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Aims: Digital image analysis (DIA) is used increasingly as an assisting tool to evaluate biomarkers, including human epidermal growth factor receptor 2 (HER2) in invasive breast cancer (BC). DIA can assist pathologists in HER2 evaluation by presenting quantitative information about the HER2 staining in APP assisted reading (AR). Concurrently, the HER2-low category (HER2-1+/2+ without HER2 gene amplification) has gained prominence due to newly developed antibody-drug conjugates. However, major inter- and intraobserver variability have been observed for the entity. The present quality assurance study investigated the concordance between DIA and AR in clinical use, especially concerning the HER2-low category. Methods and results: HER2 immunohistochemistry (IHC) in 761 tumours from 727 patients was evaluated in tissue microarray (TMA) cores by DIA (Visiopharm HER2-CONNECT) and AR. Overall concordance between HER2-scores were 73% (n = 552, weighted-κ: 0.66), and 88% (n = 669, weighted-κ: 0.70), when combining HER2-0/1+. A total of 205 scores were discordant by one category, while four were discordant by two categories. A heterogeneous HER2 pattern was relatively common in the discordant cases and a pitfall in the categorisation of HER2-low BC. AR more commonly reassigned a lower HER2 score (from HER2-1+ to HER2-0) within the HER2-low subgroup (n = 624) compared with DIA. Conclusion: DIA and AR display moderate agreement with heterogeneous and aberrant staining, representing a source of discordance and a pitfall in the evaluation of HER2.

Background: Superficial spreading melanomas (SSMs) are the most common type of melanoma and cause the majority of skin cancer deaths. More than 50% of cases harbor a mutation in the BRAF gene that activates the mitogen-activated protein kinase (MAPK) cancer signaling pathway. BRAFV600E is the most common BRAF mutation, and it represents an important biomarker that guides treatment selection. However, the relationship between the BRAFV600E gene expression and intratumoral protein distribution, on one side, and clinicopathological factors and patient outcomes, on the other, is not fully described. Additionally, whether MAPK cancer signaling activation in melanoma is due to increased biochemical activity of BRAFV600E, increased mRNA levels, or both requires further investigation. Here, we addressed these questions by examining expression patterns of BRAFV600E in primary treatment-naive melanomas and correlating them to clinicopathological factors and patient outcomes. Methods: In 166 SSM cases, we performed immunohistochemical staining to investigate the protein expression of BRAFV600E, and we measured BRAF mRNA levels using NanoString nCounter system. Results: Ninety-seven (49%) melanomas stained positive for BRAFV600E, with nearly 100% intratumoral homogeneity observed. Positive BRAFV600E expression was significantly associated with nonrecurrent disease and was found to be an independent predictor of better prognosis in univariate and multivariable analyses. Furthermore, presence of tumor-infiltrating lymphocytes, sentinel lymph node biopsy negativity, and low Breslow thickness were all independent predictors of better prognosis. We observed no difference in the BRAF mRNA levels in BRAFV600E-negative and BRAFV600E-positive melanomas, respectively. Validation in a larger publicly available cohort confirmed that there is only a weak correlation (Spearman 0.4) between BRAFV600E mRNA and protein levels and no differences in mRNA between BRAFV600E mutated and non-mutated patients. Conclusion: Our findings indicated that BRAFV600E is homogeneously present throughout the whole tumor and is associated with nonrecurrent disease and better survival in primary melanoma. We also showed that BRAFV600E mutation does not result in higher transcriptional levels, suggesting that activation of the MAPK signaling pathway in BRAFV600E mutated patients can be attributed to the increased biochemical activity caused by the mutation.

Estrogen receptor positive (ER+) breast cancer constitutes almost 85% of all breast cancer patients and are a genetically highly heterogenic group. Data on the association of somatic alterations to outcome and prognosis are however sparse. In this neoadjuvant endocrine phase II trial including postmenopausal breast cancer patients with ER+, HER2 normal breast cancer, we investigated the rate of pathogenic mutations before and after treatment as well as the association with treatment response and survival. Pretreatment and posttreatment tumour samples from 109 patients treated with neoadjuvant letrozole were collected and analysed with Next Generation Sequencing utilizing a panel of 12 genes (ALK, BRAF, EGFR, ERBB2, ERBB3, ESR1, KIT, KRAS, NRAS, PDGFRA, PIK3CA, and RAF1). Residual disease was assessed by a modified Miller Payne scale and the Residual Cancer Burden index. Survival data were collected prospectively. Among the 109 patients, 52 had at least one pathogenic mutation in the pretreatment sample and 60 in the posttreatment sample. The most frequently mutated gene was PIK3CA, followed by EGFR and KRAS. Twelve different pathogenic PIK3CA mutations were identified, primarily in exon 20 and exon 9. An altered PIK3CA mutation profile from the pre- to the posttreatment specimen was significantly associated to improved pathological outcome. Overall and Disease-Free Survival benefits in PIK3CA mutated patients was observed. Considerable heterogeneity was identified both among patients and between pre- and posttreatment samples. PIK3CA has the potential to be a predictive biomarker. To further assess the implications of a treatment related altered PIK3CA mutation profile, more data are needed.

Purpose The Prosigna-PAM50 risk of recurrence (ROR) score has documented clinical utility for the prediction of 10-year distant recurrence (DR). The present study investigated the value of Prosigna-PAM50 for predicting 10-year DR and overall survival after 5 years of endocrine treatment for postmenopausal patients with invasive lobular carcinoma. Patients and Methods Using the Danish Breast Cancer Group database, we identified patients with a diagnosis from 2000 to 2003 of estrogen receptor-positive, human epidermal growth factor receptor 2-negative invasive ductal (n = 1570) or lobular (n = 341) cancer > 20 mm or 1 to 3 positive lymph nodes and applied multivariate Cox models. Results The median follow-up for DR was 9.3 years and for overall survival 15.2 years. Of the 341 lobular and 1570 ductal cases, 140 (41%) and 349 (22%) were classified as low ROR, with a 10-year DR rate of 7.7% (95% confidence interval [CI], 3.7%-13.6%) and 3.5% (95% CI, 1.8%-6.2%), respectively. The 10-year DR rate for the intermediate ROR group for those with lobular cancer was 18% (95% CI, 10.1%-27.9%) compared with 9.7% (95% CI, 6.7%-13.4%) for those with ductal cancer. Luminal B tumors had a significantly worse outcome than luminal A tumors in both lobular (hazard ratio, 1.89; 95% CI, 1.03%-3.45%; P = .04) and ductal (hazard ratio, 3.18; 95% CI, 2.29%-4.43%; P Conclusion Prosigna PAM-50 provides significant prognostic information beyond the clinicopathologic factors in patients with invasive lobular breast cancer. Those with lobular cancer had worse 10-year DR rates compared with those with ductal cancer in the same ROR category. Our results could have an effect on the treatment decisions regarding the addition of chemotherapy for those in the intermediate ROR group.

Background: Patients with a BRCA1 or BRCA2 mutation (BRCA-mutated breast cancer) are frequently diagnosed with low differentiated and highly proliferating breast cancer characterized by high amounts of tumor-infiltrating lymphocytes (Tils). Stromal Tils (sTils) are highly prognostic in sporadic triple-negative and HER2 positive breast cancer however, their prognostic importance in BRCA-mutated breast cancers is unknown. Material and methods: Formalin-fixed paraffin-embedded primary tumor tissue from 411 patients with a germline BRCA1 or BRCA2 mutation and diagnosed with early breast cancer was included. The percentage of sTils was quantified on full HE sections according to guidelines proposed by the Immuno-Oncology Biomarker in Breast Cancer Working Group. Distribution of sTils and associates with patient and tumor characteristics were assessed according to categorical sTils groups defined as low (

The Prosigna-PAM50 risk of recurrence (ROR) score has been validated in randomized clinical trials to predict 10-year distant recurrence (DR) in hormone receptor-positive breast cancer. Here, we examine the ability of Prosigna for predicting DR at 10 years in a subgroup of postmenopausal breast cancer patients with special histological subtypes.Using the population based Danish Breast Cancer Group database, follow-up data were collected on all patients diagnosed from 2000 to 2003 with estrogen receptor (ER)-positive and human epidermal growth factor receptor 2 (HER2) normal breast cancer who by nationwide guidelines were treated with 5 year of endocrine therapy (N = 2558). Among patients with 1 to 3 positive lymph nodes or a tumor size20 mm, we identified 1570 with invasive ductal carcinoma (IDC) and 89 with special histological subtypes (apocrine, medullary, mucinous, papillary, secretory, tubular, neuroendocrine) who were tested with Prosigna. Fine and Gray models were applied to determine the prognostic value of the Prosigna-PAM50 ROR score for DR special subtypes as compared to IDC.Median follow-up for DR was 9.2 year and for OS 15.2 year. The 10-year DR rate for the special subtypes was 9.2% (95% CI: 4.0% to 17.2%) as compared to 13.7% (95% CI: 11.9% to 15.7%) for IDC. The 10-year OS was 74.2% (95% CI: 63.7% to 82.0%) for the special subtypes and 75.4% (95% CI: 73.2% to 77.4%) for IDC. Prosigna showed a statistical significant association of the continuous ROR score with risk of DR for both IDC and the special subtypes (IDC: p .0001; special subtypes: p = .01).In the present study, we demonstrated that Prosigna-PAM50 continuous ROR score added significant prognostic information for 10-year DR in postmenopausal patients with special subtypes (tumor size20 mm or 1 to 3 positive lymph nodes) and ER-positive, HER2-normal early breast cancer.

In breast cancer (BC) patients a cancer predisposing BRCA1/2 mutation is associated with adverse tumor characteristics, risk assessment and treatment allocation. We aimed to estimate overall- (OS) and disease-free survival (DFS) according to tumor characteristics and treatment among women who within two years of definitive surgery for primary BC were shown to carry a mutation in BRCA1/2 .From the clinical database of the Danish Breast Cancer Group we included 141 BRCA1 and 96 BRCA2 BC patients. Estrogen receptor and HER2 status were centrally reviewed on paraffin-embedded tumor tissue. Information on risk reducing surgery was obtained from the Danish Pathology and Patient Registries and included as time-dependent variables in Cox proportional hazard models.Ten-year OS and DFS for BRCA1 BC patients were 78% (95% CI 69-85) and 74% (95% CI 64-81). Ten-year OS and DFS for BRCA2 BC were 88% (95% CI 78-94) and 84% (95% CI 74-91). BRCA1 BC patients as compared to BRCA2 BC patients had a higher risk of BC relapse or non-breast cancer within ten years of follow-up, independent of ER status (adjusted HR 2.78 95% CI 1.28-6.05, p = .01), but BRCA mutation was not associated with OS (adjusted HR 1.98, 95% CI 0.87-4.52, p = .10). In multivariate analysis, including both BRCA1 and BRCA2 carriers, no chemotherapy was associated with a higher risk of death (adjusted OS HR 3.58, 95% CI 1.29-9.97, p = .01) and risk reducing contralateral mastectomy (RRCM) was associated with a significantly reduced risk of death (adjusted OS HR 0.42, 95% CI =0.21-0.84, p = .01).Difference in OS between BRCA1 and BRCA2 BC patients could be ascribed to tumor-biology. BRCA1 BC patients may have a shorter ten-year DFS than BRCA2 BC patients. Chemotherapy and risk reducing contralateral mastectomy reduce mortality for both BRCA1 and BRCA2 BC patients.

Background: The Prosigna (PAM50) risk of recurrence (ROR) score predicts 10yr DR in early breast cancer patients treated with ET alone (level 1 evidence). Invasive lobular breast cancer (ILBC) accounts for 10-15% of all breast cancer histological subtypes. Sporadic ILBC is characterized by somatic CDH1 mutations with loss of E-cadherin and a majority of low proliferative ER positive/HER2 negative tumors consistent with Luminal A subtype. Here we examine the ability of PAM50/ROR to predict 10yr DR in postmenopausal women who following a diagnosis of HR+ early ILBC were allocated to 5yr of ET alone. Methods: Using the population based clinical DBCG database FFPE primary tumor blocks, treatment, and follow-up data were collected from all patients diagnosed from 2000-2003 with HR+, postmenopausal EBC (N0-N1) who by nationwide guidelines were allocated to 5yr of ET alone, N=2,722 (1256 N0, 1466 N1). PAM50 intrinsic subtype classification (Luminal A, Luminal B, HER2-enriched, Basal-like) was conducted using the NanoString nCounter®Analysis System. Univariate and multivariate analyses tested the ability of PAM50 to predict DR. Patients were categorized as Low or High Risk based upon pre-specified ROR cutoff value of 40. HER2 positive tumors by immunohistochemistry were excluded from analysis. Results: Median follow-up was 9.25 years. Risk of 10yr DR by ROR and PAM50 (Luminal A and Luminal B) is shown in the table by ILBC as compared to invasive ductal breast cancer (IDBC) type. Cumulative incidence for 10 yr distant recurrence (DR)Histological subtypeRisk Group % [95% CI](N row%)Intrinsic Subtype % [95% CI](N row%)Histological subtype% [95% CI](N) LowHighLuminal ALuminal BAllDuctal3.8 [2.5-5.6] (738 35%)16.6 [14.4-18.8] (1388 65%)6.6 [5.1-8.4] (1174 59%)18.1 [15.2-21.3] (832 41%)12.1% [10.6-13.7] (2126)Lobular9.7 [5.5-15.4] (181 53%)23.9 [16.8-31.6] (159 47%)12.7 [8.6-17.8] (256 77%)24.1 [14.5-35.1] (77 23%)16.4 [12.2-21.1] (340) In this specific cohort the ILBC subgroup had a worse 10yr DR of 16.4% [12.2-21.1] as compared to IDBC of 12.1% [10.6-13.7] (Gray´s test, p = 0.046). A significant difference regarding the distribution of ILBC into both High and Low Risk Group and intrinsic subtypes as compared to IDBC was identified (p < 0.0001). Molecular intrinsic subtype analysis for ILBC by Prosigna (PAM50) showed DR 12.7 [8.6-17.8] for Luminal A vs 24.1 [14.5-35.1] for Luminal B. The difference between ILBC subtypes was not significant (p = 0.09). Adding ROR to a Fine and Gray's proportional sub-hazards model containing clinical and pathological variables significantly improved the model for ILBC, (likelihood ratio: p = 0.0001); HR for a 20-point change in ROR = 1.84 [1.37-2.48] notable with DR 9.7 [5.5-15.4] for the Low Risk Group. Conclusions: Following 5-yr of endocrine treatment alone patients with ILBC in this large population-based study had an inferior DR as compared to patients with IDBC and the apparent difference between ILBC and IDBC must be interpreted with caution. Citation Format: Laenkholm A-V, Jensen M-B, Buckingham W, Schaper C, Knoop A, Eriksen JO, Rasmussen BB, Ferree S, Haffner T, Kiboel T, Ejlertsen B. Prediction of 10yr distant recurrence (DR) using the Prosigna® (PAM50) assay in histological subgroups of a Danish breast cancer group (DBCG) cohort of postmenopausal Danish women with hormone receptor-positive (HR+) early breast cancer (EBC) allocated to 5yr of endocrine therapy (ET) alone [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-07-10.

Summary Approximately 3% to 5% of breast cancer patients are BRCA1 or BRCA2 germline mutation carriers. In this study, we correlated the distribution of intrinsic molecular subtypes according to failure pattern in a Danish cohort of BRCA germline–mutated breast cancer patients. Tumor tissues from 425 BRCA germline–mutated breast cancer patients were analyzed by immunohistochemistry for hormone receptor status, proliferation index (Ki-67), and HER2. Surrogate intrinsic molecular subtypes were assigned according to approximated St Gallen criteria. Annual hazard rates (AHR) were calculated for death and local or distant relapse, contralateral breast cancer, new primary cancer other than breast cancer, or death as first event (disease-free event). Luminal A–like subtype was observed with a frequency of 9% and 35% for BRCA1 and BRCA2, respectively, and for both BRCA1 and BRCA2 patients, the luminal B–like subtype was more frequent than the luminal A–like subtype (BRCA1 21% and BRCA2 40% luminal B–like). No events or deaths were observed for luminal A–like subtype during the first 2.5 and 0 to 5 years, respectively. AHRs for luminal B–like tumors were 5.34% (95% confidence interval [CI], 1.49-1.19) and 1.76% (95% CI, 0.36-3.16) for disease-free event and death, respectively, and those for basal-like were 6.58% (95% CI, 2.98-10.18) and 4.54% (95% CI, 2.69-6.40). A substantial proportion of BRCA carriers had luminal A–like subtype, and these were mainly BRCA2 carriers. Luminal A–like subtype was significantly associated with low AHR the first 5 years after surgery. This study warrants further exploration of the impact of the molecular intrinsic subtypes on survival in BRCA-mutated breast cancer patients.

Background: The PAM50-based (Prosigna) risk of recurrence (ROR) score and intrinsic subtypes are prognostic for women with high-risk breast cancer. We investigate the predictive ability of Prosigna regarding the effectiveness of cyclophosphamide-based adjuvant chemotherapy in premenopausal patients with high-risk breast cancer. Methods: Prosigna assays were performed on the NanoString platform in tumors from participants in Danish Breast Cancer Group (DBCG) 77B, a four-arm trial that randomized premenopausal women with high-risk early breast cancer to no systemic treatment, levamisole, oral cyclophosphamide (C) or cyclophosphamide, methotrexate and fluorouracil (CMF). Results: In total, this retrospective analysis included 460 women (40% of the 1146 randomized patients). The continuous Prosigna ROR score was prognostic in the no systemic treatment group (unadjusted P

Purpose The PAM50-based Prosigna risk of recurrence (ROR) score has been validated in randomized clinical trials to predict 10-year distant recurrence (DR). The value of Prosigna for predicting DR was examined in a comprehensive nationwide Danish cohort consisting of postmenopausal women with hormone receptor–positive early breast cancer treated with 5 years of endocrine therapy alone. Patients and Methods Using the population-based Danish Breast Cancer Cooperative Group database, follow-up data were collected on all patients diagnosed from 2000 through 2003 who, by nationwide guidelines, were treated with endocrine therapy for 5 years. Primary tumor blocks from 2,740 patients were tested with Prosigna and, after determination of human epidermal growth factor receptor 2 (HER2) status, data from 2,558 hormone receptor–positive/HER2-negative samples were analyzed, including 1,395 node-positive patients. Fine and Gray models were applied to determine the prognostic value of ROR for DR. Results Median follow-up for recurrence was 9.2 years. Twenty-six percent of the node-positive patients were classified as low ROR (n = 359) with a DR risk of 3.5% (95% confidence interval [CI], 1.9% to 6.1%) versus a DR risk of 22.1% (95% CI, 18.6% to 25.8%) at 10 years for patients classified as high ROR (n = 648). Node-negative patients classified as low and high ROR had a risk of DR of 5.0% (95% CI, 2.9% to 8.0%) and 17.8% (95% CI, 14.0% to 22.0%), respectively. Luminal B tumors (n = 947; DR risk, 18.4% [95% CI: 15.7% to 21.3%]) had a significantly worse outcome than luminal A tumors (n = 1,474,;DR risk, 7.6% [95% CI: 6.1% to 9.2%]; P < .001). Conclusion Prosigna ROR score improved the prediction of outcome in this nationwide Danish population. In a real-world setting, Prosigna can reliably identify node-negative patients and a significant proportion of patients with one to three positive nodes who can be spared treatment with adjuvant chemotherapy.

1533Background: Gene expression profiling assays are not commonly used for therapy selection for breast cancer patients with germline BRCA mutations but a complete molecular characterization could ...

544 Background: Accurate assessment of the risk of late DR (5-10 yr after surgery) may be used to tailor the duration of ET for patients with HR+ EBC. The Prosigna (PAM50) risk of recurrence (ROR) ...

513 Background: N1, HR+ EBC patients are routinely treated with both ET and chemotherapy (CT). Identification of N1 patients with good outcome who may be spared CT remains an elusive goal. In a com...