Long-Term Ultrasound Twinkling Detectability and Safety of a Polymethyl Methacrylate Soft Tissue Marker Compared to Conventional Breast Biopsy Markers-A Preclinical Study in a Porcine Model.

OBJECTIVE: We have studied the use of polymethyl methacrylate (PMMA) as an alternative biopsy marker that is readily detectable with ultrasound Doppler twinkling in cases of in vitro, ex vivo, or limited duration in vivo settings. This study investigates the long-term safety and ultrasound Doppler twinkling detectability of a PMMA breast biopsy marker following local perturbations and different dwell times in a 6-mo animal experiment. METHODS: This study, which was approved by our Institutional Animal Care and Use Committee, involved three pigs and utilized various markers, including PMMA (Zimmer Biomet), 3D-printed, and Tumark Q markers. Markers were implanted at different times for each pig. Mesh material or ethanol was used to induce a local inflammatory reaction near certain markers. A semiquantitative twinkling score assessed twinkling for actionable localization during monthly ultrasounds. At the primary endpoint, ultrasound-guided localization of lymph nodes with detectable markers was performed. Following surgical resection of the localized nodes, histomorphometric analysis was conducted to evaluate for tissue ingrowth and the formation of a tissue rind around the markers. RESULTS: No adverse events occurred. Twinkling scores of all markers for all three pigs decreased gradually over time. The Q marker exhibited the highest mean twinkling score followed by the PMMA marker, PMMA with mesh, and Q with ethanol. The 3D-printed marker with mesh and PMMA with ethanol had the lowest scores. All wire-localized lymph nodes were successfully resected. Despite varying percentages of tissue rind around the markers and a significant reduction in overall twinkling (p < 0.001) over time, mean PMMA twinkling scores remained clinically actionable at 6 and 5 mo using a General Electric C1-6 probe and 9L-probe, respectively. CONCLUSIONS: In this porcine model, the PMMA marker demonstrates an acceptable safety profile. Clinically actionable twinkling aids PMMA marker detection even after 6 mo of dwell time in porcine lymph nodes. The Q marker maintained the greatest twinkling over time compared to all the other markers studied.

Association of Residual Ductal Carcinoma In Situ With Breast Cancer Recurrence in the Neoadjuvant I-SPY2 Trial.

Importance: Pathologic complete response (pCR) after neoadjuvant chemotherapy (NAC) in breast cancer strongly correlates with overall survival and has become the standard end point in neoadjuvant trials. However, there is controversy regarding whether the definition of pCR should exclude or permit the presence of residual ductal carcinoma in situ (DCIS). Objective: To examine the association of residual DCIS in surgical specimens after neoadjuvant chemotherapy for breast cancer with survival end points to inform standards for the assessment of pathologic complete response. Design, Setting, and Participants: The study team analyzed the association of residual DCIS after NAC with 3-year event-free survival (EFS), distant recurrence-free survival (DRFS), and local-regional recurrence (LRR) in the I-SPY2 trial, an adaptive neoadjuvant platform trial for patients with breast cancer at high risk of recurrence. This is a retrospective analysis of clinical specimens and data from the ongoing I-SPY2 adaptive platform trial of novel therapeutics on a background of standard of care for early breast cancer. I-SPY2 participants are adult women diagnosed with stage II/III breast cancer at high risk of recurrence. Interventions: Participants were randomized to receive taxane and anthracycline-based neoadjuvant therapy with or without 1 of 10 investigational agents, followed by definitive surgery. Main Outcomes and Measures: The presence of DCIS and EFS, DRFS, and LRR. Results: The study team identified 933 I-SPY2 participants (aged 24 to 77 years) with complete pathology and follow-up data. Median follow-up time was 3.9 years; 337 participants (36%) had no residual invasive disease (residual cancer burden 0, or pCR). Of the 337 participants with pCR, 70 (21%) had residual DCIS, which varied significantly by tumor-receptor subtype; residual DCIS was present in 8.5% of triple negative tumors, 15.6% of hormone-receptor positive tumors, and 36.6% of ERBB2-positive tumors. Among those participants with pCR, there was no significant difference in EFS, DRFS, or LRR based on presence or absence of residual DCIS. Conclusions and Relevance: The analysis supports the definition of pCR as the absence of invasive disease after NAC regardless of the presence or absence of DCIS. Trial Registration: ClinicalTrials.gov Identifier NCT01042379.

Standardizing Pathologic Evaluation of Breast Carcinoma After Neoadjuvant Chemotherapy.

CONTEXT.­: Neoadjuvant systemic therapy refers to the use of systemic agent(s) for malignancy prior to surgical treatment and has recently emerged as an option for most breast cancer patients eligible for adjuvant systemic therapy. Consequently, treated breast carcinomas have become routine specimens in pathology practices. A standard protocol has not yet been universally adopted for the evaluation and reporting of these specimens. The American Joint Committee on Cancer staging system recognizes the challenges in staging breast carcinomas after neoadjuvant treatment and provides important data points but does not currently provide detailed guidance in estimating the residual tumor burden in the breast and lymph nodes. The Residual Cancer Burden system is the only Web-based system that quantifies treatment response as a continuous variable using residual tumor burden in the breast and the lymph nodes. OBJECTIVE.­: To provide clarifications and guidance for evaluation and reporting of postneoadjuvant breast specimens, discuss issues with the current staging and reporting systems, and provide specific suggestions for future modifications to the American Joint Committee on Cancer system and the Residual Cancer Burden calculator. DATA SOURCES.­: English-language literature on the subject and the data from the I-SPY 2, a multicenter, adaptive randomization phase 2 neoadjuvant platform trial for early-stage, high-risk breast cancer patients. CONCLUSIONS.­: This article highlights challenges in the pathologic evaluation and reporting of treated breast carcinomas and provides recommendations and clarifications for pathologists and clinicians. It also provides specific recommendations for staging and discusses future directions.

The breast tissue microbiome, stroma, immune cells and breast cancer.

BACKGROUND: Stromal and immune cell composition alterations in benign breast tissue associate with future cancer risk. Pilot data suggest the innate microbiome of normal breast tissue differs between women with and without breast cancer. Microbiome alterations might explain tissue microenvironment variations associated with disease status. METHODS: Prospectively-collected sterile normal breast tissues from women with benign (n=16) or malignant (n=17) disease underwent 16SrRNA sequencing with Illumina MiSeq and Hybrid-denovo pipeline processing. Breast tissue was scored for fibrosis and fat percentages and immune cell infiltrates (lobulitis) classified as absent/mild/moderate/severe. Alpha and beta diversity were calculated on rarefied OTU data and associations analyzed with multiple linear regression and PERMANOVA. RESULTS: Breast tissue stromal fat% was lower and fibrosis% higher in benign disease versus cancer (median 30% versus 60%, p=0.01, 70% versus 30%, p=0.002, respectively). The microbiome varied with stromal composition. Alpha diversity (Chao1) correlated with fat% (r=0.38, p=0.02) and fibrosis% (r=-0.32, p=0.05) and associated with different microbial populations as indicated by beta diversity metrics (weighted UniFrac, p=0.08, fat%, p=0.07, fibrosis%). Permutation testing with FDR control revealed taxa differences for fat% in Firmicutes, Bacilli, Bacillales, Staphylococcaceae and genus Staphylococcus, and fibrosis% in Firmicutes, Spirochaetes, Bacilli, Bacillales, Spirochaetales, Proteobacteria RF32, Sphingomonadales, Staphylococcaceae, and genera Clostridium, Staphylococcus, Spirochaetes, Actinobacteria Adlercreutzia. Moderate/severe lobulitis was more common in cancer (73%) than benign disease (13%), p=0.003, but no significant microbial associations were seen. CONCLUSION: These data suggest a link between breast tissue stromal alterations and its microbiome, further supporting a connection between the breast tissue microenvironment and breast cancer.

Randomized Phase II Study of Two Doses of Pixantrone in Patients with Metastatic Breast Cancer (NCCTG N1031, Alliance).

BACKGROUND: Anthracycline use in metastatic breast cancer (MBC) is hindered by cumulative exposure limits and risk of cardiotoxicity. Pixantrone, a novel aza-anthracenedione with structural similarities to mitoxantrone and anthracyclines, is theorized to exhibit less cardiotoxicity, mainly due to lack of iron binding. We conducted a randomized phase II study to evaluate the efficacy and safety of 2 dosing schedules of pixantrone in patients with refractory HER2-negative MBC. METHODS: Intravenous pixantrone was administered at 180 mg/m2 every 3 weeks (group A) versus 85 mg/m2 on days 1, 8, and 15 of a 28-day cycle (group B). Primary endpoint was objective response rate (ORR) and secondary endpoints included progression-free survival (PFS), median 6-month PFS, overall survival (OS), safety, quality of life, and serial assessment of circulating tumor cells. A 20% ORR was targeted as sufficient for further testing of pixantrone in this patient population. RESULTS: Forty-five patients were evaluable, with 2 confirmed partial responses in group A and 1 in group B. The trial was terminated due to insufficient activity. Overall median PFS and OS were 2.8 (95% confidence interval [CI]: 2.0-4.1) and 16.8 (95% CI: 8.9-21.6) months, respectively. Notable overall grade 3-4 adverse events were the following: neutrophil count decrease (62%), fatigue (16%), and decrease in ejection fraction (EF) (4%). CONCLUSION: Pixantrone has insufficient activity in the second- and third-line MBC setting. It appears, however, to have limited cardiotoxicity. (ClinicalTrials.gov ID: NCT01086605).

Assessment of Residual Cancer Burden and Event-Free Survival in Neoadjuvant Treatment for High-risk Breast Cancer: An Analysis of Data From the I-SPY2 Randomized Clinical Trial.

IMPORTANCE: Residual cancer burden (RCB) distributions may improve the interpretation of efficacy in neoadjuvant breast cancer trials. OBJECTIVE: To compare RCB distributions between randomized control and investigational treatments within subtypes of breast cancer and explore the relationship with survival. DESIGN, SETTING, AND PARTICIPANTS: The I-SPY2 is a multicenter, platform adaptive, randomized clinical trial in the US that compares, by subtype, investigational agents in combination with chemotherapy vs chemotherapy alone in adult women with stage 2/3 breast cancer at high risk of early recurrence. Investigational treatments graduated in a prespecified subtype if there was 85% or greater predicted probability of higher rate of pathologic complete response (pCR) in a confirmatory, 300-patient, 1:1 randomized, neoadjuvant trial in that subtype. Evaluation of a secondary end point was reported from the 10 investigational agents tested in the I-SPY2 trial from March 200 through 2016, and analyzed as of September 9, 2020. The analysis plan included modeling of RCB within subtypes defined by hormone receptor (HR) and ERBB2 status and compared control treatments with investigational treatments that graduated and those that did not graduate. INTERVENTIONS: Neoadjuvant paclitaxel plus/minus 1 of several investigational agents for 12 weeks, then 12 weeks of cyclophosphamide/doxorubicin chemotherapy followed by surgery. MAIN OUTCOMES AND MEASURES: Residual cancer burden (pathological measure of residual disease) and event-free survival (EFS). RESULTS: A total of 938 women (mean [SD] age, 49 [11] years; 66 [7%] Asian, 103 [11%] Black, and 750 [80%] White individuals) from the first 10 investigational agents were included, with a median follow-up of 52 months (IQR, 29 months). Event-free survival worsened significantly per unit of RCB in every subtype of breast cancer (HR-positive/ERBB2-negative: hazard ratio [HZR], 1.75; 95% CI, 1.45-2.16; HR-positive/ERBB2-positive: HZR, 1.55; 95% CI, 1.18-2.05; HR-negative/ERBB2-positive: HZR, 2.39; 95% CI, 1.64-3.49; HR-negative/ERBB2-negative: HZR, 1.99; 95% CI, 1.71-2.31). Prognostic information from RCB was similar from treatments that graduated (HZR, 2.00; 95% CI, 1.57-2.55; 254 [27%]), did not graduate (HZR, 1.87; 95% CI, 1.61-2.17; 486 [52%]), or were control (HZR, 1.79; 95% CI, 1.42-2.26; 198 [21%]). Investigational treatments significantly lowered RCB in HR-negative/ERBB2-negative (graduated and nongraduated treatments) and ERBB2-positive subtypes (graduated treatments), with improved EFS (HZR, 0.61; 95% CI, 0.41-0.93) in the exploratory analysis. CONCLUSIONS AND RELEVANCE: In this randomized clinical trial, the prognostic significance of RCB was consistent regardless of subtype and treatment. Effective neoadjuvant treatments shifted the distribution of RCB in addition to increasing pCR rate and appeared to improve EFS. Using a standardized quantitative method to measure response advances the interpretation of efficacy. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT01042379.

Detailed Reanalysis of 500 Breast Cancers With Equivocal HER2 Immunohistochemistry and Borderline ERBB2 Fluorescence In Situ Hybridization Results.

OBJECTIVES: We investigated the impact of our laboratory's reflex testing process for resolving ERBB2 (HER2) status on breast cancer samples that require additional workup after fluorescence in situ hybridization (FISH), per guideline recommendations published in 2018 by the American Society of Clinical Oncology (ASCO) and the College of American Pathologists (CAP). METHODS: In total, 500 breast cancer specimens with ERBB2 FISH results in groups 2 through 4 (all reported as immunohistochemistry [IHC] equivocal [2+] at external laboratories) were resubmitted for IHC testing in our laboratory. Per the ASCO/CAP guideline, FISH was rescored when internal IHC was also equivocal (2+), targeted to tumor areas demonstrating more intense IHC staining, if observed. RESULTS: Reflex IHC/FISH testing changed the final reported ERBB2 status in 185 of 500 (37.0%) samples. Result changes included discordant IHC (n = 4 score 0, n = 132 score 1+, and n = 16 score 3+) and discordant FISH (n = 33). Numerical differences in FISH scores were comparable for targeted vs nontargeted FISH rescoring (P = .086 for ERBB2 copy number; P = .49 for ERBB2 ratio). Two cases showed larger differences in FISH scores, suggesting heterogeneity. CONCLUSIONS: Retesting of breast cancer samples with equivocal IHC frequently changes IHC results, but targeted reanalysis of borderline FISH results rarely identifies significant differences in ERBB2 copy number or ratio.