A scoping review of statistical methods in studies of biomarker-related treatment heterogeneity for breast cancer.

BACKGROUND: Many scientific papers are published each year and substantial resources are spent to develop biomarker-based tests for precision oncology. However, only a handful of tests is currently used in daily clinical practice, since development is challenging. In this situation, the application of adequate statistical methods is essential, but little is known about the scope of methods used. METHODS: A PubMed search identified clinical studies among women with breast cancer comparing at least two different treatment groups, one of which chemotherapy or endocrine treatment, by levels of at least one biomarker. Studies presenting original data published in 2019 in one of 15 selected journals were eligible for this review. Clinical and statistical characteristics were extracted by three reviewers and a selection of characteristics for each study was reported. RESULTS: Of 164 studies identified by the query, 31 were eligible. Over 70 different biomarkers were evaluated. Twenty-two studies (71%) evaluated multiplicative interaction between treatment and biomarker. Twenty-eight studies (90%) evaluated either the treatment effect in biomarker subgroups or the biomarker effect in treatment subgroups. Eight studies (26%) reported results for one predictive biomarker analysis, while the majority performed multiple evaluations, either for several biomarkers, outcomes and/or subpopulations. Twenty-one studies (68%) claimed to have found significant differences in treatment effects by biomarker level. Fourteen studies (45%) mentioned that the study was not designed to evaluate treatment effect heterogeneity. CONCLUSIONS: Most studies evaluated treatment heterogeneity via separate analyses of biomarker-specific treatment effects and/or multiplicative interaction analysis. There is a need for the application of more efficient statistical methods to evaluate treatment heterogeneity in clinical studies.

Limiting systemic endocrine overtreatment in postmenopausal breast cancer patients with an ultralow classification of the 70-gene signature.

PURPOSE: Guidelines recommend endocrine treatment for estrogen receptor-positive (ER+) breast cancers for up to 10 years. Earlier data suggest that the 70-gene signature (MammaPrint) has potential to select patients that have an excellent survival without chemotherapy and limited or no tamoxifen treatment. The aim was to validate the 70-gene signature ultralow-risk classification for endocrine therapy decision making. METHODS: In the IKA trial, postmenopausal patients with non-metastatic breast cancer had been randomized between no or limited adjuvant tamoxifen treatment without receiving chemotherapy. For this secondary analysis, FFPE tumor material was obtained of ER+HER2- patients with 0-3 positive lymph nodes and tested for the 70-gene signature. Distant recurrence-free interval (DRFI) long-term follow-up data were collected. Kaplan-Meier curves were used to estimate DRFI, stratified by lymph node status, for the three predefined 70-gene signature risk groups. RESULTS: A reliable 70-gene signature could be obtained for 135 patients. Of the node-negative and node-positive patients, respectively, 20% and 13% had an ultralow-risk classification. No DRFI events were observed for node-negative patients with an ultralow-risk score in the first 10 years. The 10-year DRFI was 90% and 66% in the low-risk (but not ultralow) and high-risk classified node-negative patients, respectively. CONCLUSION: These survival analyses indicate that the postmenopausal node-negative ER+HER2- patients with an ultralow-risk 70-gene signature score have an excellent 10-year DRFI after surgery with a median of 1 year of endocrine treatment. This is in line with published results of the STO-3-randomized clinical trial and supports the concept that it is possible to reduce the duration of endocrine treatment in selected patients.

Assessment of structural chromosomal instability phenotypes as biomarkers of carboplatin response in triple negative breast cancer: the TNT trial.

BACKGROUND: In the TNT trial of triple negative breast cancer (NCT00532727), germline BRCA1/2 mutations were present in 28% of carboplatin responders. We assessed quantitative measures of structural chromosomal instability (CIN) to identify a wider patient subgroup within TNT with preferential benefit from carboplatin over docetaxel. PATIENTS AND METHODS: Copy number aberrations (CNAs) were established from 135 formalin-fixed paraffin-embedded primary carcinomas using Illumina OmniExpress SNP-arrays. Seven published [allelic imbalanced CNA (AiCNA); allelic balanced CNA (AbCNA); copy number neutral loss of heterozygosity (CnLOH); number of telomeric allelic imbalances (NtAI); BRCA1-like status; percentage of genome altered (PGA); homologous recombination deficiency (HRD) scores] and two novel [Shannon diversity index (SI); high-level amplifications (HLAMP)] CIN-measurements were derived. HLAMP was defined based on the presence of at least one of the top 5% amplified cytobands located on 1q, 8q and 10p. Continuous CIN-measurements were divided into tertiles. All nine CIN-measurements were used to analyse objective response rate (ORR) and progression-free survival (PFS). RESULTS: Patients with tumours without HLAMP had a numerically higher ORR and significantly longer PFS in the carboplatin (C) than in the docetaxel (D) arm [56% (C) versus 29% (D), PHLAMP,quiet = 0.085; PFS 6.1 months (C) versus 4.1 months (D), Pinteraction/HLAMP = 0.047]. In the carboplatin arm, patients with tumours showing intermediate telomeric NtAI and AiCNA had higher ORR [54% (C) versus 20% (D), PNtAI,intermediate = 0.03; 62% (C) versus 33% (D), PAiCNA,intermediate = 0.076]. Patients with high AiCNA and PGA had shorter PFS in the carboplatin arm [3.4 months (high) versus 5.7 months (low/intermediate); and 3.8 months (high) versus 5.6 months (low/intermediate), respectively; Pinteraction/AiCNA = 0.027, Padj.interaction/AiCNA = 0.125 and Pinteraction/PGA = 0.053, Padj.interaction/PGA = 0.176], whilst no difference was observed in the docetaxel arm. CONCLUSIONS: Patients with tumours lacking HLAMP and demonstrating intermediate CIN-measurements formed a subgroup benefitting from carboplatin relative to docetaxel treatment within the TNT trial. This suggests a complex and paradoxical relationship between the extent of genomic instability in primary tumours and treatment response in the metastatic setting.

A real or apparent decrease in glomerular filtration rate in patients using olaparib?

PURPOSE: Olaparib is a poly (ADP-ribose) polymerase (PARP) inhibitor indicated for ovarian and metastatic breast cancer. Increased serum creatinine levels have been observed in patients taking olaparib, but the underlying mechanism is unknown. This study aimed to investigate if patients receiving olaparib have increased creatinine levels during olaparib treatment and whether this actually relates to a declined glomerular filtration rate (GFR). METHODS: We retrospectively identified patients using olaparib at the Netherlands Cancer Institute - Antoni van Leeuwenhoek (NKI-AVL) from 2012 until 2020. Patients with at least one plasma or serum sample available at baseline/off treatment and during olaparib treatment were included. Cystatin C levels were measured, creatinine levels were available and renal function was determined by calculating the estimated glomerular filtration rate (eGFR) using the Creatinine Equation (CKD-EPI 2009) and the Cystatin C Equation (CKD-EPI 2012). RESULTS: In total, 66 patients were included. Olaparib treatment was associated with a 14% increase in median creatinine from 72 (inter quartile range (IQR): 22) µmol/L before/off treatment to 82 (IQR: 20) µmol/L during treatment (p < 0.001) and a 13% decrease in median creatinine-derived eGFR from 86 (IQR: 26) mL/min/1.73 m2 before/off treatment to 75 (IQR: 29) mL/min/1.73 m2 during treatment (p < 0.001). Olaparib treatment had no significant effect on median cystatin C levels (p = 0.520) and the median cystatin C-derived eGFR (p = 0.918). CONCLUSIONS: This study demonstrates that olaparib likely causes inhibition of renal transporters leading to a reversible and dose-dependent increase in creatinine and does not affect GFR, since the median cystatin C-derived eGFR was comparable before/off treatment and during treatment of olaparib. Using the creatinine-derived eGFR can give an underestimation of GFR in patients taking olaparib. Therefore, an alternative renal marker such as cystatin C should be used to accurately calculate eGFR in patients taking olaparib.

An Antiestrogenic Activity Score for tamoxifen and its metabolites is associated with breast cancer outcome.

PURPOSE: Endoxifen concentrations have been associated with breast cancer recurrence in tamoxifen-treated patients. However, tamoxifen itself and other metabolites also show antiestrogenic anti-tumor activity. Therefore, the aim of this study was to develop a comprehensive Antiestrogenic Activity Score (AAS), which accounts for concentration and antiestrogenic activity of tamoxifen and three metabolites. An association between the AAS and recurrence-free survival was investigated and compared to a previously published threshold for endoxifen concentrations of 5.97 ng/mL. PATIENTS AND METHODS: The antiestrogenic activities of tamoxifen, (Z)-endoxifen, (Z)-4-hydroxytamoxifen, and N-desmethyltamoxifen were determined in a cell proliferation assay. The AAS was determined by calculating the sum of each metabolite concentration multiplied by an IC50 ratio, relative to tamoxifen. The AAS was calculated for 1370 patients with estrogen receptor alpha (ERα)-positive breast cancer. An association between AAS and recurrence was investigated using Cox regression and compared with the 5.97 ng/mL endoxifen threshold using concordance indices. RESULTS: An AAS threshold of 1798 was associated with recurrence-free survival, hazard ratio (HR) 0.67 (95% confidence interval (CI) 0.47-0.96), bias corrected after bootstrap HR 0.69 (95% CI 0.48-0.99). The concordance indices for AAS and endoxifen did not significantly differ; however, using the AAS threshold instead of endoxifen led to different dose recommendations for 5.2% of the patients. CONCLUSIONS: Endoxifen concentrations can serve as a proxy for the antiestrogenic effect of tamoxifen and metabolites. However, for the aggregate effect of tamoxifen and three metabolites, defined by an integrative algorithm, a trend towards improving treatment is seen and moreover, is significantly associated with breast cancer recurrence.

BRCA1-like profile is not significantly associated with survival benefit of non-myeloablative intensified chemotherapy in the GAIN randomized controlled trial.

PURPOSE: The BRCA1-like profile identifies tumors with a defect in homologous recombination due to inactivation of BRCA1. This profile has been shown to predict which stage III breast cancer patients benefit from myeloablative, DNA double-strand-break-inducing chemotherapy. We tested the predictive potential of the BRCA1-like profile for adjuvant non-myeloablative, intensified dose-dense chemotherapy in the GAIN trial. METHODS: Lymph node positive breast cancer patients were randomized to 3 × 3 dose-dense cycles of intensified epirubicin, paclitaxel, and cyclophosphamide (ETC) or 4 cycles concurrent epirubicin and cyclophosphamide followed by 10 cycles of weekly paclitaxel combined with 4 cycles capecitabine (EC-TX). Only triple negative breast cancer patients (TNBC) for whom tissue was available were included in these planned analyses. BRCA1-like or non-BRCA1-like copy number profiles were derived from low coverage sequencing data. RESULTS: 119 out of 163 TNBC patients (73%) had a BRCA1-like profile. After median follow-up of 83 months, disease free survival (DFS) was not significantly different between BRCA1-like and non-BRCA1-like patients [adjusted hazard ratio (adj.HR) 1.02; 95% confidence interval (CI) 0.55-1.86], neither was overall survival (OS; adj.HR 1.26; 95% CI 0.58-2.71). When split by BRCA1-like status, DFS and OS were not significantly different between treatments. However, EC-TX seemed to result in a trend to an improvement in DFS in patients with a BRCA1-like tumor, while the reverse accounted for ETC treatment in patients with a non-BRCA1-like tumor (p for interaction = 0.094). CONCLUSIONS: The BRCA1-like profile is not associated with survival benefit for a non-myeloablative, intensified regimen in this study population. Considering the limited cohort size, capecitabine might have additional benefit for TNBC patients.

Clinically node negative breast cancer patients undergoing breast conserving therapy, sentinel lymph node procedure versus follow-up: a Dutch randomized controlled multicentre trial (BOOG 2013-08).

BACKGROUND: Studies showed that axillary lymph node dissection can be safely omitted in presence of positive sentinel lymph node(s) in breast cancer patients treated with breast conserving therapy. Since the outcome of the sentinel lymph node biopsy has no clinical consequence, the value of the procedure itself is being questioned. The aim of the BOOG 2013-08 trial is to investigate whether the sentinel lymph node biopsy can be safely omitted in clinically node negative breast cancer patients treated with breast conserving therapy. METHODS: The BOOG 2013-08 is a Dutch prospective non-inferiority randomized multicentre trial. Women with pathologically confirmed clinically node negative T1-2 invasive breast cancer undergoing breast conserving therapy will be randomized for sentinel lymph node biopsy versus no sentinel lymph node biopsy. Endpoints include regional recurrence after 5 (primary endpoint) and 10 years of follow-up, distant-disease free and overall survival, quality of life, morbidity and cost-effectiveness. Previous data indicate a 5-year regional recurrence free survival rate of 99% for the control arm and 96% for the study arm. In combination with a non-inferiority limit of 5% and probability of 0.8, this result in a sample size of 1.644 patients including a lost to follow-up rate of 10%. Primary and secondary endpoints will be reported after 5 and 10 years of follow-up. DISCUSSION: If the sentinel lymph node biopsy can be safely omitted in clinically node negative breast cancer patients undergoing breast conserving therapy, this study will cost-effectively lead to a decreased axillary morbidity rate and thereby improved quality of life with non-inferior regional control, distant-disease free survival and overall survival. TRIAL REGISTRATION: The BOOG 2013-08 study is registered in ClinicalTrials.gov since October 20, 2014, Identifier: NCT02271828. https://clinicaltrials.gov/ct2/show/NCT02271828.

Using a gene expression signature when controversy exists regarding the indication for adjuvant systemic treatment reduces the proportion of patients receiving adjuvant chemotherapy: a nationwide study.

PURPOSE: The Dutch national guideline advises use of gene-expression signatures, such as the 70-gene signature (70-GS), in case of ambivalence regarding the benefit of adjuvant chemotherapy (CT). In this nationwide study, the impact of 70-GS use on the administration of CT in early breast cancer patients with a dubious indication for CT is assessed. METHODS: Patients within a national guideline directed indication area for 70-GS use who were surgically treated between November 2011 and April 2013 were selected from the Netherlands Cancer Registry database. The effect of 70-GS use on the administration of CT was evaluated in guideline- and age-delineated subgroups addressing potential effect of bias by linear mixed-effect modeling and instrumental variable (IV) analyses. RESULTS: A total of 2,043 patients within the indicated area for 70-GS use were included, of whom 298 received a 70-GS. Without use of the 70-GS, 45% of patients received CT. The 70-GS use was associated with a 9.5% decrease in CT administration (95% confidence interval (CI): -15.7 to -3.3%) in linear mixed-effect model analyses and IV analyses showed similar results (-9.9%; 95% CI: -19.3 to -0.4). CONCLUSION: In patients in whom the Dutch national guidelines suggest the use of a gene-expression profile, 70-GS use is associated with a 10% decrease in the administration of adjuvant CT.Genet Med 18 7, 720-726.Genetics in Medicine (2016); 18 7, 720-726. doi:10.1038/gim.2015.152.

The prognostic value of the neoadjuvant response index in triple-negative breast cancer: validation and comparison with pathological complete response as outcome measure.

The Neoadjuvant response index (NRI) has been proposed as a simple measure of downstaging by neoadjuvant treatment in breast cancer. It was previously found to predict recurrence-free survival (RFS) in triple-negative (TN) breast cancer. It was at least as accurate as the standard binary system, the absence or presence of a pathological complete remission (pCR), which is the commonly employed outcome measure. The NRI was evaluated in an independent consecutive series of patients to validate the previous findings. Univariable and multivariable analyses were done to assess the predictive value of clinical parameters and of the NRI for RFS. We combined the original and validation series of patients to build a multivariable predictive model for RFS after neoadjuvant chemotherapy in TN breast cancer. The validation set (N = 108) confirmed that patients with a higher-than-median NRI (>0.7) had excellent RFS (P = 0.002), similar to that of patients who had achieved a pCR. Multivariable analysis in 191 patients showed that the NRI was a strong independent predictor of RFS (P = 0.0002), with N-stage (P = 0.001) and T-stage (P = 0.014) ranking second and third, respectively. Importantly, among patients who did not achieve a pCR (NRI values below 1), higher NRI values were still associated with better RFS. The NRI is a simple method and a practical tool to predict RFS in TN breast cancer patients treated with neoadjuvant chemotherapy. It adds prognostic information to the presence or absence of pCR and could be useful to compare the efficacies of different chemotherapy regimens.

The value of completion axillary treatment in sentinel node positive breast cancer patients undergoing a mastectomy: a Dutch randomized controlled multicentre trial (BOOG 2013-07).

BACKGROUND: Trials failed to demonstrate additional value of completion axillary lymph node dissection in case of limited sentinel lymph node metastases in breast cancer patients undergoing breast conserving therapy. It has been suggested that the low regional recurrence rates in these trials might partially be ascribed to accidental irradiation of part of the axilla by whole breast radiation therapy, which precludes extrapolation of results to mastectomy patients. The aim of the randomized controlled BOOG 2013-07 trial is therefore to investigate whether completion axillary treatment can be safely omitted in sentinel lymph node positive breast cancer patients treated with mastectomy. DESIGN: This study is designed as a non-inferiority randomized controlled multicentre trial. Women aged 18 years or older diagnosed with unilateral invasive clinically T1-2 N0 breast cancer who are treated with mastectomy, and who have a maximum of three axillary sentinel lymph nodes containing micro- and/or macrometastases, will be randomized for completion axillary treatment versus no completion axillary treatment. Completion axillary treatment can consist of completion axillary lymph node dissection or axillary radiation therapy. Primary endpoint is regional recurrence rate at 5 years. Based on a 5-year regional recurrence free survival rate of 98 % among controls and 96 % for study subjects, the sample size amounts 439 per arm (including 10 % lost to follow-up), to be able to reject the null hypothesis that the rate for study and control subjects is inferior by at least 5 % with a probability of 0.8. Results will be reported after 5 and 10 years of follow-up. DISCUSSION: We hypothesize that completion axillary treatment can be safely omitted in sentinel node positive breast cancer patients undergoing mastectomy. If confirmed, this study will significantly decrease the number of breast cancer patients receiving extensive treatment of the axilla, thereby diminishing the risk of morbidity and improving quality of life, while maintaining excellent regional control and without affecting survival. TRIAL REGISTRATION: The BOOG 2013-07 study is registered in the register of ClinicalTrials.gov since April 10, 2014, Identifier: NCT02112682 .

Tamoxifen dose and serum concentrations of tamoxifen and six of its metabolites in routine clinical outpatient care.

A sensitive and selective HPLC-MS/MS assay was used to analyze steady-state serum concentrations of tamoxifen, N-desmethyltamoxifen (E)-endoxifen, (Z)-endoxifen, N-desmethyl-4'-hydroxytamoxifen, 4-hydroxytamoxifen, and 4'-hydroxytamoxifen to support therapeutic drug monitoring (TDM) in patients treated with tamoxifen according to standard of care. When the (Z)-endoxifen serum concentration was below the predefined therapeutic threshold concentration of 5.9 ng/mL, the clinician was advised to increase the tamoxifen dose and to collect another serum sample. Paired serum samples from patients at one dose level at different time points during the tamoxifen treatment were used to assess the intra-patient variability. A total of 251 serum samples were analyzed, obtained from 205 patients. Of these patients, 197 used 20 mg tamoxifen per day and 8 patients used 10 mg/day. There was wide variability in tamoxifen and metabolite concentrations within the dosing groups. The threshold concentration for (Z)-endoxifen was reached in one patient (12 %) in the 10 mg group, in 153 patients (78 %) in the 20 mg group, and in 26 (96 %) of the patients who received a dose increase to 30 or 40 mg/day. Dose increase from 20 to 30 or 40 mg per day resulted in a significant increase in the mean serum concentrations of all analytes (p < 0.001). The mean intra-patient variability was between 10 and 20 % for all analytes. These results support the suitability of TDM for optimizing the tamoxifen treatment. It is shown that tamoxifen dose is related to (Z)-endoxifen exposure and increasing this dose leads to a higher serum concentration of tamoxifen and its metabolites. The low intra-patient variability suggests that only one serum sample is needed for TDM, making this a relatively noninvasive way to optimize the patient's treatment.

Use of dried blood spots for the determination of serum concentrations of tamoxifen and endoxifen.

The anti-estrogenic effect of tamoxifen is suggested to be mainly attributable to its metabolite (Z)-endoxifen, and a minimum therapeutic threshold for (Z)-endoxifen in serum has been proposed. The objective of this research was to establish the relationship between dried blood spot (DBS) and serum concentrations of tamoxifen and (Z)-endoxifen to allow the use of DBS sampling, a simple and patient-friendly alternative to venous sampling, in clinical practice. Paired DBS and serum samples were obtained from 50 patients using tamoxifen and analyzed using HPLC-MS/MS. Serum concentrations were calculated from DBS concentrations using the formula calculated serum concentration = DBS concentration/([1-haematocrit (Hct)] + blood cell-to-serum ratio × Hct). The blood cell-to-serum ratio was determined ex vivo by incubating a batch of whole blood spiked with both analytes. The average Hct for female adults was imputed as a fixed value. Calculated and analyzed serum concentrations were compared using weighted Deming regression. Weighted Deming regression analysis comparing 44 matching pairs of DBS and serum samples showed a proportional bias for both analytes. Serum concentrations were calculated using [Tamoxifen] serum, calculated  = [Tamoxifen] DBS /0.779 and [(Z)-Endoxifen] serum, calculated = [(Z)-Endoxifen] DBS /0.663. Calculated serum concentrations were within 20 % of analyzed serum concentrations in 84 and 100 % of patient samples for tamoxifen and (Z)-endoxifen, respectively. In conclusion, DBS concentrations of tamoxifen and (Z)-endoxifen were equal to serum concentrations after correction for Hct and blood cell-to-serum ratio. DBS sampling can be used in clinical practice.

Prognostic factors for survival in metastatic breast cancer by hormone receptor status.

Hormone receptor (HR) status is an important prognostic factor for patients with metastatic breast cancer (MBC) and is also correlated with other prognostic factors, such as initial lymph node status, HER2-Neu status and age. The prognostic value of these other factors, however, is unknown when stratified by HR positive versus HR negative patients. The aim of this study was to evaluate prognostic factors for MBC survival in relation to HR status. Dutch women diagnosed with breast cancer in 2003-2006 treated with curative intent who developed MBC within 5 years of follow-up were selected from the Netherlands cancer registry (N = 2,001). Independent prognostic factors for survival after metastatic occurrence were determined by multivariable Cox survival analyses stratified by HR status. Interactions between HR status and prognostic factors were determined. Median survival for MBC patients with HR negative (HR-) tumours was 8 months, compared to 19 months for HR positive (HR+) patients. The prognostic value of lymph node status, HER2-Neu status, adjuvant endocrine treatment and first-line palliative chemotherapy was dependent on HR status. Initial lymph node status was independently associated with survival in HR- patients, but not in HR+ patients. HER2-Neu positive status was associated with better survival in both HR+ and HR- patients, although the association was stronger in HR- patients. Similarly, patients treated with first-line palliative chemotherapy fared better, especially HR- patients. HR+ patients had worse survival if they had received adjuvant endocrine treatment. This study shows that the prognostic value of various factors depends on HR status in MBC. This information may help physicians to determine individual prognostic profiles and therapeutic strategies for MBC patients.

Optimized outcome prediction in breast cancer by combining the 70-gene signature with clinical risk prediction algorithms.

Clinical guidelines for breast cancer treatment differ in their selection of patients at a high risk of recurrence who are eligible to receive adjuvant systemic treatment (AST). The 70-gene signature is a molecular tool to better guide AST decisions. The aim of this study was to evaluate whether adding the 70-gene signature to clinical risk prediction algorithms can optimize outcome prediction and consequently treatment decisions in early stage, node-negative breast cancer patients. A 70-gene signature was available for 427 patients participating in the RASTER study (cT1-3N0M0). Median follow-up was 61.6 months. Based on 5-year distant-recurrence free interval (DRFI) probabilities survival areas under the curve (AUC) were calculated and compared for risk estimations based on the six clinical risk prediction algorithms: Adjuvant! Online (AOL), Nottingham Prognostic Index (NPI), St. Gallen (2003), the Dutch National guidelines (CBO 2004 and NABON 2012), and PREDICT plus. Also, survival AUC were calculated after adding the 70-gene signature to these clinical risk estimations. Systemically untreated patients with a high clinical risk estimation but a low risk 70-gene signature had an excellent 5-year DRFI varying between 97.1 and 100 %, depending on the clinical risk prediction algorithms used in the comparison. The best risk estimation was obtained in this cohort by adding the 70-gene signature to CBO 2012 (AUC: 0.644) and PREDICT (AUC: 0.662). Clinical risk estimations by all clinical algorithms improved by adding the 70-gene signature. Patients with a low risk 70-gene signature have an excellent survival, independent of their clinical risk estimation. Adding the 70-gene signature to clinical risk prediction algorithms improves risk estimations and therefore might improve the identification of early stage node-negative breast cancer patients for whom AST has limited value. In this cohort, the PREDICT plus tool in combination with the 70-gene signature provided the best risk prediction.

Cox proportional hazards regression in small studies of predictive biomarkers.

Predictive biomarkers are essential for personalized medicine since they select the best treatment for a specific patient. However, of all biomarkers that are evaluated, only few are eventually used in clinical practice. Many promising biomarkers may be erroneously abandoned because they are investigated in small studies using standard statistical techniques which can cause small sample bias or lack of power. The standard technique for failure time endpoints is Cox proportional hazards regression with a multiplicative interaction term between binary variables of biomarker and treatment. Properties of this model in small studies have not been evaluated so far, therefore we performed a simulation study to understand its small sample behavior. As a remedy, we applied a Firth correction to the score function of the Cox model and obtained confidence intervals (CI) using a profile likelihood (PL) approach. These methods are generally recommended for small studies of different design. Our results show that a Cox model estimates the biomarker-treatment interaction term and the treatment effect in one of the biomarker subgroups with bias, and overestimates their standard errors. Bias is however reduced and power is increased with Firth correction and PL CIs. Hence, the modified Cox model and PL CI should be used instead of a standard Cox model with Wald based CI in small studies of predictive biomarkers.

Intensified alkylating chemotherapy for patients with oligometastatic breast cancer harboring homologous recombination deficiency: Primary outcomes from the randomized phase III OLIGO study.

BACKGROUND: Oligometastatic breast cancer (OMBC) is a clinical entity with a prospect of long-term survival, but uncertainty remains on its optimal treatment. We studied whether intensified alkylating chemotherapy (IACT) improves long-term outcome compared to conventional-dose chemotherapy (CDCT) as part of a multimodality approach for patients with OMBC harboring homologous recombination deficiency (HRD). PATIENTS AND METHODS: Eligible patients had HER2-negative OMBC, harboring HRD, with ≤ 3 distant metastases, pathologic proof of distant disease and a favorable response to three cycles CDCT. Participants were randomized 1:1 to continue with either CDCT or IACT. IACT consisted of one mobilization course followed by two cycles of mini-CTC (carboplatin, thiotepa and cyclophosphamide) supported by peripheral blood progenitor cell reinfusion. Primary outcome was event-free survival (EFS). Secondary endpoints included overall survival (OS), quality of life and safety. RESULTS: Seventy-five patients were randomized to either IACT (n = 36) or CDCT (n = 39). Twenty-three (31 %) patients had hormone receptor-positive disease and 52 (69 %) had triple-negative disease. Median EFS in the IACT-group was 28 months (95 % confidence interval [CI] 21-not reached [NR]) versus 25 months (95 %CI 14-NR) in the CDCT-group (hazard ratio [HR] for recurrence or death 0.78, 95 %CI 0.42-1.42). Median OS was 67 months (95 %CI 37-NR) in the IACT-group and 36 (95 %CI 26-NR) in the CDCT-group (HR 0.74, 95 %CI 0.37-1.48). CONCLUSIONS: The entire study population experienced long-term survival, with median OS well over five years. IACT compared to CDCT did not improve outcome in patients with OMBC harboring study-defined HRD. The optimal therapy for patients with OMBC requires further study. TRIAL REGISTRATION: ClinicalTrials.gov: NCT01646034.

Quality assurance of radiation therapy after breast-conserving surgery among patients in the BOOG 2013-08 trial.

BACKGROUND AND PURPOSE: In the BOOG 2013-08 trial (NCT02271828), cT1-2N0 breast cancer patients were randomized between breast conserving surgery with or without sentinel lymph node biopsy (SLNB) followed by whole breast radiotherapy (WBRT). While awaiting primary endpoint results (axillary recurrence rate), this study aims to perform a quality assurance analysis on protocol adherence and (incidental) axillary radiation therapy (RT) dose. MATERIALS AND METHODS: Patients were enrolled between 2015 and 2022. Data on prescribed RT and (in 25% of included patients) planning target volumes (PTV) parameters were recorded for axillary levels I-IV and compared between treatment arms. Multivariable linear regression analysis was performed to determine prognostic variables for incidental axillary RT dose. RESULTS: 1,439/1,461 included patients (98.5%) were treated according to protocol and 87 patients (5.9%) received regional RT (SLNB 10.9%, no-SLNB 1.5 %). In 326 patients included in the subgroup analysis, the mean incidental PTV dose at axilla level I was 59.5% of the prescribed breast RT dose. In 5 patients (1.5%) the mean PTV dose at level I was ≥95% of the prescribed breast dose. No statistically or clinically significant differences regarding incidental axillary RT dose were found between treatment arms. Tumour bed boost (yes/no) was associated with a higher incidental mean dose in level I (R2 = 0.035, F(6, 263) = 1.532, p 0.168). CONCLUSION: The results indicate that RT-protocol adherence was high, and that incidental axillary RT dose was low in the BOOG 2013-08 trial. Potential differences between treatmentarms regarding the primary endpoint can thus not be attributed to different axillary radiation doses.

Prognostic value of histopathologic traits independent of stromal tumor-infiltrating lymphocyte levels in chemotherapy-naïve patients with triple-negative breast cancer.

BACKGROUND: In the absence of prognostic biomarkers, most patients with early-stage triple-negative breast cancer (eTNBC) are treated with combination chemotherapy. The identification of biomarkers to select patients for whom treatment de-escalation or escalation could be considered remains an unmet need. We evaluated the prognostic value of histopathologic traits in a unique cohort of young, (neo)adjuvant chemotherapy-naïve patients with early-stage (stage I or II), node-negative TNBC and long-term follow-up, in relation to stromal tumor-infiltrating lymphocytes (sTILs) for which the prognostic value was recently reported. MATERIALS AND METHODS: We studied all 485 patients with node-negative eTNBC from the population-based PARADIGM cohort which selected women aged <40 years diagnosed between 1989 and 2000. None of the patients had received (neo)adjuvant chemotherapy according to standard practice at the time. Associations between histopathologic traits and breast cancer-specific survival (BCSS) were analyzed with Cox proportional hazard models. RESULTS: With a median follow-up of 20.0 years, an independent prognostic value for BCSS was observed for lymphovascular invasion (LVI) [adjusted (adj.) hazard ratio (HR) 2.35, 95% confidence interval (CI) 1.49-3.69], fibrotic focus (adj. HR 1.61, 95% CI 1.09-2.37) and sTILs (per 10% increment adj. HR 0.75, 95% CI 0.69-0.82). In the sTILs <30% subgroup, the presence of LVI resulted in a higher cumulative incidence of breast cancer death (at 20 years, 58%; 95% CI 41% to 72%) compared with when LVI was absent (at 20 years, 32%; 95% CI 26% to 39%). In the ≥75% sTILs subgroup, the presence of LVI might be associated with poor survival (HR 11.45, 95% CI 0.71-182.36, two deaths). We confirm the lack of prognostic value of androgen receptor expression and human epidermal growth factor receptor 2 -low status. CONCLUSIONS: sTILs, LVI and fibrotic focus provide independent prognostic information in young women with node-negative eTNBC. Our results are of importance for the selection of patients for de-escalation and escalation trials.

A prospective evaluation of a breast cancer prognosis signature in the observational RASTER study.

The 70-gene signature (MammaPrint™) has been developed on retrospective series of breast cancer patients to predict the risk of breast cancer distant metastases. The microarRAy-prognoSTics-in-breast-cancER (RASTER) study was the first study designed to prospectively evaluate the performance of the 70-gene signature, which result was available for 427 patients (cT1-3N0M0). Adjuvant systemic treatment decisions were based on the Dutch CBO 2004 guidelines, the 70-gene signature and doctors' and patients' preferences. Five-year distant-recurrence-free-interval (DRFI) probabilities were compared between subgroups based on the 70-gene signature and Adjuvant! Online (AOL) (10-year survival probability <90% was defined as high-risk). Median follow-up was 61.6 months. Fifteen percent (33/219) of the 70-gene signature low-risk patients received adjuvant chemotherapy (ACT) versus 81% (169/208) of the 70-gene signature high-risk patients. The 5-year DRFI probabilities for 70-gene signature low-risk (n = 219) and high-risk (n = 208) patients were 97.0% and 91.7%. The 5-year DRFI probabilities for AOL low-risk (n = 132) and high-risk (n = 295) patients were 96.7% and 93.4%. For 70-gene signature low-risk-AOL high-risk patients (n = 124), of whom 76% (n = 94) had not received ACT, 5-year DRFI was 98.4%. In the AOL high-risk group, 32% (94/295) less patients would be eligible to receive ACT if the 70-gene signature was used. In this prospective community-based observational study, the 5-year DRFI probabilities confirmed the additional prognostic value of the 70-gene signature to clinicopathological risk estimations such as AOL. Omission of adjuvant chemotherapy as judged appropriate by doctors and patients and instigated by a low-risk 70-gene signature result, appeared not to compromise outcome.