues that need to be considered before reliable computational reporting of TILs can be incorporated into the trial and routine clinical management of patients with triple-negative breast cancer. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Prediction of Pathologic Complete Response in Breast Cancer Patients Comparing Magnetic Resonance Imaging with Ultrasound in Neoadjuvant Setting.

BACKGROUND: Some subgroups of breast cancer patients receiving neoadjuvant chemotherapy (NACT) show high rates of pathologic complete response (pCR) in the breast, proposing the possibility of omitting surgery. Prediction of pCR is dependent on accurate imaging methods. This study investigated whether magnetic resonance imaging (MRI) is better than ultrasound (US) in predicting pCR in breast cancer patients receiving NACT. METHODS: This institutional, retrospective study enrolled breast cancer patients receiving NACT who were examined by either MRI or combined US and mammography before surgery from 2016 to 2019. Imaging findings were compared with pathologic response evaluation of the tumor. Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy for prediction of pCR were calculated and compared between MRI and US. RESULTS: Among 307 patients, 151 were examined by MRI and 156 by US. In the MRI group, 37 patients (24.5 %) had a pCR compared with 51 patients (32.7 %) in the US group. Radiologic complete response (rCR) was found in 35 patients (23.2 %) in the MRI group and 26 patients (16.7 %) in the US group. In the MRI and US groups, estimates were calculated respectively for sensitivity (87.7 % vs 91.4 %), specificity (56.8 % vs 33.3 %), PPV (86.2 % vs 73.8 %), NPV (60.0 % vs 65.4 %), and accuracy (80.1 % vs 72.4 %). CONCLUSIONS: In predicting pCR, MRI was more specific than US, but not sufficiently specific enough to be a valid predictor of pCR for omission of surgery. As an imaging method, MRI should be preferred when future studies investigating prediction of pCR in NACT patients are planned.

Synchronous bilateral breast cancer: a nationwide study on histopathology and etiology.

PURPOSE: The aim of the present study was to describe histopathologic characteristics of synchronous bilateral breast cancer (SBBC), and by comparing SBBC to unilateral breast cancer (UBC), identify possible etiological mechanisms of SBBC. METHODS: Patients with primary SBBC (diagnosed within 4 months) and UBC diagnosed in Denmark between 1999 and 2015 were included. Detailed data on histopathology were retrieved from the Danish Breast Cancer Group database and the Danish Pathology Register. Associations between bilateral disease and the different histopathologic characteristics were evaluated by odds ratios and estimated by multinomial regression models. RESULTS: 1214 patients with SBBC and 59,221 with UBC were included. Patients with SBBC more often had invasive lobular carcinomas (OR 1.29; 95% CI 1.13-1.47), a clinically distinct subtype of breast cancer, than UBC patients. Further, they were older than UBC patients, more often had multifocal cancer (OR 1.13; 95% CI 1.01-1.26), and a less aggressive subtype than UBC patients. Invasive lobular carcinoma was associated with having multiple tumors in breast tissue-both in the form of bilateral disease and multifocal disease, and this association was independent of laterality. No similar pattern was observed for other tumor characteristics. CONCLUSION: We identified two etiological mechanisms that could explain some of the occurrence of SBBC. The high proportion of less aggressive carcinomas and higher age of SBBC compared to UBC patients suggests that many are diagnosed at a subclinical stage as slow-growing tumors have a higher probability of simultaneous diagnosis. The high proportion of invasive lobular carcinoma observed in bilateral and multifocal disease, being independent of laterality, suggests that these patients have an increased propensity to malignant tumor formation in breast tissue.

Prognostic value of histopathological DCIS features in a large-scale international interrater reliability study.

PURPOSE: For optimal management of ductal carcinoma in situ (DCIS), reproducible histopathological assessment is essential to distinguish low-risk from high-risk DCIS. Therefore, we analyzed interrater reliability of histopathological DCIS features and assessed their associations with subsequent ipsilateral invasive breast cancer (iIBC) risk. METHODS: Using a case-cohort design, reliability was assessed in a population-based, nationwide cohort of 2767 women with screen-detected DCIS diagnosed between 1993 and 2004, treated by breast-conserving surgery with/without radiotherapy (BCS ± RT) using Krippendorff's alpha (KA) and Gwet's AC2 (GAC2). Thirty-eight raters scored histopathological DCIS features including grade (2-tiered and 3-tiered), growth pattern, mitotic activity, periductal fibrosis, and lymphocytic infiltrate in 342 women. Using majority opinion-based scores for each feature, their association with subsequent iIBC risk was assessed using Cox regression. RESULTS: Interrater reliability of grade using various classifications was fair to moderate, and only substantial for grade 1 versus 2 + 3 when using GAC2 (0.78). Reliability for growth pattern (KA 0.44, GAC2 0.78), calcifications (KA 0.49, GAC2 0.70) and necrosis (KA 0.47, GAC2 0.70) was moderate using KA and substantial using GAC2; for (type of) periductal fibrosis and lymphocytic infiltrate fair to moderate estimates were found and for mitotic activity reliability was substantial using GAC2 (0.70). Only in patients treated with BCS-RT, high mitotic activity was associated with a higher iIBC risk in univariable analysis (Hazard Ratio (HR) 2.53, 95% Confidence Interval (95% CI) 1.05-6.11); grade 3 versus 1 + 2 (HR 2.64, 95% CI 1.35-5.14) and a cribriform/solid versus flat epithelial atypia/clinging/(micro)papillary growth pattern (HR 3.70, 95% CI 1.34-10.23) were independently associated with a higher iIBC risk. CONCLUSIONS: Using majority opinion-based scores, DCIS grade, growth pattern, and mitotic activity are associated with iIBC risk in patients treated with BCS-RT, but interrater variability is substantial. Semi-quantitative grading, incorporating and separately evaluating nuclear pleomorphism, growth pattern, and mitotic activity, may improve the reliability and prognostic value of these features.

Basal biomarkers nestin and INPP4B predict gemcitabine benefit in metastatic breast cancer: Samples from the phase III SBG0102 clinical trial.

In a formal prospective-retrospective analysis of the phase III SBG0102 clinical trial randomizing metastatic breast cancer patients to gemcitabine-docetaxel or to single agent docetaxel, patients with basal-like tumors by PAM50 gene expression had significantly better overall survival in the gemcitabine arm. By immunohistochemistry (IHC), triple negative status was not predictive, but more specific biomarkers have since become available defining basal-like by nestin positivity or loss of inositol-polyphosphate-4-phosphate (INPP4B). Here, we evaluate their capacity to identify which patients benefit from gemcitabine in the metastatic setting. Nestin and INPP4B staining and interpretation followed published methods. A prespecified statistical plan evaluated the primary hypothesis that patients with basal-like breast cancer, defined as "nestin+ or INPP4B-", would have superior overall survival on gemcitabine-docetaxel when compared to docetaxel. Interaction tests, Kaplan-Meier curves and forest plots were used to assess prognostic and predictive capacities of biomarkers relative to treatment. Among 239 cases evaluable for our study, 36 (15%) had been classified as basal-like by PAM50. "Nestin+ or INPP4B-" was observed in 41 (17%) of the total cases and was significantly associated with PAM50 basal-like subtype. Within an estimated median follow-up of 13 years, patients assigned as IHC basal "nestin+ or INPP4B-" had significantly better overall survival on gemcitabine-docetaxel versus docetaxel monotherapy (HR = 0.31, 95%CI: 0.16-0.60), whereas no differences were observed for other patients (HR = 0.99), p-interaction < 0.01. In the metastatic setting, women with IHC basal breast cancers defined as "nestin+ or INPP4B-" have superior overall survival when randomized to gemcitabine-containing chemotherapy compared to docetaxel alone. These findings need to be validated using larger prospective-retrospective phase III clinical trials series.

Breast cancer mortality in synchronous bilateral breast cancer patients.

BACKGROUND: Evidence suggests that patients with synchronous bilateral breast cancer (SBBC), diagnosed within 4 months, have an inferior prognosis compared to unilateral breast cancer (UBC) patients. Using data from nationwide Danish clinical databases, this cohort study investigated whether the inferior prognosis could be explained by SBBC patients having a more aggressive disease, or whether the prognosis could be explained by the fact that they have two simultaneous cancers. METHODS: Patients were diagnosed from 1999-2015. The main outcome was excess mortality, subtracting background population mortality from observed mortality. Differences between SBBC and UBC patients were evaluated by rate ratios (RR) and estimated by Poisson regression. RESULTS: In total, 1214 SBBC and 59 177 UBC patients were included. SBBC patients had a significantly higher excess mortality than UBC patients after adjustment for age and period (RR = 1.73; 95% CI:1.44-2.08; p < 0.01) and after adjusting for characteristics of the worst tumour as traditionally done (RR = 1.31; 95% CI:1.08-1.57; p = 0.01). However, adjusting for characteristics of both tumours, using a more advanced competing risks model, no difference was observed (RR = 1.01; 95% CI:0.83-1.22; p = 0.93). CONCLUSIONS: Our study does not support that the inferior prognosis in SBBC patients is due to having more aggressive tumours per se, but rather the combined effect of having two simultaneous cancers.

PD-L1 expression in breast cancer: expression in subtypes and prognostic significance: a systematic review.

PURPOSE: To systematically review the literature on the expression of PD-L1 in primary BC, variation of expression between subtypes and effect on overall survival (OS), disease-free survival (DFS), and recurrence-free survival (RFS). Additionally, for studies in the neoadjuvant setting, we have reviewed the ability of PD-L1 to predict pathological complete response (pCR). METHODS: Articles included in this review were retrieved by searching PubMed (1966-2018) and EMBASE (1980-2018). The following search terms were used: "PD-L1 expression" and "breast cancer" (PubMed234; EMBASE 161). RESULTS: Thirty-seven articles were found relevant to this study. We summarize important findings from these works, and show that the observed PD-L1 expression in the studies varies greatly, with expression rates ranging from 0 to 83% across subtypes. PD-L1 expression in relation to prognosis both in the adjuvant and neoadjuvant chemotherapy setting remains controversial, with studies finding better, worse, or no effect on prognosis. We also show that a wide variety of strategies are used when evaluating PD-L1 immunohistochemically, e.g., different cut-off points, different cell types evaluated, and different perceptions of when a cell is positive for PD-L1 (cytoplasmic vs membrane staining). CONCLUSION: Further investigation of PD-L1 expression in breast cancer and its effect on prognosis is required. There is little consensus on the methods used to evaluate PD-L1 expression immunohistochemically, and this may contribute to the diverging results found in this study.

Sentinel and non-sentinel lymph node metastases in patients with microinvasive breast cancer: a nationwide study.

PURPOSE: To determine the incidence and risk factors of sentinel lymph node (SN) and non-SN metastases in patients with microinvasive breast cancer (MIBC, T1mic). This to identify MIBC patients in whom axillary staging can be safely omitted. METHODS: The Danish Breast Cancer Group database was used to identify a total of 409 women with breast cancer ≤ 1 mm who underwent sentinel lymph node biopsy (SLNB) between 2002 and 2015. After validation, 233 patients were eligible for the analysis. The incidence rates of SN and non-SN metastases were determined. The associations between clinicopathological variables and a positive SN [pN1, pN1mi, or pN0(i+)] were analyzed using univariate and multivariate designs. RESULTS: Of 233 patients with MIBC, only 9 (3.9%) had SN macrometastases. An additional 18 (7.7%) and 23 (9.9%) had SN micrometastases and isolated tumor cells (ITCs), respectively. Of patients with SN macrometastases, two (22.2%) had non-SN macrometastases. In the adjusted analysis, a positive SN was associated with younger age (P = 0.0001) and a positive human epidermal growth factor 2 receptor (HER2) status (P = 0.03). CONCLUSIONS: The low incidence of SN macrometastases < 4% suggests omission of axillary staging in MIBC patients without staging at primary surgery, especially in older (≥ 50 years) HER2- patients. Still, the relatively high proportion of patients with non-SN macrometastases indicates that axillary treatment might be considered in SN positive patients, especially in younger HER2+ MIBC patients.

Two open-label, single arm, non-randomized phase II studies of irinotecan for the treatment of metastatic breast cancer in patients with increased copy number of the topoisomerase I gene.

BACKGROUND: Treatment options in metastatic breast cancer are limited. New therapies preferable with predictive biomarkers are needed. The aim of these trials was to investigate if gene copy number of the topoisomerase 1 gene was predictive of response to the topoisomerase inhibitor irinotecan. METHODS: Two open-label, single-arm phase II studies including HER2 positive and negative patients were conducted. Patients were eligible for inclusion if the primary tumor or a metastatic lesion had increased expression of the topoisomerase 1 gene defined as a TOP1 gene copy number of ≥4 or a TOP1/CEN20 ratio of ≥2. Patients were treated with irinotecan +/- trastuzumab weekly for 4 weeks following 2 weeks break, until progression or unacceptable toxicities. Evaluation scans were performed every 6 weeks. Primary endpoint was clinical benefit rate defined as the fraction of patients with stable disease for ≥4 months. RESULTS: The pre-planned number of 18 patients in each trial was not reached, thus no formal statistical analysis could be performed. Nine patients with HER2 negative disease and three patients with HER2 positive disease were included. Three patients obtained a partial remission and two patients had SD. CONCLUSIONS: The trials did not include the planned number of patients. No association between gene copy number of the topoisomerase 1 gene and response to irinotecan could be proved, however a clinical benefit was found in 5/12 patients and in 2/3 patients with HER2 positive disease. This could call for further investigation of the drug in the metastatic setting, especially in HER2 positive BC. TRIAL REGISTRATION: Eudract registration numbers 2012-002348-26 and 2012-002347-23 . Registration date August 20th 2012.

Predicting efficacy of epirubicin by a multigene assay in advanced breast cancer within a Danish Breast Cancer Cooperative Group (DBCG) cohort: a retrospective-prospective blinded study.

PURPOSE: Anthracyclines remain a cornerstone in the treatment of primary and advanced breast cancer (BC). This study has evaluated the predictive value of a multigene mRNA-based drug response predictor (DRP) in the treatment of advanced BC with epirubicin. The DRP is a mathematical method combining in vitro sensitivity and gene expression with clinical genetic information from > 3000 clinical tumor samples. METHODS: From a DBCG cohort, 140 consecutive patients were treated with epirubicin between May 1997 and November 2016. After patient informed consent, mRNA was isolated from archival formalin-fixed paraffin-embedded primary breast tumor tissue and analyzed using Affymetrix arrays. Using time to progression (TTP) as primary endpoint, the efficacy of epirubicin was analyzed according to DRP combined with clinicopathological data collected retrospectively from patients' medical records. Statistical analysis was done using Cox proportional hazards model stratified by treatment line. RESULTS: Median TTP was 9.3 months. The DRP was significantly associated to TTP (P = 0.03). The hazard ratio for DRP scores differing by 50 percentage points was 0.55 (95% CI -0.93, one-sided). A 75% DRP was associated with a median TTP of 13 months compared to 7 months following a 25% DRP. Multivariate analysis showed that DRP was independent of age and number of metastases. CONCLUSION: The current study prospectively validates the predictive capability of DRP regarding epirubicin previously shown retrospectively allowing the patients predicted to be poor responders to choose more effective alternatives. Randomized prospective studies are needed to demonstrate if such an approach will lead to increased overall survival.

Triple negative breast cancer - prognostic role of immune-related factors: a systematic review.

PURPOSE: Treatment of breast cancer has been increasingly successful in recent years with the advent of HER2-receptor targeted treatment and endocrine treatment. However, the triple negative subgroup of breast cancer (TNBC) (estrogen-, progesterone- and HER2-receptor negative) still lacks targeted treatment options. TNBC is a type of breast cancer that often affects younger women, and generally has a worse prognosis than other types of breast cancer. Recently, the complex role of the immune system in cancer growth, elimination and metastasis has been the object of increased attention. There is hope that a more detailed understanding of the intricate roles of the constituents of the immune system, will hold potential both as prognostic or predictive markers of cancer progression, but also as treatment targets for a wide range of tumors, including TNBC. The aim of this review is to provide an overview of the cellular immune microenvironment in TNBC, and to highlight areas in which TNBC may differ from other types of breast cancer. MATERIAL AND METHODS: A search of PubMed was made using the terms 'triple negative breast cancer' and 'tumor infiltrating lymphocytes', 'CD8', 'CD4', 'B cells', 'natural killer cells', 'macrophages', myeloid derived suppressor cells', 'dendritic cells', 'immune check point inhibitor', 'CTLA-4' and 'PD-L1'. RESULTS: We find that whilst factors such as TILs and certain subgroups of TILs (e.g., CD8 + and regulator T-cells) have been extensively researched, none of these markers are currently applicable to routine clinical practice. Also, TNBC differs from other types of breast cancer with regards to cellular composition of the immune infiltrate and PD-L1 expression, and the prognostic significance of these. CONCLUSIONS: Immune-related factors have the potential as both prognostic and predictive biomarkers for new treatments targeting the immune system in breast cancer. However, multivariate analyses, taking other well-known factors into account, are required to determine the true value of these biomarkers. Also, differences between TNBC and other types of breast cancer may have implications for treatment and use of immune-related factors as biomarkers.

Mortality and recurrence rates among systemically untreated high risk breast cancer patients included in the DBCG 77 trials.

BACKGROUND: Following loco-regional treatment for early breast cancer accurate prognostication is essential for communicating benefits of systemic treatment. The aim of this study was to determine time to recurrence and long-term mortality rates in high risk patients according to patient characteristics and subtypes as assigned by immunohistochemistry panels. PATIENTS AND METHODS: In November 1977 through January 1983, 2862 patients with tumors larger than 5 cm or positive axillary nodes were included in the DBCG 77 trials. Archival tumor tissue from patients randomly assigned to no systemic treatment was analyzed for ER, PR, Ki67, EGFR and HER2. Intrinsic subtypes were defined as follows: Luminal A, ER or PR >0%, HER2-negative, PR >10% and Ki67 < 14%; Luminal B, ER or PR >0%, (PR ≤10% or HER2-positive or Ki67 ≥ 14%); HER2E, ER 0%, PR 0%, HER2 positive; Core basal, ER 0%, PR 0%, HER2 negative and EGFR positive. Multivariate categorical and fractional polynomials (MFP) models were used to construct prognostic subsets by clinicopathologic characteristics. RESULTS: In a multivariate model, mortality rate was significantly associated with age, tumor size, nodal status, invasion, histological type and grade, as well as subtype classification. CONCLUSIONS: With 35 years of follow-up, in this population of high-risk patients with no systemic therapy, no subgroup based on a composite prognostic score and/or molecular subtypes could be identified without excess mortality as compared to the background population.

An inter-observer Ki67 reproducibility study applying two different assessment methods: on behalf of the Danish Scientific Committee of Pathology, Danish breast cancer cooperative group (DBCG).

INTRODUCTION: In 2011, the St. Gallen Consensus Conference introduced the use of pathology to define the intrinsic breast cancer subtypes by application of immunohistochemical (IHC) surrogate markers ER, PR, HER2 and Ki67 with a specified Ki67 cutoff (>14%) for luminal B-like definition. Reports concerning impaired reproducibility of Ki67 estimation and threshold inconsistency led to the initiation of this quality assurance study (2013-2015). The aim of the study was to investigate inter-observer variation for Ki67 estimation in malignant breast tumors by two different quantification methods (assessment method and count method) including measure of agreement between methods. MATERIAL AND METHODS: Fourteen experienced breast pathologists from 12 pathology departments evaluated 118 slides from a consecutive series of malignant breast tumors. The staining interpretation was performed according to both the Danish and Swedish guidelines. Reproducibility was quantified by intra-class correlation coefficient (ICC) and Lights Kappa with dichotomization of observations at the larger than (>) 20% threshold. The agreement between observations by the two quantification methods was evaluated by Bland-Altman plot. RESULTS: For the fourteen raters the median ranged from 20% to 40% by the assessment method and from 22.5% to 36.5% by the count method. Light's Kappa was 0.664 for observation by the assessment method and 0.649 by the count method. The ICC was 0.82 (95% CI: 0.77-0.86) by the assessment method vs. 0.84 (95% CI: 0.80-0.87) by the count method. CONCLUSION: Although the study in general showed a moderate to good inter-observer agreement according to both ICC and Lights Kappa, still major discrepancies were identified in especially the mid-range of observations. Consequently, for now Ki67 estimation is not implemented in the DBCG treatment algorithm.

Risk of non-sentinel node metastases in patients with symptomatic cancers compared to screen-detected breast cancers.

BACKGROUND: Symptomatic breast cancers may be more aggressive as compared to screen-detected breast cancers. This could favor axillary lymph node dissection (ALND) in patients with symptomatic breast cancer and positive sentinel nodes. METHOD: We identified 955 patients registered in the Danish Breast Cancer Cooperative Group (DBCG) Database in 2008 - 2010 with micrometastases (773) or isolated tumor cells (ITC) (182) in the sentinel node. Patients were cross-checked in the Danish Quality Database of Mammography Screening and 481 patients were identified as screen-detected cancers. The remaining 474 patients were considered as having symptomatic cancers. Multivariate analyses of the risk of non-sentinel node metastases were performed including known risk factors for non-sentinel node metastases as well as method of detection. RESULTS: 18% of the patients had metastases in non-sentinel nodes. This was evenly distributed between patients with symptomatic and screen-detected cancers; 18.5% vs 17.5% (OR 1.07; 95% CI 0.77-1.49; p = 0.69). In patients with micrometastases 21% had non-sentinel node metastases in the group with symptomatic cancers compared to 19% of patients with screen-detected cancers. This difference was not significant (OR 1.16; 95% CI 0.81-1.65, p = 0.43). Neither the multivariate analysis showed an increased risk of non-sentinel node metastases in patients with symptomatic cancers compared to screen-detected cancers (OR 1.12, CI 0.77-1.62, p = 0.55). In patients with ITCs 8% of patients with symptomatic cancers had non-sentinel node metastases compared to 13% of patients with screen-detected cancers. This difference was not significant (OR 0.58; 95% CI 0.22-1.54, p = 0.27). In the multivariate analysis, the risk of non-sentinel node metastases was still not significantly increased in patients with symptomatic cancers compared to screen-detected cancers (OR 0.45; 95% CI 0.16-1.27, p = 0.13). CONCLUSION: We did not find any clinically relevant difference in the risk of non-sentinel node metastases between patients with symptomatic and screen-detected cancers with micrometastases or ITC in the sentinel node.

Time trends in axilla management among early breast cancer patients: Persisting major variation in clinical practice across European centers.

Background We examined time trends in axilla management among patients with early breast cancer in European clinical settings. Material and methods EUROCANPlatform partners, including population-based and cancer center-specific registries, provided routinely available clinical cancer registry data for a comparative study of axillary management trends among patients with first non-metastatic breast cancer who were not selected for neoadjuvant therapy during the last decade. We used an additional short questionnaire to compare clinical care patterns in 2014. Results Patients treated in cancer centers were younger than population-based registry populations. Tumor size and lymph node status distributions varied little between settings or over time. In 2003, sentinel lymph node biopsy (SLNB) use varied between 26% and 81% for pT1 tumors, and between 2% and 68% for pT2 tumors. By 2010, SLNB use increased to 79-96% and 49-92% for pT1 and pT2 tumors, respectively. Axillary lymph node dissection (ALND) use for pT1 tumors decreased from between 75% and 27% in 2003 to 47% and 12% in 2010, and from between 90% and 55% to 79% and 19% for pT2 tumors, respectively. In 2014, important differences in axillary management existed for patients with micrometastases only, and for patients fulfilling the ACOSOG Z0011 criteria for omitting ALND. Conclusion This study demonstrates persisting differences in important aspects of axillary management throughout the recent decade. The results highlight the need for international comparative patterns of care studies in oncology, which may help to identify areas where further studies and consensus building may be necessary.

Topoisomerase-1 gene copy aberrations are frequent in patients with breast cancer.

Topoisomerase-1 (Top1) targeting drugs have shown promising efficacy in patients with metastatic breast cancer (BC). However, these drugs are rather toxic calling for development and validation of predictive biomarkers to increase the therapeutic index. As these drugs are targeting the Top1 protein and since no validated anti-Top1 antibodies for immunohistochemistry have been reported, we raised the hypothesis that TOP1 gene amplifications may serve as a proxy for the Top1 protein and thereby a biomarker of response to treatment with Top1 inhibitors in BC. The aim was to determine the prevalence of TOP1 gene copy gain in BC. The prevalence of TOP1 gene copy gain was investigated by fluorescence in situ hybridization with a TOP1/CEN-20 probemix in normal breast tissue (N = 100) and in tissue from patients with metastatic BC in a discovery (N = 100) and a validation cohort (N = 205). As amplification of 20q including CEN-20 is common in BC a TOP1/CEN-2 probemix was applied to the validation cohort. More than 30% of the patients had gene copy numbers of ≥ 4 and ∼20% of the patients had TOP1/CEN-20 ratios ≥ 1.5. The CEN-2 probe did not add any information. Gain of the TOP1 gene appears to be common in BC making the gene a potential biomarker for response to treatment with Top1 inhibitors. As 20q amplification is a common finding in BC and as no other suitable reference gene has yet been identified, TOP1 copy number may be a more valid method of detecting gain than using a gene/centromere ratio.

Prognostic significance of axillary dissection in breast cancer patients with micrometastases or isolated tumor cells in sentinel nodes: a nationwide study.

We estimated the impact of axillary lymph node dissection (ALND) on the risk of axillary recurrence (AR) and overall survival (OS) in breast cancer patients with micrometastases or isolated tumor cells (ITC) in sentinel nodes. We used the Danish Breast Cancer Cooperative Group (DBCG) database to identify patients with micrometastases or ITC in sentinel nodes following surgery for primary breast cancer between 2002 and 2008. A Cox proportional hazard regression model was developed to assess the hazard ratios (HR) for AR and OS between patients with and without ALND. We identified 2074 patients, of which 240 did not undergo further axillary surgery. The 5-year cumulated incidence for AR was 1.58 %. No significant difference in AR was seen between patients with and without ALND. The age adjusted HR for AR if ALND was omitted was 1.79 (95 % CI 0.41-7.80, P = 0.44) in patients with micrometastases and 2.21 (95 % CI 0.54-8.95, P = 0.27), in patients with ITC after a median follow-up of 6 years and 3 months. There was no significant difference in overall survival between patients with and without ALND, when adjusting for age, co-morbidity, tumor size, histology type, malignancy grade, lymphovascular invasion, hormone receptor status, adjuvant systemic treatment and radiotherapy, with a HR for death if ALND was omitted of 1.21 (95 % CI 0.86-1.69, P = 0.27) in patients with micrometastases and 0.96 (95 % CI 0.57-1.62, P = 0.89) in patients with ITC after a medium follow-up on 8 and 5 years. In this nationwide study, we found a low risk of AR on 1.58 % and we did not find a significantly increased risk of AR if ALND was omitted in patients with micrometastases or ITC in sentinel nodes. Furthermore, no significant difference in overall survival was seen between patients with and without ALND when adjusting for adjuvant treatment.

Reduced risk of axillary lymphatic spread in triple-negative breast cancer.

We examined the association between the hormone receptor (HR) and human epidermal growth factor receptor 2 (HER2) status of women with primary breast cancer and the risk of axillary lymph node (ALN) involvement at the time of diagnosis. Information on 20,009 women diagnosed with primary breast cancer between 2008 and 2012 was retrieved from the Danish Breast Cancer Cooperative Group database. The associations between clinical and pathological variables and ALN involvement at the time of diagnosis were evaluated in univariate and multivariate regression analyses, as well as the significance of tumor subtypes in ALN involvement. The risk of ALN metastases at the time of diagnosis was significantly reduced in HR-negative patients compared to HR-positive patients [adjusted odds ratio (OR) 0.69; 95 % CI 0.63-0.76; P = 0.0009]. A HER2-positive status was associated with an increased risk of ALN involvement at diagnosis compared to a HER2-negative status (OR 1.37; 95 % CI 1.24-1.50; P < 0.0001). An interaction between HER2 and HR was observed, with a HER2-positive status significantly associated with ALN involvement at the time of diagnosis only in HR-negative patients (P < 0.0001). The triple-negative breast cancer (TNBC) patients showed a significantly reduced risk of ALN involvement at the time of diagnosis compared to patients with HR-positive/HER2-negative tumors (OR 0.55; 95 % CI 0.49-0.62; P < 0.0001). The HR and HER2 statuses are significantly associated with ALN involvement at the time of diagnosis. Despite the poor prognosis, TNBC patients have a reduced risk of ALN involvement at the time of diagnosis compared to patients with other subtypes, when adjusting for other risk factors. This may indicate that TNBC tends to spread hematogenously rather than lymphogenously.

A phase II study of weekly irinotecan in patients with locally advanced or metastatic HER2- negative breast cancer and increased copy numbers of the topoisomerase 1 (TOP1) gene: a study protocol.

BACKGROUND: About 20% of patients with primary breast cancer develop metastatic disease during the course of the disease. At this point the disease is considered incurable and thus treatment is aimed at palliation and life prolongation. As many patients will have received both an anthracycline and a taxane in the adjuvant setting, treatment options for metastatic breast cancer are limited. Furthermore response rates for the most commonly used drugs range from around 30% to 12% . Thus new treatment options are needed and preferably coupled to biomarkers predictive of response. Irinotecan is a topoisomerase 1 inhibitor used for decades for the treatment of colorectal cancer. Four studies have investigated the efficacy of irinotecan monotherapy in breast cancer and all have included non-biomarker selected patients. In these studies response rates for irinotecan ranged from 5%-23% and are thus comparable to response rates obtained with drugs commonly used in the metastatic setting. If a predictive biomarker could be identified for irinotecan, response rates might be even higher. METHODS/DESIGN: This multi-centre phase II single arm trial was designed to investigate if patients with metastatic breast cancer and increased expression of the topoisomerase 1 gene have a high likelihood of obtaining a clinical benefit from treatment with irinotecan. Trial recruitment is two-staged as 19 patients are planned to participate in the first part. If less than 7 patients have clinical benefit the trial stops, if more than 7 patients have clinical benefit a total of 40 patients will be included. DISCUSSION: This ongoing trial is the first to prospectively test copy number of the topoisomerase I gene as a predictive biomarker of response to irinotecan. TRIAL REGISTRATION: EudraCT number 2012-002348-26 .

Prognosis of synchronous bilateral breast cancer: a review and meta-analysis of observational studies.

Currently, no consistent evidence-based guidelines for the management of synchronous bilateral breast cancer (SBBC) exist and it is uncertain how presenting with SBBC affects patients' prognosis. We conducted a review of studies analyzing the association between SBBC and prognosis. The studies that reported adjusted effect measures were included in meta-analyses of effect of bilaterality on breast cancer mortality. From 57 initially identified records 17 studies from 11 different countries including 8,050 SBBC patients were included. The quality of the studies varied but was generally low with small sample sizes, and lack of consistent, detailed histo-pathological information. When doing meta-analysis on the subgroup of studies that provided adjusted effect estimates on breast cancer mortality (nine studies including 3,631 SBBC cases), we found that bilaterality in itself had a negative impact on prognosis after adjustment for known prognostic factors (pooled HR 1.37, 95 % CI 1.24-1.50, p < 0.0001). Multiple sensitivity analyses indicated robustness of the overall estimate. This review summarizes the current evidence of the association between SBBC and prognosis. The previously accepted convention that appropriate adjuvant treatment can be determined by considering the higher risk cancer was not confirmed in this review; rather it seems that being diagnosed with two tumors simultaneously entails a worse prognosis above and beyond that of the unilateral cancers of the same stage. To determine the true association between SBBC and breast cancer prognosis, studies of large and updated samples of SBBC should be done and include thorough histo-pathologic information.