Tumor-Infiltrating Lymphocytes in Triple-Negative Breast Cancer.

Importance: The association of tumor-infiltrating lymphocyte (TIL) abundance in breast cancer tissue with cancer recurrence and death in patients with early-stage triple-negative breast cancer (TNBC) who are not treated with adjuvant or neoadjuvant chemotherapy is unclear. Objective: To study the association of TIL abundance in breast cancer tissue with survival among patients with early-stage TNBC who were treated with locoregional therapy but no chemotherapy. Design, Setting, and Participants: Retrospective pooled analysis of individual patient-level data from 13 participating centers in North America (Rochester, Minnesota; Vancouver, British Columbia, Canada), Europe (Paris, Lyon, and Villejuif, France; Amsterdam and Rotterdam, the Netherlands; Milan, Padova, and Genova, Italy; Gothenburg, Sweden), and Asia (Tokyo, Japan; Seoul, Korea), including 1966 participants diagnosed with TNBC between 1979 and 2017 (with follow-up until September 27, 2021) who received treatment with surgery with or without radiotherapy but no adjuvant or neoadjuvant chemotherapy. Exposure: TIL abundance in breast tissue from resected primary tumors. Main Outcomes and Measures: The primary outcome was invasive disease-free survival [iDFS]. Secondary outcomes were recurrence-free survival [RFS], survival free of distant recurrence [distant RFS, DRFS], and overall survival. Associations were assessed using a multivariable Cox model stratified by participating center. Results: This study included 1966 patients with TNBC (median age, 56 years [IQR, 39-71]; 55% had stage I TNBC). The median TIL level was 15% (IQR, 5%-40%). Four-hundred seventeen (21%) had a TIL level of 50% or more (median age, 41 years [IQR, 36-63]), and 1300 (66%) had a TIL level of less than 30% (median age, 59 years [IQR, 41-72]). Five-year DRFS for stage I TNBC was 94% (95% CI, 91%-96%) for patients with a TIL level of 50% or more, compared with 78% (95% CI, 75%-80%) for those with a TIL level of less than 30%; 5-year overall survival was 95% (95% CI, 92%-97%) for patients with a TIL level of 50% or more, compared with 82% (95% CI, 79%-84%) for those with a TIL level of less than 30%. At a median follow-up of 18 years, and after adjusting for age, tumor size, nodal status, histological grade, and receipt of radiotherapy, each 10% higher TIL increment was associated independently with improved iDFS (hazard ratio [HR], 0.92 [0.89-0.94]), RFS (HR, 0.90 [0.87-0.92]), DRFS (HR, 0.87 [0.84-0.90]), and overall survival (0.88 [0.85-0.91]) (likelihood ratio test, P < 10e-6). Conclusions and Relevance: In patients with early-stage TNBC who did not undergo adjuvant or neoadjuvant chemotherapy, breast cancer tissue with a higher abundance of TIL levels was associated with significantly better survival. These results suggest that breast tissue TIL abundance is a prognostic factor for patients with early-stage TNBC.

HER2-low and tumor infiltrating lymphocytes in triple-negative breast cancer: Are they connected?

Most patients with triple-negative breast cancer (TNBC) are not candidates for targeted therapy, leaving chemotherapy as the primary treatment option. Recently, immunotherapy has demonstrated promising results in TNBC, due to its immunogenicity. In addition, a novel antibody-drug conjugate, namely, trastuzumab-deruxtecan, has shown effectiveness in TNBC patients with low-HER2 expression (HER2-low). These novel treatment options raise the question about the potential association between the density of stromal tumor-infiltrating lymphocytes (sTILs) and the level of HER2 expression. We aimed to evaluate the association between the level of HER2 expression (HER2-low versus HER2-0) and density of sTILs in TNBC patients, and how they impact the response to neoadjuvant chemotherapy (NAC). This was a retrospective multicenter study including all TNBC patients diagnosed between 2018 and 2022. Central pathology review included sTILs percentages and level of HER2 expression. Tumors were reclassified as either HER2-0 (HER2 IHC 0) or HER2-low (IHC 1 + or 2 + with negative reflex test). Various clinicopathologic characteristics, including sTILs density, and response to NAC were compared between HER2-0 and HER2-low cases. In total, 753 TNBC patients were included in this study, of which 292 patients received NAC. Interobserver agreement between the original pathology report and central review was moderate (77% had the same IHC status after reclassification in either HER2-0 or HER2-low; k = 0.45). HER2-low TNBC represented about one third (36%) of the tumors. No significant difference in sTILs density or complete pathologic response rate was found between HER2-0 and HER2-low cases (p = 0.476 and p = 0.339, respectively). The density of sTILs (≥ 10% sTILs vs. < 10%) was independently associated with achieving a pCR (p = 0.011). In conclusion, no significant association was found between HER2-low status and density of sTILs nor response to NAC. Nonetheless, sTILs could be an independent biomarker for predicting NAC response in TNBC patients.

HER2-low breast cancer and response to neoadjuvant chemotherapy: a population-based cohort study.

About half of breast cancers (BC) without amplification of the human epidermal growth factor receptor 2 (HER2) have a low HER2 protein expression level (HER2-low). The clinical impact of HER2-low and the response to neoadjuvant chemotherapy (NAC) is unclear. This study aimed to assess the association between HER2-low BC and pathological response to NAC. Data from the Dutch Pathology Registry were collected for 11,988 BC patients treated with NAC between 2014 and 2022. HER2-low BC was defined as an immunohistochemical score of 1+ or 2+ and a negative molecular reflex test. We compared clinicopathological features of HER2-0 versus HER2-low BC and assessed the correlation between HER2 status and the pathological complete response (pCR) rate after NAC, including overall survival. Among hormone receptor (HR)-positive tumours, 67% (n=4,619) were HER2-low, compared to 47% (n=1,167) in the HR-negative group. Around 32% (n=207) of patients had a discordant HER2 status between the pre-NAC biopsy and the corresponding post-NAC resection, within which 87% (n=165) changed from HER2-0 to HER2-low or vice versa. The pCR rate was significantly lower in HER2-low BC compared to HER2-0 BC within the HR-positive group (4% versus 5%; p=0.022). However, the absolute difference was limited, so the clinical relevance is questionable. In HR-negative cases, the difference in pCR was not significant (32% versus 34%; p=0.266). No significant difference in overall survival was observed between HER2-low and HER2-0 tumours, regardless of hormone receptor status. The antibody-drug conjugate trastuzumab deruxtecan (T-DXd) has improved survival outcomes of patients with HER2-low metastatic BC. The finding that one-third of the patients in this study had a discordant HER2 status between the pre-NAC biopsy and the post-NAC resection specimen could impact clinical decision-making should T-DXd be used in early BC treatment.

The effect of (neo)adjuvant chemotherapy on long-term survival outcomes in patients with invasive lobular breast cancer treated with endocrine therapy: A retrospective cohort study.

BACKGROUND: Despite histological and molecular differences between invasive lobular carcinoma (ILC) and invasive carcinoma of no special type, according to national treatment guidelines no distinction is made regarding the use of (neo)adjuvant chemotherapy. Studies on the long-term outcome of chemotherapy in patients with ILC are scarce and show inconclusive results. METHODS: All patients with estrogen receptor (ER)-positive, human epidermal growth factor receptor 2 (HER2)-negative ILC with an indication for chemotherapy treated with adjuvant endocrine therapy were selected from the Erasmus Medical Center Breast Cancer database. Cox proportional hazards models were used to estimate the effect of chemotherapy on recurrence-free survival (RFS), breast cancer-specific survival (BCSS), and overall survival (OS). RESULTS: A total of 520 patients were selected, of whom 379 were treated with chemotherapy and 141 were not. Patients in the chemotherapy group were younger (51 vs. 61 years old; p < .001), had a higher T status (T3+, 33% vs. 14%; p < .001), and more often had lymph node involvement (80% vs. 49%; p < .001) in comparison to the no-chemotherapy group. After adjusting for confounders, chemotherapy treatment was not associated with better RFS (hazard ratio [HR], 1.20; 95% confidence interval [CI], 0.63-2.31), BCSS (HR, 1.24; 95% CI, 0.60-2.58), or OS (HR, 0.97; 95% CI, 0.56-1.66). This was also reflected by adjusted Cox survival curves in the chemotherapy versus no-chemotherapy group for RFS (75% vs. 79%), BCSS (80% vs. 84%), and OS (72% vs. 71%). CONCLUSIONS: Chemotherapy is not associated with improved RFS, BCSS, or OS for patients with ER+/HER2- ILC treated with adjuvant endocrine therapy and with an indication for chemotherapy.

External validation and clinical utility assessment of PREDICT breast cancer prognostic model in young, systemic treatment-naïve women with node-negative breast cancer.

BACKGROUND: The validity of the PREDICT breast cancer prognostic model is unclear for young patients without adjuvant systemic treatment. This study aimed to validate PREDICT and assess its clinical utility in young women with node-negative breast cancer who did not receive systemic treatment. METHODS: We selected all women from the Netherlands Cancer Registry who were diagnosed with node-negative breast cancer under age 40 between 1989 and 2000, a period when adjuvant systemic treatment was not standard practice for women with node-negative disease. We evaluated the calibration and discrimination of PREDICT using the observed/expected (O/E) mortality ratio, and the area under the receiver operating characteristic curve (AUC), respectively. Additionally, we compared the potential clinical utility of PREDICT for selectively administering chemotherapy to the chemotherapy-to-all strategy using decision curve analysis at predefined thresholds. RESULTS: A total of 2264 women with a median age at diagnosis of 36 years were included. Of them, 71.2% had estrogen receptor (ER)-positive tumors and 44.0% had grade 3 tumors. Median tumor size was 16 mm. PREDICT v2.2 underestimated 10-year all-cause mortality by 33% in all women (O/E ratio:1.33, 95%CI:1.22-1.43). Model discrimination was moderate overall (AUC10-year:0.65, 95%CI:0.62-0.68), and poor for women with ER-negative tumors (AUC10-year:0.56, 95%CI:0.51-0.62). Compared to the chemotherapy-to-all strategy, PREDICT only showed a slightly higher net benefit in women with ER-positive tumors, but not in women with ER-negative tumors. CONCLUSIONS: PREDICT yields unreliable predictions for young women with node-negative breast cancer. Further model updates are needed before PREDICT can be routinely used in this patient subset.

Proof-of-concept study linking ex vivo sensitivity testing to neoadjuvant anthracycline-based chemotherapy response in breast cancer patients.

We developed a functional ex vivo anthracycline-based sensitivity test. Surgical resection material of primary breast cancer (BC) was used to determine criteria for the ex vivo sensitivity assay based on morphology, proliferation and apoptosis. Subsequently, a proof-of-concept study was performed correlating results of this assay on primary BC biopsies with in vivo response after treatment with anthracycline-containing neoadjuvant chemotherapy (NAC). Cut off values for the ex vivo anthracycline-based sensitivity test were established based on analysis of 21 primary breast tumor samples obtained after surgery. In the proof-of-concept study based on a new set of tumor biopsies, 41 patients were included. Eight biopsies did not contain tumor cells and three patients could not be biopsied for various reasons. In the remaining 30 biopsies, the success rate of the ex vivo test was 77% (23/30); six out of seven failed tests were due to excessive apoptosis, our pre-specified test criteria. Of the 23 patients with a successful ex vivo test result, three patients did not undergo NAC after the biopsy. Here we report the ex vivo anthracycline-based sensitivity assay is feasible on biopsy material and shows 75% concordance between ex vivo outcomes and in vivo MRI response. Unfortunately, the percentage of unsuccessful tests is rather high. This study provides the foundation for further development of ex vivo sensitivity assays.

Interobserver Variation in the Assessment of Immunohistochemistry Expression Levels in HER2-Negative Breast Cancer: Can We Improve the Identification of Low Levels of HER2 Expression by Adjusting the Criteria? An International Interobserver Study.

The classification of human epidermal growth factor receptor 2 (HER2) expression is optimized to detect HER2-amplified breast cancer (BC). However, novel HER2-targeting agents are also effective for BCs with low levels of HER2. This raises the question whether the current guidelines for HER2 testing are sufficiently reproducible to identify HER2-low BC. The aim of this multicenter international study was to assess the interobserver agreement of specific HER2 immunohistochemistry scores in cases with negative HER2 results (0, 1+, or 2+/in situ hybridization negative) according to the current American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) guidelines. Furthermore, we evaluated whether the agreement improved by redefining immunohistochemistry (IHC) scoring criteria or by adding fluorescent in situ hybridization (FISH). We conducted a 2-round study of 105 nonamplified BCs. During the first assessment, 16 pathologists used the latest version of the ASCO/CAP guidelines. After a consensus meeting, the same pathologists scored the same digital slides using modified IHC scoring criteria based on the 2007 ASCO/CAP guidelines, and an extra "ultralow" category was added. Overall, the interobserver agreement was limited (4.7% of cases with 100% agreement) in the first round, but this was improved by clustering IHC categories. In the second round, the highest reproducibility was observed when comparing IHC 0 with the ultralow/1+/2+ grouped cluster (74.3% of cases with 100% agreement). The FISH results were not statistically different between HER2-0 and HER2-low cases, regardless of the IHC criteria used. In conclusion, our study suggests that the modified 2007 ASCO/CAP criteria were more reproducible in distinguishing HER2-0 from HER2-low cases than the 2018 ASCO/CAP criteria. However, the reproducibility was still moderate, which was not improved by adding FISH. This could lead to a suboptimal selection of patients eligible for novel HER2-targeting agents. If the threshold between HER2 IHC 0 and 1+ is to be clinically actionable, there is a need for clearer, more reproducible IHC definitions, training, and/or development of more accurate methods to detect this subtle difference in protein expression levels.

HER2-Low Breast Cancer: Incidence, Clinicopathologic Features, and Survival Outcomes From Real-World Data of a Large Nationwide Cohort.

Patients with breast cancer (BC) with low levels of human epidermal growth factor receptor 2 (HER2) expression (HER2-low) could benefit from novel antibody-drug conjugates. However, there is conflicting information regarding the characteristics of HER2-low BC and its outcome. We assessed the clinicopathologic characteristics and outcomes of HER2-low BC using real-world data from the Dutch National Pathology Registry. This retrospective study incorporated all patients with primary invasive BC, without neoadjuvant therapy, reported in the Dutch National Pathology Registry synoptic reporting module between 2014 and 2022. HER2 status was categorized as HER2-0 (defined as an immunohistochemistry score of 0 according to the current American Society of Clinical Oncology/College of American Pathologists guidelines) or HER2-low (immunohistochemistry score 1+ or 2+ without amplification). Clinicopathologic characteristics and overall survival of HER2-low BC were compared with HER2-0, adjusted for estrogen receptor (ER) status. We included 65,035 patients with BC, resulting in 69,424 tumors. The proportion of HER2-low BC was 62% in the ER+ cohort and 38% in the ER- cohort. A substantial number of patients had a different HER2 category between the needle biopsy and the corresponding surgical resection (28%) or among multiple tumors (28%). After multivariable logistic analysis, HER2-low tumors were significantly associated with histologic subtype, a higher ER, and lower progesterone receptor expression in the ER+ cohort, whereas within the ER-cohort, HER2-low tumors were associated with a lower tumor grade. However, the absolute differences were limited, and there was no significant difference in overall survival between HER2-low and HER2-0 tumors within the ER+ or ER- cohort. The classification of HER2 expression (HER2-0 vs HER2-low) varies between biopsies and corresponding resection specimens and within multiple tumors in the same patient, which could affect clinical decision making in case only HER2-low cases are eligible for novel HER2-targeting agents. The limited follow-up time and the lack of substantial clinicopathologic differences between HER2-low and HER2-0-cases could explain the lack of differences in overall survival.

Triple-Negative Breast Cancer and Predictive Markers of Response to Neoadjuvant Chemotherapy: A Systematic Review.

Around 40-50% of all triple-negative breast cancer (TNBC) patients achieve a pathological complete response (pCR) after treatment with neoadjuvant chemotherapy (NAC). The identification of biomarkers predicting the response to NAC could be helpful for personalized treatment. This systematic review provides an overview of putative biomarkers at baseline that are predictive for a pCR following NAC. Embase, Medline and Web of Science were searched for articles published between January 2010 and August 2022. The articles had to meet the following criteria: patients with primary invasive TNBC without distant metastases and patients must have received NAC. In total, 2045 articles were screened by two reviewers resulting in the inclusion of 92 articles. Overall, the most frequently reported biomarkers associated with a pCR were a high expression of Ki-67, an expression of PD-L1 and the abundance of tumor-infiltrating lymphocytes, particularly CD8+ T cells, and corresponding immune gene signatures. In addition, our review reveals proteomic, genomic and transcriptomic markers that relate to cancer cells, the tumor microenvironment and the peripheral blood, which also affect chemo-sensitivity. We conclude that a prediction model based on a combination of tumor and immune markers is likely to better stratify TNBC patients with respect to NAC response.

Risk of metachronous contralateral breast cancer in patients with primary invasive lobular breast cancer: Results from a nationwide cohort.

Lobular primary breast cancer (PBC) histology has been proposed as a risk factor for contralateral breast cancer (CBC), but results have been inconsistent. We investigated CBC risk and the impact of systemic therapy in lobular versus ductal PBC. Further, CBC characteristics following these histologic subtypes were explored. We selected 74,373 women diagnosed between 2003 and 2010 with stage I-III invasive PBC from the nationwide Netherlands Cancer Registry. We assessed absolute risk of CBC taking into account competing risks among those with lobular (n = 8903), lobular mixed with other types (n = 3512), versus ductal (n = 62,230) histology. Hazard ratios (HR) for CBC were estimated in a cause-specific Cox model, adjusting for age at PBC diagnosis, radiotherapy, chemotherapy and/or endocrine therapy. Multivariable HRs for CBC were 1.18 (95% CI: 1.04-1.33) for lobular and 1.37 (95% CI: 1.16-1.63) for lobular mixed versus ductal PBC. Ten-year cumulative CBC incidences in patients with lobular, lobular mixed versus ductal PBC were 3.2%, 3.6% versus 2.8% when treated with systemic therapy and 6.6%, 7.7% versus 5.6% in patients without systemic therapy, respectively. Metachronous CBCs were diagnosed in a less favourable stage in 19%, 26% and 23% and less favourable differentiation grade in 22%, 33% and 27% than the PBCs of patients with lobular, lobular mixed and ductal PBC, respectively. In conclusion, lobular and lobular mixed PBC histology are associated with modestly increased CBC risk. Personalised CBC risk assessment needs to consider PBC histology, including systemic treatment administration. The impact on prognosis of CBCs with unfavourable characteristics warrants further evaluation.

Expression and Localization of Ferritin-Heavy Chain Predicts Recurrence for Breast Cancer Patients with a BRCA1/2 Mutation.

The ferritin-heavy chain (FTH1) is the catalytic subunit of the ferroxidase ferritin, which prevents oxidative DNA damage via intracellular iron storage. FTH1 was shown to be a prognostic marker for triple-negative breast cancer (BC) patients and associated with an enrichment of CD8+ effector T cells. However, whether the expression and localization of FTH1 are also associated with clinical outcome in other BC subtypes is unknown. Here, we investigated the association of FTH1 with time to survival in BCs from 222 BRCA1/2 mutation carriers by immunohistochemistry on tissue microarrays. In addition, for 51 of these patients, the association between FTH1 and specific subsets of T cells was evaluated on whole slides using automatic scoring algorithms. We revealed that nuclear FTH1 (nFTH1) expression, in multivariable analyses, was associated with a shorter disease-free (HR = 2.71, 95% CI = 1.49-4.92, p = 0.001) and metastasis-free survival (HR = 3.54, 95% CI = 1.45-8.66, p = 0.006) in patients carrying a BRCA1/2 mutation. However, we found no relation between cytoplasmic FTH1 expression and survival of BRCA1/2 mutation carriers. Moreover, we did not detect an association between FTH1 expression and the amount of CD45+ (p = 0.13), CD8+ (p = 0.18), CD4+ (p = 0.20) or FOXP3+ cells (p = 0.17). Consequently, the mechanism underlying the worse recurrence-free survival of nFTH1 expression in BRCA1/2 mutation carriers needs further investigation.

Real-world data of HER2-low metastatic breast cancer: A population based cohort study.

BACKGROUND: With the introduction of investigational human epidermal growth factor receptor 2 (HER2) targeting treatments, thorough understanding of breast cancer with different HER2 expression levels is critical. The aim of this study was to compare clinicopathologic characteristics and survival of patients with metastatic breast cancer according to the level of HER2 expression. METHODS: Women with distant metastatic breast cancer during 2008-2016 were selected from PALGA, the Dutch Pathology Registry, and linked to the PHARMO Database Network. Breast cancer samples were categorised as HER2 immunohistochemistry score 0 (IHC0), HER2-low or HER2+. RESULTS: Among women with hormone receptor (HR) positive metastatic breast cancer (n = 989), 373 (38%) cancers were HER2 IHC0, 472 (48%) were HER2-low and 144 (15%) were HER2+. Among HR negative patients (n = 272), the proportion of HER2 IHC0, HER2-low and HER2+ was 110 (40%), 104 (38%) and 58 (21%) respectively. Within the HR + cohort, patients with HER2 IHC0 or HER2-low cancer were significantly older compared to HER2+ patients. This age difference was not seen in the HR-cohort. The localisation of distant metastases differed significantly between HER2 IHC0 or HER2-low versus HER2+ cases. Survival rates did not differ markedly by subtypes. CONCLUSION: Substantial proportion of patients had a HER2-low breast cancer. No clear differences in survival were found when comparing HER2 and HR status. Getting more granular insights in the level of HER2 expression and addressing HER2-low as a separate category could help to assess the impact of emerging treatment strategies. Therefore, more detailed information on HER2 expression should be routinely reported.

Selecting patients with HER2-low breast cancer: Getting out of the tangle.

The promising effect of antibody-drug conjugates on breast cancer with low expression of HER2 (HER2-low) raises many questions regarding the optimal selection of patients for this treatment. A key question is whether HER2 immunohistochemistry, an assay optimised to detect HER2 amplification, is reliable enough to assess HER2 protein levels to select patients with HER2-low breast cancer in daily pathology practices worldwide. Moreover, whether this assessment can be performed with sufficient reproducibility between pathologists in daily practices is debatable. Herein, we address the historical track record of the CAP-ASCO HER2 Guidelines, the reported limited reproducibility by pathologists of HER2 immunohistochemistry in the non-amplified cases, and the performance variation of different antibodies. Based on this summary, we propose solutions to improve the robustness to enable reliable identification of patients with HER2-low breast cancer.

External Quality Assessment 2.0: The Importance of a Standardized Implementation of TILs for Daily and Trial Practices.

Increasing data suggests that an intact immune system is required for improvedoutcomes in patients with Human Epidermal Growth Factor Receptor 2 (HER2+) and Triple Negative Breast Cancer (TNBC) [...].

HER2-low breast cancer shows a lower immune response compared to HER2-negative cases.

Currently, the human epidermal growth factor receptor 2 (HER2) status of breast cancer is classified dichotomously as negative or positive to select patients for HER2-targeted therapy. However, with the introduction of novel treatment options, it is important to get more insight in the biology of cancers with low HER2 expression. Therefore, we studied several clinicopathologic characteristics in relation to the level of HER2 expression (HER2- versus HER2low). We used a well-documented cohort of breast cancer patients (n = 529), with available tissue microarrays and Affymetrix mRNA expression data. HER2 status was scored as negative (immunohistochemistry 0) or low (immunohistochemistry 1 + or 2 + without amplification). We associated HER2 status with several clinicopathologic characteristics, gene-expression data and survival, stratified for estrogen receptor (ER) status. Overall, breast cancers were scored as HER2- (n = 429) or HER2low (n = 100). Within the ER+ cohort (n = 305), no significant associations were found between the HER2 groups and clinicopathologic features. However, HER2low tumors showed several differentially expressed genes compared to HER2- cases, including genes that are associated with worse outcome and depletion of immunity. In ER- cases (n = 224), HER2low status was significantly associated with increased regional nodal positivity, lower density of tumor infiltrating lymphocyte and a lower protein expression of Ki-67 and EGFR compared to HER2- cases. After multivariate analysis, only density of tumor infiltrating lymphocytes remained significantly associated with HER2low status (P = 0.035). No difference in survival was observed between HER2low and HER2- patients, neither in the ER+ nor ER- cohort. In conclusion, our data suggests that HER2low breast cancer is associated with a lower immune response compared to HER2- breast cancer.

Functional RECAP (REpair CAPacity) assay identifies homologous recombination deficiency undetected by DNA-based BRCAness tests.

Germline BRCA1/2 mutation status is predictive for response to Poly-[ADP-Ribose]-Polymerase (PARP) inhibitors in breast cancer (BC) patients. However, non-germline BRCA1/2 mutated and homologous recombination repair deficient (HRD) tumors are likely also PARP-inhibitor sensitive. Clinical validity and utility of various HRD biomarkers are under investigation. The REpair CAPacity (RECAP) test is a functional method to select HRD tumors based on their inability to form RAD51 foci. We investigated whether this functional test defines a similar group of HRD tumors as DNA-based tests. An HRD enriched cohort (n = 71; 52 primary and 19 metastatic BCs) selected based on the RECAP test (26 RECAP-HRD; 37%), was subjected to DNA-based HRD tests (i.e., Classifier of HOmologous Recombination Deficiency (CHORD) and BRCA1/2-like classifier). Whole genome sequencing (WGS) was carried out for 38 primary and 19 metastatic BCs. The RECAP test identified all bi-allelic BRCA deficient samples (n = 15) in this cohort. RECAP status partially correlated with DNA-based HRD test outcomes (70% concordance for both RECAP-CHORD and RECAP-BRCA1/2-like classifier). RECAP selected additional samples unable to form RAD51 foci, suggesting that this functional assay identified deficiencies in other DNA repair genes, which could also result in PARP-inhibitor sensitivity. Direct comparison of these HRD tests in clinical trials will be required to evaluate the optimal predictive test for clinical decision making.

Prognostic Value of Stromal Tumor-Infiltrating Lymphocytes in Young, Node-Negative, Triple-Negative Breast Cancer Patients Who Did Not Receive (neo)Adjuvant Systemic Therapy.

PURPOSE: Triple-negative breast cancer (TNBC) is considered aggressive, and therefore, virtually all young patients with TNBC receive (neo)adjuvant chemotherapy. Increased stromal tumor-infiltrating lymphocytes (sTILs) have been associated with a favorable prognosis in TNBC. However, whether this association holds for patients who are node-negative (N0), young (< 40 years), and chemotherapy-naïve, and thus can be used for chemotherapy de-escalation strategies, is unknown. METHODS: We selected all patients with N0 TNBC diagnosed between 1989 and 2000 from a Dutch population-based registry. Patients were age < 40 years at diagnosis and had not received (neo)adjuvant systemic therapy, as was standard practice at the time. Formalin-fixed paraffin-embedded blocks were retrieved (PALGA: Dutch Pathology Registry), and a pathology review including sTILs was performed. Patients were categorized according to sTILs (< 30%, 30%-75%, and ≥ 75%). Multivariable Cox regression was performed for overall survival, with or without sTILs as a covariate. Cumulative incidence of distant metastasis or death was analyzed in a competing risk model, with second primary tumors as competing risk. RESULTS: sTILs were scored for 441 patients. High sTILs (≥ 75%; 21%) translated into an excellent prognosis with a 15-year cumulative incidence of a distant metastasis or death of only 2.1% (95% CI, 0 to 5.0), whereas low sTILs (< 30%; 52%) had an unfavorable prognosis with a 15-year cumulative incidence of a distant metastasis or death of 38.4% (32.1 to 44.6). In addition, every 10% increment of sTILs decreased the risk of death by 19% (adjusted hazard ratio: 0.81; 95% CI, 0.76 to 0.87), which are an independent predictor adding prognostic information to standard clinicopathologic variables (χ2 = 46.7, P < .001). CONCLUSION: Chemotherapy-naïve, young patients with N0 TNBC with high sTILs (≥ 75%) have an excellent long-term prognosis. Therefore, sTILs should be considered for prospective clinical trials investigating (neo)adjuvant chemotherapy de-escalation strategies.

Functional Ex Vivo Tissue-Based Chemotherapy Sensitivity Testing for Breast Cancer.

Background chemotherapy is part of most breast cancer (BC) treatment schedules. However, a substantial fraction of BC tumors does not respond to the treatment. Unfortunately, no standard biomarkers exist for response prediction. Therefore, we aim to develop ex vivo sensitivity assays for two types of commonly used cytostatics (i.e., platinum derivates and taxanes) on organotypic BC tissue slices. METHODS: Ex vivo cisplatin sensitivity assays were established using organotypic tissue slices derived from the surgical resection material of 13 primary BCs and 20 fresh histological biopsies obtained from various metastatic sites. Furthermore, tissue slices of 10 primary BCs were used to establish a docetaxel ex vivo sensitivity assay. RESULTS: Cisplatin sensitivity was assessed by tissue morphology, proliferation and apoptosis, while the relative increase in the mitotic index was discriminative for docetaxel sensitivity. Based on these read-outs, a scoring system was proposed to discriminate sensitive from resistant tumors for each cytostatic. We successful completed the cisplatin sensitivity assay on 12/16 (75%) biopsies as well. CONCLUSIONS: We developed an ex vivo cisplatin and docetaxel assay on BC slices. We also adapted the assay for biopsy-sized specimens as the next step towards the correlation of ex vivo test results and in vivo responses.

Interobserver Agreement of PD-L1/SP142 Immunohistochemistry and Tumor-Infiltrating Lymphocytes (TILs) in Distant Metastases of Triple-Negative Breast Cancer: A Proof-of-Concept Study. A Report on Behalf of the International Immuno-Oncology Biomarker Working Group.

Patients with advanced triple-negative breast cancer (TNBC) benefit from treatment with atezolizumab, provided that the tumor contains ≥1% of PD-L1/SP142-positive immune cells. Numbers of tumor-infiltrating lymphocytes (TILs) vary strongly according to the anatomic localization of TNBC metastases. We investigated inter-pathologist agreement in the assessment of PD-L1/SP142 immunohistochemistry and TILs. Ten pathologists evaluated PD-L1/SP142 expression in a proficiency test comprising 28 primary TNBCs, as well as PD-L1/SP142 expression and levels of TILs in 49 distant TNBC metastases with various localizations. Interobserver agreement for PD-L1 status (positive vs. negative) was high in the proficiency test: the corresponding scores as percentages showed good agreement with the consensus diagnosis. In TNBC metastases, there was substantial variability in PD-L1 status at the individual patient level. For one in five patients, the chance of treatment was essentially random, with half of the pathologists designating them as positive and half negative. Assessment of PD-L1/SP142 and TILs as percentages in TNBC metastases showed poor and moderate agreement, respectively. Additional training for metastatic TNBC is required to enhance interobserver agreement. Such training, focusing on metastatic specimens, seems worthwhile, since the same pathologists obtained high percentages of concordance (ranging from 93% to 100%) on the PD-L1 status of primary TNBCs.