MYC Protein Interactome Profiling Reveals Functionally Distinct Regions that Cooperate to Drive Tumorigenesis.

Transforming members of the MYC family (MYC, MYCL1, and MYCN) encode transcription factors containing six highly conserved regions, termed MYC homology boxes (MBs). By conducting proteomic profiling of the MB interactomes, we demonstrate that half of the MYC interactors require one or more MBs for binding. Comprehensive phenotypic analyses reveal that two MBs, MB0 and MBII, are universally required for transformation. MBII mediates interactions with acetyltransferase-containing complexes, enabling histone acetylation, and is essential for MYC-dependent tumor initiation. By contrast, MB0 mediates interactions with transcription elongation factors via direct binding to the general transcription factor TFIIF. MB0 is dispensable for tumor initiation but is a major accelerator of tumor growth. Notably, the full transforming activity of MYC can be restored by co-expression of the non-transforming MB0 and MBII deletion proteins, indicating that these two regions confer separate molecular functions, both of which are required for oncogenic MYC activity.

Reliability and Variability of Ki-67 Digital Image Analysis Methods for Clinical Diagnostics in Breast Cancer.

Ki-67 is a nuclear protein associated with proliferation, and a strong potential biomarker in breast cancer, but is not routinely measured in current clinical management owing to a lack of standardization. Digital image analysis (DIA) is a promising technology that could allow high-throughput analysis and standardization. There is a dearth of data on the clinical reliability as well as intra- and interalgorithmic variability of different DIA methods. In this study, we scored and compared a set of breast cancer cases in which manually counted Ki-67 has already been demonstrated to have prognostic value (n = 278) to 5 DIA methods, namely Aperio ePathology (Lieca Biosystems), Definiens Tissue Studio (Definiens AG), Qupath, an unsupervised immunohistochemical color histogram algorithm, and a deep-learning pipeline piNET. The piNET system achieved high agreement (interclass correlation coefficient: 0.850) and correlation (R = 0.85) with the reference score. The Qupath algorithm exhibited a high degree of reproducibility among all rater instances (interclass correlation coefficient: 0.889). Although piNET performed well against absolute manual counts, none of the tested DIA methods classified common Ki-67 cutoffs with high agreement or reached the clinically relevant Cohen's κ of at least 0.8. The highest agreement achieved was a Cohen's κ statistic of 0.73 for cutoffs 20% and 25% by the piNET system. The main contributors to interalgorithmic variation and poor cutoff characterization included heterogeneous tumor biology, varying algorithm implementation, and setting assignments. It appears that image segmentation is the primary explanation for semiautomated intra-algorithmic variation, which involves significant manual intervention to correct. Automated pipelines, such as piNET, may be crucial in developing robust and reproducible unbiased DIA approaches to accurately quantify Ki-67 for clinical diagnosis in the future.

Primary MALT lymphoma of the breast: pathological and radiological characteristics.

PURPOSE: Primary Mucosa-associated lymphoid tissue (MALT) lymphoma is a rare diagnosis in the breast, and clinical diagnosis based on radiological features is often challenging. This study aimed to evaluate the clinicopathological, and radiological characteristics of the patients diagnosed with primary breast MALT lymphoma. METHODS: This study examined 18 cases of primary MALT lymphoma of the breast diagnosed at a single tertiary center between January 2002 to December 2020. Medical charts, radiological imaging and original pathology slides were reviewed for each case. RESULTS: All cases were female (gender assigned at birth) and presented with a palpable mass or an incidental imaging finding. Imaging presentation ranged from mammographic asymmetries, circumscribed masses, and ultrasound masses lacking suspicious features. Seventeen cases were biopsied under ultrasound; one received a diagnostic excision biopsy. Microscopic examination of the breast specimens demonstrated atypical small lymphocyte infiltration with plasmacytoid differentiation and rare lymphoepithelial lesions. Immunohistochemistry was performed in all cases and established the diagnosis. Most patients were treated with radiotherapy, and only three were treated with chemotherapy. The median follow-up period was 4 years and 7.5 months, and all patients were alive at the last follow-up. CONCLUSION: Primary MALT breast lymphomas are usually indolent and non-systemic, and local radiotherapy may effectively alleviate local symptoms. Radiological findings show overlap with benign morphological features, which can delay the diagnosis of this unusual etiology. Although further studies involving a larger cohort could help establish the clinical and radiological characteristics of primary breast MALT lymphomas, pathology remains the primary method of diagnosis. TRIAL REGISTRATION NUMBER: University Health Network Ethics Committee (CAPCR/UHN REB number 19-5844), retrospectively registered.

Therapeutic inhibition of USP9x-mediated Notch signaling in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is a breast cancer subtype that lacks targeted treatment options. The activation of the Notch developmental signaling pathway, which is a feature of TNBC, results in the secretion of proinflammatory cytokines and the recruitment of protumoral macrophages to the tumor microenvironment. While the Notch pathway is an obvious therapeutic target, its activity is ubiquitous, and predictably, anti-Notch therapies are burdened with significant on-target side effects. Previously, we discovered that, under conditions of cellular stress commonly found in the tumor microenvironment, the deubiquitinase USP9x forms a multiprotein complex with the pseudokinase tribbles homolog 3 (TRB3) that together activate the Notch pathway. Herein, we provide preclinical studies that support the potential of therapeutic USP9x inhibition to deactivate Notch. Using a murine TNBC model, we show that USP9x knockdown abrogates Notch activation, reducing the production of the proinflammatory cytokines, C-C motif chemokine ligand 2 (CCL2) and interleukin-1 beta (IL-1ß). Concomitant with these molecular changes, a reduction in tumor inflammation, the augmentation of antitumor immune response, and the suppression of tumor growth were observed. The pharmacological inhibition of USP9x using G9, a partially selective, small-molecule USP9x inhibitor, reduced Notch activity, remodeled the tumor immune landscape, and reduced tumor growth without associated toxicity. Proving the role of Notch, the ectopic expression of the activated Notch1 intracellular domain rescued G9-induced effects. This work supports the potential of USP9x inhibition to target Notch in metabolically vulnerable tissues like TNBC, while sparing normal Notch-dependent tissues.

Annual Mammographic Screening Reduces the Risk of Interval or Higher Stage Invasive Breast Cancers Among Postmenopausal Women in the Ontario Breast Screening Program.

Purpose: In the Ontario Breast Screening Program (OBSP) annual screening improved breast cancer detection for women 50-74 years with a family/personal history compared to biennial, while detection was equivalent for women screened annually for mammographic density ≥75%. This study compares the risk of interval or higher stage invasive cancers among postmenopausal women screened annually vs biennially by age and estrogen use. Methods: A retrospective design identified 4247 invasive breast cancers diagnosed among concurrent cohorts of women 50-74 screened in the OBSP with digital mammography between 2011 and 2014, followed until 2016. Polytomous logistic regression estimated the risk of interval or higher stage breast cancers by age and estrogen use between women screened annually because of first-degree relative with breast or ovarian cancer or personal history of ovarian cancer, or mammographic density ≥75%, and those screened biennially. Results: The risk of interval vs screen-detected cancers was significantly reduced in women screened annually for family/personal history (OR=.64; 95%CI:0.51-.80), particularly those 60-74 years (OR=.59; 95%CI:0.45-.77) or not currently using estrogen (OR=.66; 95%CI:0.52-.83) compared to those screened biennially. The risk of stage II-IV vs stage I tumors was also lower in women 60-74 years screened annually for family/personal history (OR=.79; 95%CI:0.64-.97) and in those screened annually for mammographic density ≥75% currently using estrogen (OR=.51; 95%CI:0.26-1.01) compared to women screened biennially. Conclusion: Postmenopausal women at increased risk screened annually had equivalent or reduced risks of interval or higher stage invasive breast cancers than those screened biennially, further supporting risk-based screening in this population.

Intraoperative fluorescence imaging with aminolevulinic acid detects grossly occult breast cancer: a phase II randomized controlled trial.

BACKGROUND: Re-excision due to positive margins following breast-conserving surgery (BCS) negatively affects patient outcomes and healthcare costs. The inability to visualize margin involvement is a significant challenge in BCS. 5-Aminolevulinic acid hydrochloride (5-ALA HCl), a non-fluorescent oral prodrug, causes intracellular accumulation of fluorescent porphyrins in cancer cells. This single-center Phase II randomized controlled trial evaluated the safety, feasibility, and diagnostic accuracy of a prototype handheld fluorescence imaging device plus 5-ALA for intraoperative visualization of invasive breast carcinomas during BCS. METHODS: Fifty-four patients were enrolled and randomized to receive no 5-ALA or oral 5-ALA HCl (15 or 30 mg/kg). Forty-five patients (n = 15/group) were included in the analysis. Fluorescence imaging of the excised surgical specimen was performed, and biopsies were collected from within and outside the clinically demarcated tumor border of the gross specimen for blinded histopathology. RESULTS: In the absence of 5-ALA, tissue autofluorescence imaging lacked tumor-specific fluorescent contrast. Both 5-ALA doses caused bright red tumor fluorescence, with improved visualization of tumor contrasted against normal tissue autofluorescence. In the 15 mg/kg 5-ALA group, the positive predictive value (PPV) for detecting breast cancer inside and outside the grossly demarcated tumor border was 100.0% and 55.6%, respectively. In the 30 mg/kg 5-ALA group, the PPV was 100.0% and 50.0% inside and outside the demarcated tumor border, respectively. No adverse events were observed, and clinical feasibility of this imaging device-5-ALA combination approach was confirmed. CONCLUSIONS: This is the first known clinical report of visualization of 5-ALA-induced fluorescence in invasive breast carcinoma using a real-time handheld intraoperative fluorescence imaging device. TRIAL REGISTRATION: Clinicaltrials.gov identifier NCT01837225 . Registered 23 April 2013.

Lobular neoplasia occult on conventional imaging and diagnosed on MRI-guided biopsy: can we estimate upgrade on surgical pathology?

PURPOSE: The goal of this study is to evaluate the frequency and imaging features of lobular neoplasia (LN) diagnosed on MRI-guided biopsy, determine the upgrade rate to malignancy, and assess for any features that may be associated with an upgrade on surgical excision. MATERIALS AND METHODS: Research ethical board approved the review of consecutive patients with MRI-detected LN between January 2009 and December 2018 with differentiation between pure LN and LN with associated other high-risk lesions. The final outcome was determined by final pathology results from surgical excision or 24 months of follow-up. Appropriate statistical tests were used. RESULTS: Out of 1250 MRI-guided biopsies performed, 76 lesions (6%) fulfilled the inclusion criteria and formed the study cohort. Of the 76 lesions, 54 (71%) were pure LN while the rest had coexistent high-risk lesion. Non-mass enhancement (NME) was the most common lesion type (62, 82%). Fifty-nine lesions (78%) were surgically excised, the other 17 had benign follow-up. Overall, 8 lesions (11%) were upgraded to malignancy on final pathology. Malignant outcome was associated with larger lesion size (5.5 versus 1.9 cm, P < 0.001) and a clumped NME pattern (75% versus 24%, P = 0.006). Lesion size and clumped NME remained significantly associated with upgrade on sub-analysis of the pure LN group. CONCLUSION: Larger lesion size and clumped NME are imaging findings associated with upgrade of LN diagnosed by MRI-guided biopsy. This may influence patient management in this clinical setting. Additional larger studies are needed to consolidate our results and to potentially detect additional factors associated with upgrade.

Cellular fibroepithelial lesions diagnosed on core needle biopsy: Is there any role of clinical-sonography features helping to differentiate fibroadenomas and phyllodes tumor?

OBJECTIVE: The purpose of this study is to determine the role of clinico-sonographic features of breast cellular fibroepithelial lesions (CFELs) diagnosed on core needle biopsy (CNB) in the differentiation between fibroadenoma (FA) and phyllodes. MATERIALS AND METHODS: Results of consecutive women with a CNB showing CFEL from 2005 to 2010 were retrospectively reviewed. Clinical and sonographic findings were compared with surgical outcomes. Chi-square and Fisher's exact tests were used followed by a regression model for statistical analysis. RESULTS: A total of 131 women with 134 CFEL were included in the study; 89 (66%) were FAs and 45 (34%) were phyllodes (32 benign; 13 malignant). Significant predictors of increased risk of phyllodes tumor were patient age equal to or greater than 50 years (P = .021) and lesion size less than 2 cm at sonography (P = .043). No other imaging or clinical features were able to differentiate FA from phyllodes tumors. CONCLUSION: CFEL with a larger size in older women is associated with the surgical pathological result of phyllodes tumor and management should be tailored accordingly. Younger patients with small size nodules might be approached less aggressively, depending on a personalized discussion with the surgeons, taking into account the results obtained in this study.

Molecular Profiling and Significance of Circulating Tumor Cell Based Genetic Signatures.

Cancer kills by metastasizing beyond the primary site. Early detection, surgical intervention and other treatments have improved the survival rates of patients with cancer, however, once metastasis occurs, responses to conventional therapies become significantly less effective, and this remains the leading cause of death. Circulating tumor cells (CTCs) are tumor cells that have preferentially disseminated from the primary tumor mass into the hematological system, and are en route to favorable distant sites where if they survive, can develop into metastases. They may be the earliest detectable cells with metastatic ability, and are gaining increasing attention because of their prognostic value in many types of cancers including breast, prostate, colon and lung. Recent technological advances have removed barriers that previously hindered the detection and isolation of these rare cells from blood, and have exponentially improved the genetic resolution at which we can characterize signatures that define CTCs. Some of the most significant observations from such examinations are described here. Firstly, aberrations that were thought to be unique to CTCs are detected at subclonal frequencies within primary tumors with measurable heterogeneity, indicating pre-existing genetic signatures for metastasis. Secondly, these subclonal events are enriched in CTCs and metastases, pointing towards the selection of a more 'fit' component of tumor cells with survival advantages. Lastly, this component of cancer cells may also be the chemoresistant portion that escapes systemic treatment, or acquires resistance during progression of the disease. The future of cancer management may include a standardized method of measuring intratumor heterogeneity of the primary as well as matched CTCs. This will help identify and target rare aberrations within primary tumors that make them more adept to disseminate, and also to monitor the development of treatment resistant subclones as cancer progresses.

Digital Compared with Screen-Film Mammography: Measures of Diagnostic Accuracy among Women Screened in the Ontario Breast Screening Program.

PURPOSE: To compare measures of diagnostic accuracy between large concurrent cohorts of women screened with digital computed radiography (CR), direct radiography (DR), and screen-film mammography (SFM). MATERIALS AND METHODS: This study was approved by the University of Toronto Research Ethics Board; informed consent was not required. Three concurrent cohorts of women aged 50-74 years who were screened from 2008-2009 in the Ontario Breast Screening Program with SFM (487,334 screening examinations, 403,688 women), DR (254,758 screening examinations, 220,520 women), or CR (74,140 screening examinations, 64,210 women) were followed for 2 years or until breast cancer diagnosis. Breast cancers were classified as screening-detected or interval on the basis of the woman's final screening and assessment results. Interval cancer rate (per 10 000 negative screening examinations), sensitivity, and specificity were compared across the cohorts by using mixed-effects logistic regression analysis. RESULTS: Interval cancer rates were higher, although not significantly so, for CR (15.2 per 10,000; 95% confidence interval [CI]: 12.8, 17.8) and were similar for DR (13.7 per 10,000; 95% CI: 12.4, 15.0) compared with SFM (13.0 per 10,000; 95% CI: 12.1, 13.9). For CR versus SFM, specificity was similar while sensitivity was significantly lower (odds ratio [OR] = 0.62; 95% CI: 0.47, 0.83; P = .001), particularly for invasive cancers detected at a rescreening examination, for women with breast density of less than 75%, for women with no family history, and for postmenopausal women. For DR versus SFM, sensitivity was similar while specificity was lower (OR = 0.92; 95% CI: 0.87, 0.98; P = .01), particularly for rescreening examinations, for women aged 60-74 years, for women with breast density of less than 75%, for women with a family history, and for women who were postmenopausal. CONCLUSION: Given the 38% lower sensitivity of CR imaging systems compared with SFM, programs should assess the continued use of this technology for breast screening.

Circulating Tumor Cells (CTC) and Cell-Free DNA (cfDNA) Workshop 2016: Scientific Opportunities and Logistics for Cancer Clinical Trial Incorporation.

Despite the identification of circulating tumor cells (CTCs) and cell-free DNA (cfDNA) as potential blood-based biomarkers capable of providing prognostic and predictive information in cancer, they have not been incorporated into routine clinical practice. This resistance is due in part to technological limitations hampering CTC and cfDNA analysis, as well as a limited understanding of precisely how to interpret emergent biomarkers across various disease stages and tumor types. In recognition of these challenges, a group of researchers and clinicians focused on blood-based biomarker development met at the Canadian Cancer Trials Group (CCTG) Spring Meeting in Toronto, Canada on 29 April 2016 for a workshop discussing novel CTC/cfDNA technologies, interpretation of data obtained from CTCs versus cfDNA, challenges regarding disease evolution and heterogeneity, and logistical considerations for incorporation of CTCs/cfDNA into clinical trials, and ultimately into routine clinical use. The objectives of this workshop included discussion of the current barriers to clinical implementation and recent progress made in the field, as well as fueling meaningful collaborations and partnerships between researchers and clinicians. We anticipate that the considerations highlighted at this workshop will lead to advances in both basic and translational research and will ultimately impact patient management strategies and patient outcomes.

Changes in insulin receptor signaling underlie neoadjuvant metformin administration in breast cancer: a prospective window of opportunity neoadjuvant study.

INTRODUCTION: The antidiabetic drug metformin exhibits potential anticancer properties that are believed to involve both direct (insulin-independent) and indirect (insulin-dependent) actions. Direct effects are linked to activation of AMP-activated protein kinase (AMPK) and an inhibition of mammalian target of rapamycin mTOR signaling, and indirect effects are mediated by reductions in circulating insulin, leading to reduced insulin receptor (IR)-mediated signaling. However, the in vivo impact of metformin on cancer cell signaling and the factors governing sensitivity in patients remain unknown. METHODS: We conducted a neoadjuvant, single-arm, "window of opportunity" trial to examine the clinical and biological effects of metformin on patients with breast cancer. Women with untreated breast cancer who did not have diabetes were given 500 mg of metformin three times daily for ≥2 weeks after diagnostic biopsy until surgery. Fasting blood and tumor samples were collected at diagnosis and surgery. Blood glucose and insulin were assayed to assess the physiologic effects of metformin, and immunohistochemical analysis of tumors was used to characterize cellular markers before and after treatment. RESULTS: Levels of IR expression decreased significantly in tumors (P = 0.04), as did the phosphorylation status of protein kinase B (PKB)/Akt (S473), extracellular signal-regulated kinase 1/2 (ERK1/2, T202/Y204), AMPK (T172) and acetyl coenzyme A carboxylase (S79) (P = 0.0001, P < 0.0001, P < 0.005 and P = 0.02, respectively). All tumors expressed organic cation transporter 1, with 90% (35 of 39) exhibiting an Allred score of 5 or higher. CONCLUSIONS: Reduced PKB/Akt and ERK1/2 phosphorylation, coupled with decreased insulin and IR levels, suggest insulin-dependent effects are important in the clinical setting. These results are consistent with beneficial anticancer effects of metformin and highlight key factors involved in sensitivity, which could be used to identify patients with breast cancer who may be responsive to metformin-based therapies. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT00897884. Registered 8 May 2009.

Identification of genomic signatures in circulating tumor cells from breast cancer.

Levels of circulating tumor cells (CTCs) in blood have prognostic value in early and metastatic breast cancer. CTCs also show varying degrees of concordance with molecular markers of primary tumors they originate from. It is expected that individual cells reflect the heterogeneity and evolution of tumor cells as they acquire new functions and differential responses to chemotherapy. However, a degree of commonality is also plausible, highlighting alterations that allow tumor cells to perform CTC-defining activities such as invasion and intravasation. Using a matched tumor-normal approach, we performed high-resolution copy number profiling of CTCs from breast cancer to identify occult changes occurring during progression to metastasis. We identified a signature of recurrent gain in CTCs, consisting of 90 minimal common regions (MCRs) of copy number gain. These were predominantly found across chromosome 19 and were identified at low frequencies (3-4%) in 787 primary breast carcinomas examined. CTC genomic signatures clustered into two groups independent of subtype: a dormancy-related signature with 16 MCRs (AKT2, PTEN, CADM2); and a tumor-aggressiveness related signature with 358 MCRs (ANGPTL4, BSG, MIR-373). There were two MCRs in common between the groups on 19q13 and 21q21, containing genes involved in resistance to anoikis, TGFß-signaling and metastasis (TFF3, LTBP4, NUMBL). Furthermore, a region harboring the ERBB2 gene was gained in a majority of patients. Regions 20q13 and 15q24 were associated with distant metastasis. The distinctiveness of CTC signatures highlights cell populations with different functional or metastatic potential. Such novel targets could help to specifically identify and block dissemination.

Digital versus screen-film mammography: impact of mammographic density and hormone therapy on breast cancer detection.

Most studies that have examined the effects of mammographic density and hormone therapy use on breast cancer detection have included screen-film mammography. This study further examines this association in post-menopausal women screened by digital mammography. Approved by the University of Toronto Research Ethics Board, this study identified 688,418 women of age 50-74 years screened with digital or screen-film mammography from 2008 to 2009 within the Ontario Breast Screening Program. Of 2993 eligible women with invasive breast cancer, 2450 were contacted and 1421 participated (847 screen-film mammography, 574 digital direct radiography). Mammographic density was measured by study radiologists using the standard BI-RADS classification system and by a computer-assisted method. Information on hormone therapy use was collected by a telephone-administered questionnaire. Logistic regression and two-tailed tests for significance evaluated associations between factors and detection method by mammography type. Women with >75 % radiologist-measured mammographic density compared to those with <25 % were more likely to be diagnosed with an interval than screen-detected cancer, with the difference being greater for those screened with screen-film (OR = 6.40, 95 % CI 2.30-17.85) than digital mammography (OR = 2.41, 95 % CI 0.67-8.58) and aged 50-64 years screened with screen-film mammography (OR = 10.86, 95 % CI 2.96-39.57). Recent former hormone therapy users were also at an increased risk of having an interval cancer with the association being significant for women screened with digital mammography (OR = 2.08, 95 % CI 1.17-3.71). Breast screening using digital mammography lowers the risk of having an interval cancer for post-menopausal women aged 50-64 with greater mammographic density.

Evidence for biological effects of metformin in operable breast cancer: biomarker analysis in a pre-operative window of opportunity randomized trial.

Metformin has therapeutic potential against breast cancer, but the mechanisms of action in vivo remain uncertain. This study examined biomarker effects of metformin in primary breast cancer in a preoperative window of opportunity trial. Non-diabetic women with operable invasive breast cancer were randomized to receive open label pre-operative metformin (500 mg daily for 1 week then 1 g twice daily for a further week) or as controls, not receiving metformin. Patients in both arms had a core biopsy pre-randomisation and again at the time of surgery. Immunohistochemistry for phospho-AMPK (pAMPK), phospho-Akt (pAkt), insulin receptor, cleaved caspase-3, and Ki67 was performed on formalin-fixed paraffin-embedded cores, scored blinded to treatment and analysed by paired t test. In metformin-treated patients, significant up-regulation of pAMPK (paired t test, p = 0.04) and down-regulation of pAkt (paired t test, p = 0.043) were demonstrated compared to the control group. Insulin receptor and serum insulin remained similar following metformin treatment compared with a rise in insulin receptor and insulin in controls. Significant falls in Ki67 and cleaved caspase-3 (paired t test, p = 0.044) were seen in the metformin-treated patients but not in the control group. Changes were independent of body mass index. These biomarker data suggest mechanisms for metformin action in vivo in breast cancer patients via up-regulation of tumor pAMPK, down-regulation of pAkt, and suppression of insulin responses reflecting cytostatic rather than cytotoxic mechanisms.

Validation of the prognostic gene portfolio, ClinicoMolecular Triad Classification, using an independent prospective breast cancer cohort and external patient populations.

INTRODUCTION: Using genome-wide expression profiles of a prospective training cohort of breast cancer patients, ClinicoMolecular Triad Classification (CMTC) was recently developed to classify breast cancers into three clinically relevant groups to aid treatment decisions. CMTC was found to be both prognostic and predictive in a large external breast cancer cohort in that study. This study serves to validate the reproducibility of CMTC and its prognostic value using independent patient cohorts. METHODS: An independent internal cohort (n = 284) and a new external cohort (n = 2,181) were used to validate the association of CMTC between clinicopathological factors, 12 known gene signatures, two molecular subtype classifiers, and 19 oncogenic signalling pathway activities, and to reproduce the abilities of CMTC to predict clinical outcomes of breast cancer. In addition, we also updated the outcome data of the original training cohort (n = 147). RESULTS: The original training cohort reached a statistically significant difference (p < 0.05) in disease-free survivals between the three CMTC groups after an additional two years of follow-up (median = 55 months). The prognostic value of the triad classification was reproduced in the second independent internal cohort and the new external validation cohort. CMTC achieved even higher prognostic significance when all available patients were analyzed (n = 4,851). Oncogenic pathways Myc, E2F1, Ras and ß-catenin were again implicated in the high-risk groups. CONCLUSIONS: Both prospective internal cohorts and the independent external cohorts reproduced the triad classification of CMTC and its prognostic significance. CMTC is an independent prognostic predictor, and it outperformed 12 other known prognostic gene signatures, molecular subtype classifications, and all other standard prognostic clinicopathological factors. Our results support further development of CMTC portfolio into a guide for personalized breast cancer treatments.

Digital compared to screen-film mammography: breast cancer prognostic features in an organized screening program.

Our previous study found cancer detection rates were equivalent for direct radiography compared to screen-film mammography, while rates for computed radiography were significantly lower. This study compares prognostic features of invasive breast cancers by type of mammography. Approved by the University of Toronto Research Ethics Board, this study identified invasive breast cancers diagnosed among concurrent cohorts of women aged 50-74 screened by direct radiography, computed radiography, or screen-film mammography from January 1, 2008 to December 31, 2009. During the study period, 816,232 mammograms were performed on 668,418 women, and 3,323 invasive breast cancers were diagnosed. Of 2,642 eligible women contacted, 2,041 participated (77.3 %). The final sample size for analysis included 1,405 screen-detected and 418 interval cancers (diagnosed within 24 months of a negative screening mammogram). Polytomous logistic regression was performed to evaluate the association between tumour characteristics and type of mammography, and between tumour characteristics and detection method. Odds ratios (OR) and 95 % confidence intervals (CI) were recorded. Cancers detected by computed radiography compared to screen-film mammography were significantly more likely to be lymph node positive (OR 1.94, 95 %CI 1.01-3.73) and have higher stage (II:I, OR 2.14, 95 %CI 1.11-4.13 and III/IV:I, OR 2.97, 95 %CI 1.02-8.59). Compared to screen-film mammography, significantly more cancers detected by direct radiography (OR 1.64, 95 %CI 1.12-2.38) were lymph node positive. Interval cancers had worse prognostic features compared to screen-detected cancers, irrespective of mammography type. Screening with computed radiography may lead to the detection of cancers with a less favourable stage distribution compared to screen-film mammography that may reflect a delayed diagnosis. Screening programs should re-evaluate their use of computed radiography for breast screening.

AI improves accuracy, agreement and efficiency of pathologists for Ki67 assessments in breast cancer.

The Ki-67 proliferation index (PI) guides treatment decisions in breast cancer but suffers from poor inter-rater reproducibility. Although AI tools have been designed for Ki-67 assessment, their impact on pathologists' work remains understudied. 90 international pathologists were recruited to assess the Ki-67 PI of ten breast cancer tissue microarrays with and without AI. Accuracy, agreement, and turnaround time with and without AI were compared. Pathologists' perspectives on AI were collected. Using AI led to a significant decrease in PI error (2.1% with AI vs. 5.9% without AI, p < 0.001), better inter-rater agreement (ICC: 0.70 vs. 0.92; Krippendorff's α: 0.63 vs. 0.89; Fleiss' Kappa: 0.40 vs. 0.86), and an 11.9% overall median reduction in turnaround time. Most pathologists (84%) found the AI reliable. For Ki-67 assessments, 76% of respondents believed AI enhances accuracy, 82% said it improves consistency, and 83% trust it will improve efficiency. This study highlights AI's potential to standardize Ki-67 scoring, especially between 5 and 30% PI-a range with low PI agreement. This could pave the way for a universally accepted PI score to guide treatment decisions, emphasizing the promising role of AI integration into pathologist workflows.

A handheld device for intra-cavity and ex vivo fluorescence imaging of breast conserving surgery margins with 5-aminolevulinic acid.

BACKGROUND: Visualization of cancer during breast conserving surgery (BCS) remains challenging; the BCS reoperation rate is reported to be 20-70% of patients. An urgent clinical need exists for real-time intraoperative visualization of breast carcinomas during BCS. We previously demonstrated the ability of a prototype imaging device to identify breast carcinoma in excised surgical specimens following 5-aminolevulinic acid (5-ALA) administration. However, this prototype device was not designed to image the surgical cavity for remaining carcinoma after the excised lumpectomy specimen is removed. A new handheld fluorescence (FL) imaging prototype device, designed to image both excised specimens and within the surgical cavity, was assessed in a clinical trial to evaluate its clinical utility for first-in-human, real-time intraoperative imaging during index BCS. RESULTS: The imaging device combines consumer-grade imaging sensory technology with miniature light-emitting diodes (LEDs) and multiband optical filtering to capture high-resolution white light (WL) and FL digital images and videos. The technology allows for visualization of protoporphyrin IX (PpIX), which fluoresces red when excited by violet-blue light. To date, n = 17 patients have received 20 mg kg bodyweight (BW) 5-ALA orally 2-4 h before imaging to facilitate the accumulation of PpIX within tumour cells. Tissue types were identified based on their colour appearance. Breast tumours in sectioned lumpectomies appeared red, which contrasted against the green connective tissues and orange-brown adipose tissues. In addition, ductal carcinoma in situ (DCIS) that was missed during intraoperative standard of care was identified at the surgical margin at <1 mm depth. In addition, artifacts due to the surgical drape, illumination, and blood within the surgical cavity were discovered. CONCLUSIONS: This study has demonstrated the detection of a grossly occult positive margin intraoperatively. Artifacts from imaging within the surgical cavity have been identified, and potential mitigations have been proposed. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT01837225 (Trial start date is September 2010. It was registered to ClinicalTrials.gov retrospectively on April 23, 2013, then later updated on April 9, 2020, to reflect the introduction of the new imaging device.).

Can we select individuals with low risk ductal carcinoma in situ (DCIS)? A population-based outcomes analysis.

Ductal carcinoma in situ (DCIS), a non-invasive breast cancer, is usually treated by breast-conserving surgery (BCS). Randomized trials prove that the addition of radiotherapy (XRT) leads to lower rates of recurrence. Despite the evidence, half of women do not receive XRT after BCS. It is unknown how well clinicians identify women with low risk DCIS for treatment by BCS alone or to what extent women with DCIS develop recurrent cancer due to the omission of radiotherapy. We report the outcomes of a population of women with DCIS treated with BCS, alone or with radiotherapy, and evaluate the effectiveness of each therapeutic approach. All women diagnosed with DCIS and treated with BCS, alone or with radiotherapy in Ontario from 1994 to 2003 were identified. Treatments and outcomes were validated by chart review. Survival analyses were used to study the development of local recurrence (LR) in relation to patient and tumor characteristics and the use of radiotherapy. The cohort included 3,762 women treated with breast-conserving therapy; 1,895 of whom (50 %) also received radiation. At 10 years median follow-up, LR developed in 233 (12 %) women who received radiotherapy and in 363 (19 %) of women who did not (p < 0.0001). The 10-year actuarial LR rate for women who did and did not receive radiotherapy was 12.7 and 20.0 % (p < 0.0001). Differences were significant for both for invasive LR (7.0 vs. 10.0 %, p < 0.0001) and for DCIS recurrence (6.1 vs. 10.8 %, p < 0.0001). We estimate that 22 % of recurrences diagnosed in Ontario women treated for DCIS between 1994 and 2003 would have been prevented if all patients had received radiotherapy. The omission of radiotherapy after BCS for DCIS resulted in substantive recurrences that might have been avoided with treatment. Additional markers are needed to identify a low risk group in whom radiation can be safely omitted.