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Recent ASCO/CAP guidelines focus on decision making associated with the presence/absence of continuous breast biomarkers. Statistical standardization (SS) is demonstrated as a method to evaluate the effects of continuous RT-PCR biomarker expression levels on breast cancer outcomes. MA.14 allocated 667 postmenopausal patients to tamoxifen based on locally determined ER/PR. Of 299 available patient tumor samples, 292 passed internal quality control. All tumors were centrally assessed by RT-PCR ER/PR/HER2 with each biomarker's z-scores categorized: ≥1.0 standard deviation (SD) below mean;1.0 SD below mean; ≤1.0 SD above mean;1.0 SD above mean. Log-rank statistics tested univariate differences in breast cancer relapse-free survival (RFS). Continuous SS-ER/PR/HER2 were assessed in multivariate Cox step-wise forward regression, adding a factor if p ≤ 0.05. Sensitivity analyses examined an external HER2+ cut-point of 1.32. Patients whose tumors were tested were representative of the MA.14 population (p values = 0.18-0.90). At 9.8 years median follow-up, SS-ER did not univariately impact RFS (p = 0.31). SS-PR values above the mean (z ≥ 0.0) had the best univariate RFS (p = 0.03). SS-HER2 also univariately impacted RFS (p = 0.004) with lowest (z-scores ≤ -1.0) and highest (z-scores 1.0) having shortest RFS. Multivariate stratified/unstratified Cox models indicated patients with T1 tumors (p = 0.02/p = 0.0002) and higher SS-PR (p = 0.02/p = 0.01) had longer RFS; node-negative patients had better RFS (in unstratified analysis, p 0.0001). Local ER/PR status did not impact RFS (p 0.05). Patients with SS HER2+ ≥ 1.32 had worse RFS (univariate, p = 0.05; multivariate, p = 0.06). We demonstrated that higher SS-PR, and SS HER2 levels, measured by RT-PCR impacted breast cancer RFS outcomes. Evaluation in other trials may provide support for this methodology.

Chronic neuropathic pain and depression are often comorbid. Ketamine has been used to treat refractory pain. There is emerging evidence for use in depression. We present a case of a pediatric patient who was successfully treated with subanesthetic intravenous ketamine infusion for chronic neuropathic pain and suicidality.

Patients with hormone receptor–positive early breast cancer have a continuous yearly rate of recurrence extending out to 15 years after having received adjuvant endocrine therapy with tamoxifen for 5 years (1). More than half of the recurrences and about two-thirds of breast cancer deaths in this group will occur beyond 5 years from diagnosis (ie, late recurrences and death) (1). The National Cancer Institute of Canada (NCIC) Clinical Trials Group MA.17 trial was a randomized, placebo-controlled trial demonstrating that extended endocrine therapy with letrozole improves disease-free survival (DFS) (2), distant DFS (DDFS), and overall survival (OS) in disease-free postmenopausal patients with hormonal receptor–positive breast cancer after 5 years of tamoxifen (3). Although extended antihormonal therapy has become standard practice and is endorsed by international clinical practice guidelines (4,5), understanding which patients will actually benefit from longer treatment is paramount to individualized therapy. The status of primary tumor estrogen receptor (ER) and progesterone receptor (PR) expression has been suggested, but not confirmed, as a way to identify patients who have increased benefit from extended letrozole (5). Patients with ER+ and PR+ tumors have been shown to have improved DFS (P = .02) when compared with patients with ER+ and PR− tumors (6). Currently, however, there are no guideline-accepted biomarkers to stratify hormonal receptor–positive patients for prediction of benefit beyond the 5 years of adjuvant endocrine therapy. In addition, standard clinico-pathological factors and clinically available genomic signatures, including the 21-gene (Recurrence Score) assay (7) and the 70-gene assay (8), have greatest prognostic performance for recurrence risk within the first 5 years of adjuvant therapy. Identification of additional biomarkers to further stratify hormone receptor–positive tumors to predict those at risk of late recurrence and those who may or may not benefit from extended endocrine therapy would be of substantial clinical utility (9). We have previously demonstrated that the two-gene expression ratio, HOXB13/IL17BR (H/I), is a prognostic biomarker in both untreated and tamoxifen-treated early-stage ER+ breast cancer patients (10–13). However, potential assessment of H/I as a predictive biomarker for endocrine therapy has been limited by the analysis of retrospective/observational cohorts or by the use of single treatment arm cohorts from randomized trials (11–13). Herein, we have conducted a prospective–retrospective (14), nested case-control study in a subset of patients from NCIC Clinical Trials Group MA.17 trial to evaluate the performance of H/I for 1) prognostication of late disease recurrence and 2) prediction of treatment benefit from extended adjuvant letrozole therapy in patients with hormone receptor–positive early breast cancer.

Background: Poor inter-laboratory comparability of common clinically used breast cancer biomarkers led to a proposal of statistical standardization (SS) of laboratory results, similar to bone mineral density (BMD) z-scores. This analysis is the first utilization of SS in a trial where all women received TAM. Methods: MA.14 allocated 667 postmenopausal women to TAM +/− Octreotide LAR (OCT) based on locally determined ER/PR, without HER2 status. At 9.8 yrs median follow-up, the secondary endpoint of relapse-free survival (RFS) had a non-significant hazard ratio (HR) for TAM-OCT to TAM of 0.87 (95% CI 0.63–1.21; p = 0.40). 299 patients who were representative of MA.14 patients by treatment and stratification factors (exact Fisher p-values=0.19–0.90) had their tumors centrally assessed for ER, PR, and HER2 by RT-PCR. Continuous values were used for SS of each biomarker. Univariate (uni) assessment used similar categorizations as those for BMD, assigning ER/PR/HER2 values by number of standard deviations (SD) about the mean (Group 1, z-score ≥1.0 SD below mean; Group 2, z-score 1.0 SD above mean). A log-rank statistic was used to test for differences between SS biomarker groups with K-M plots for graphical description. Multivariate (multi) effects of SS biomarkers and baseline patient characteristics on RFS were examined with exploratory (un)stratified Cox step-wise forward regression, adding a factor if likelihood ratio criterion was p ≤ 0.05. Sensitivity analyses used a prior external HER2+ cut-point of ≥1.32 SD. Results: 292 patient samples passing internal analytical quality control were included in this analysis. Uni analyses indicated SS ER was not associated with RFS (p = 0.31). SS PR had a significant uni effect on RFS [p = 0.03; Group 4 compared to Group 1, HR of 0.33 (95% CI 0.12–0.90); Group 3 compared to Group 1, HR of 0.42 (95% CI 0.21–0.83); and Group 2 compared to Group 1 HR of 0.70 (95%CI 0.36–1.37)]. SS HER2 also had a significant uni effect on RFS [p = 0.004; Group 4 compared to Group 1, HR of 0.90 (95% CI 0.37–2.16)]; Group 3 compared to Group 1, HR of 0.39 (95% CI 0.18–0.84); and, Group 2 compared to Group 1, HR of 0.34 (95% CI 0.16–0.70)]. Multi stratified/unstratified Cox models indicated T1 tumours (p = 0.02/p = 0.0002) and higher SS PR (p = 0.02/0.01) were associated with significantly longer RFS; other unstratified results showed that N-ve patients had better RFS (p < .0001), while local ER/PR status did not impact RFS (p > 0.05). The HER2+ cut-point of ≥1.32 SD indicated directionally worse RFS (uni p-value=0.05; multi p-value=0.06). Discussion: In MA.14, all women received TAM. Local ER/PR status using categorical or semi-quantitative values did not impact RFS. A statistically standardized approach using continuous centralized ER, PR, HER2 by RT-PCR demonstrated that increasing PR values were associated with better RFS. Evaluation in other trials may provide support for this methodology. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-07-13.

Background Biomarkers that can be used to accurately assess the residual risk of disease recurrence in women with hormone receptor–positive breast cancer are clinically valuable. We evaluated the prognostic value of the Breast Cancer Index (BCI), a continuous risk index based on a combination of HOXB13:IL17BR and molecular grade index, in women with early breast cancer treated with either tamoxifen alone or tamoxifen plus octreotide in the NCIC MA.14 phase III clinical trial (ClinicalTrials.gov Identifier NCT00002864; registered 1 November 1999). Methods Gene expression analysis of BCI by real-time polymerase chain reaction was performed blinded to outcome on RNA extracted from archived formalin-fixed, paraffin-embedded tumor samples of 299 patients with both lymph node–negative (LN−) and lymph node–positive (LN+) disease enrolled in the MA.14 trial. Our primary objective was to determine the prognostic performance of BCI based on relapse-free survival (RFS). MA.14 patients experienced similar RFS on both treatment arms. Association of gene expression data with RFS was evaluated in univariate analysis with a stratified log-rank test statistic, depicted with a Kaplan-Meier plot and an adjusted Cox survivor plot. In the multivariate assessment, we used stratified Cox regression. The prognostic performance of an emerging, optimized linear BCI model was also assessed in a post hoc analysis. Results Of 299 samples, 292 were assessed successfully for BCI for 146 patients accrued in each MA.14 treatment arm. BCI risk groups had a significant univariate association with RFS (stratified log-rank p = 0.005, unstratified log-rank p = 0.007). Adjusted 10-year RFS in BCI low-, intermediate-, and high-risk groups was 87.5 %, 83.9 %, and 74.7 %, respectively. BCI had a significant prognostic effect [hazard ratio (HR) 2.34, 95 % confidence interval (CI) 1.33–4.11; p = 0.004], although not a predictive effect, on RFS in stratified multivariate analysis, adjusted for pathological tumor stage (HR 2.22, 95 % CI 1.22–4.07; p = 0.01). In the post hoc multivariate analysis, higher linear BCI was associated with shorter RFS (p = 0.002). Conclusions BCI had a strong prognostic effect on RFS in patients with early-stage breast cancer treated with tamoxifen alone or with tamoxifen and octreotide. BCI was prognostic in both LN− and LN+ patients. This retrospective study is an independent validation of the prognostic performance of BCI in a prospective trial. Electronic supplementary material The online version of this article (doi:10.1186/s13058-015-0660-6) contains supplementary material, which is available to authorized users.

RFS Kaplan-Meier plot of linear BCI for lymph nodeâ positive patients. (PDF 90 kb)

REMARK diagram for BCI investigations. (PDF 51 kb)

Precise proteomic profiling of limited levels of disease tissue represents an extremely challenging task. Here, we present an effective and reproducible microproteomic workflow for sample sizes of only 10,000 cells that integrates selective sample procurement via laser capture microdissection (LCM), sample clean-up and protein level fractionation using short-range SDS-PAGE, followed by ultrasensitive LC–MS/MS analysis using a 10 μm i.d. porous layer open tubular (PLOT) column. With 10,000 LCM captured mouse hepatocytes for method development and performance assessment, only 10% of the in-gel digest, equivalent to ∼1000 cells, was needed per LC–MS/MS analysis. The optimized workflow was applied to the differential proteomic analysis of 10,000 LCM collected primary and metastatic breast cancer cells from the same patient. More than 1100 proteins were identified from each injection with >1700 proteins identified from three LCM samples of 10,000 cells from the same patient (1123 with at least two unique peptides). Label free quantitation (spectral counting) was performed to identify differential protein expression between the primary and metastatic cell populations. Informatics analysis of the resulting data indicated that vesicular transport and extracellular remodeling processes were significantly altered between the two cell types. The ability to extract meaningful biological information from limited, but highly informative cell populations demonstrates the significant benefits of the described microproteomic workflow.

Background: The continuous linear Breast Cancer Index (BCI) risk index combines the ratio of genes HOXB13 to IL17BR (H/I) and the molecular grade index (MGI) (Zhang et al, Clinical Cancer Research, 2013). The BCI signature was developed for node-negative breast cancer patients treated with tamoxifen. We examine here whether linear BCI is prognostic for node-positive hormone-receptor positive tamoxifen-treated patients. Methods: MA.14 randomly assigned 667 hormone positive (HR+), postmenopausal women to 5 years of tamoxifen (TAM) +/- 2 years of octreotide LAR (TAM-OCT). A representative subgroup of 299 patients underwent gene expression profiling by RT-PCR for linear BCI. We performed exploratory analyses restricted to node positive patients. The primary objective was to assess the prognostic effect of BCI on relapse-free survival (RFS). RFS was defined as the time from randomization to the time of recurrence of the primary disease alone, including local and ipsilateral nodal recurrence and metastatic disease, and censoring at longest follow-up or death from another cause. With a median 9.8 years follow-up, the association of BCI with RFS was assessed by multivariate Cox regression including treatment, stratification factors (other than nodal status), and baseline patient and tumor characteristics. Patients were defined to be low risk based on BCI if the adjusted Cox survival was >95%, where adjustment was by trial treatment, stratification factors, and baseline patient and tumor characteristics, including IGF-1, IGFBP-3, and C-peptide. Results: 292 of 299 patient samples passed internal analytical quality control; 116 node positive ER+ve patients had 34 (29.3%) relapses, with adjusted Cox survival at 9.6 years of 87.8%. Fifty-two of the 116 patients (45%) did not receive adjuvant chemotherapy, and experienced 11 (21%) RFS events. In the 116 patients, higher continuous BCI value was associated with shorter RFS (p=0.002): hazard ratio (HR) 1.49 (95% CI 1.16-1.91). Smaller pathologic T had significantly (p=0.03) better RFS HR=0.39, (95%CI 0.17-0.90). With MA.14 patient mean BCI of 5.09532, Cox survival at 4.1 years was 95.2%; 17/34 (50%) who recurred had failed by this time. Discussion: In this subgroup analysis, we found that BCI and tumor size were significant prognostic factors for node-positive hormone-receptor positive patients who were treated with tamoxifen. Citation Format: Dennis Sgroi, Paul Goss, Judy-Anne Chapman, Elizabeth Richardson, Shemeica Binns, Yi Zhang, Cathy Schnabel, Mark Erlander, Kathy Pritchard, Lei Han, Lois Sheperd, Michael Pollack. Prognostic significance of breast cancer index (BCI) in node-positive hormone receptor positive early breast cancer: NCIC CTG MA.14 [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-01-10.

561 Background: Breast Cancer Index (BCI), a continuous risk index, combines the ratio of HOXB13 to IL17BR (H/I) and the molecular grade index (MGI) (Jerevall et al., British J Cancer, 2011). Here, the prognostic and predictive performance of BCI for BC relapse in MA.14 trial was examined. Methods: MA.14 randomly assigned 667 hormone receptor positive (HR+) women to 5 years of tamoxifen (TAM) +/- 2 years of octreotide LAR (TAM-OCT). A representative subgroup of 299 patients was profiled by RT-PCR for BCI. The primary objective was to determine the prognostic performance of BCI based on relapse-free survival (RFS) with median 9.8 years follow-up. Association of BCI was assessed with step-wise forward stratified Cox regression. Pre-defined categories of low (L), intermediate (I) and high (H) BCI risk groups were used to provide adjusted 5- and 10-year RFS. Results: 292 of 299 patient samples passed internal analytical quality control. The 292 patients contained 49% LN+ patients and had 19.9% BC relapses. Both continuous and pre-specified BCI risk groups were significant multivariate factors (p

2 Background: The MA.17 trial demonstrated that extended adjuvant endocrine therapy with letrozole after 5-y of tamoxifen markedly reduced the risk of recurrence in women with ER+ early stage breast cancer. This trial provides an opportunity to assess the ability of biomarkers to predict late recurrences in ER+ breast cancer. The Breast Cancer Index (BCI), a continuous risk index based on the combination of HOXB13:IL17BR (H:I) and the molecular grade index (MGI), estimates the individual risk of recurrence in ER+ breast cancer patients. In this study, the prognostic utility of BCI to predict late recurrences was examined. Methods: FFPE tumor blocks were collected from patients who experienced a breast cancer recurrence up to unblinding of MA.17. Controls were matched 2:1 for age, tumor size, nodal status and prior chemotherapy, and were disease free for longer than cases. All cases were reviewed for standard histopathology and evaluated using the real-time RT-PCR BCI assay. Results: Patient characteristics for the case-control study were similar to that from the overall study. Characteristics for cases (N=83) and controls (N=166) were not significantly different except for treatment. A higher percentage of controls compared to cases tended to be categorized as low risk by BCI (58% vs 43%), while a lower percentage of controls than cases tended to be categorized as high risk by BCI (34% vs 24%). In univariate analysis, treatment, BCI, H:I and HOXB13, but not tumor grade or MGI, were significant predictors of late recurrence. After adjusting for standard variables (age, tumor grade and treatment), BCI (OR 2.37; P=0.03), H:I (OR 2.55; P=0.04) and HOXB13 (OR 1.35; P=0.02) remained significant predictors of recurrence. HOXB13 expression at diagnosis predicted patient benefit from extended endocrine therapy with letrozole. Conclusions: In this case-controlled study, the data demonstrate that BCI is a significant predictor of late recurrences in ER+ patients following 5-y of tamoxifen. The prognostic performance of BCI to predict late recurrences was largely dependent on HOXB13 expression. The integration of H:I and MGI within BCI provides prognostic utility for both early and late recurrences.