The aim of this study is to evaluate the performance of the Radialis organ-targeted positron emission tomography (PET) Camera with standardized tests and through assessment of clinical-imaging ...results. Sensitivity, count-rate performance, and spatial resolution were evaluated according to the National Electrical Manufacturers Association (NEMA) NU-4 standards, with necessary modifications to accommodate the planar detector design. The detectability of small objects was shown with micro hotspot phantom images. The clinical performance of the camera was also demonstrated through breast cancer images acquired with varying injected doses of 2-fluorine-18-fluoro-2-deoxy-D-glucose (18F-FDG) and qualitatively compared with sample digital full-field mammography, magnetic resonance imaging (MRI), and whole-body (WB) PET images. Micro hotspot phantom sources were visualized down to 1.35 mm-diameter rods. Spatial resolution was calculated to be 2.3 ± 0.1 mm for the in-plane resolution and 6.8 ± 0.1 mm for the cross-plane resolution using maximum likelihood expectation maximization (MLEM) reconstruction. The system peak noise equivalent count rate was 17.8 kcps at a 18F-FDG concentration of 10.5 kBq/mL. System scatter fraction was 24%. The overall efficiency at the peak noise equivalent count rate was 5400 cps/MBq. The maximum axial sensitivity achieved was 3.5%, with an average system sensitivity of 2.4%. Selected results from clinical trials demonstrate capability of imaging lesions at the chest wall and identifying false-negative X-ray findings and false-positive MRI findings, even at up to a 10-fold dose reduction in comparison with standard 18F-FDG doses (i.e., at 37 MBq or 1 mCi). The evaluation of the organ-targeted Radialis PET Camera indicates that it is a promising technology for high-image-quality, low-dose PET imaging. High-efficiency radiotracer detection also opens an opportunity to reduce administered doses of radiopharmaceuticals and, therefore, patient exposure to radiation.
To better understand the expression pattern of programmed death-ligand 1 (PD-L1) expression in different breast cancer types, we characterized PD-L1 expression in tumor and tumor-infiltrating immune ...cells, in relation to mutation rate, BRCA1-like status and survival. We analyzed 410 primary treatment-naive breast tumors comprising 162 estrogen receptor-positive (ER+) and HER2−, 101 HER2+ and 147 triple-negative (TN) cancers. Pathologists quantified tumor-infiltrating lymphocytes (TILs) and PD-L1 expression in tumor cells and TILs using whole slides and tissue microarray. Mutation rate was assessed by DNA sequencing, BRCA1-like status using multiplex ligation-dependent probe amplification, and immune landscape by multiplex image analyses of CD4, CD68, CD8, FOXP3, cytokeratin, and PD-L1. Half of PD-L1 scores evaluated by tissue microarray were false negatives compared to whole slide evaluations. We observed at least 1% of PD-L1-positive (PD-L1+) cells in 53.1% of ER+HER2−, 73.3% of HER2+, and 84.4% of TN tumors. PD-L1 expression was higher in ductal compared to lobular carcinomas, also within ER+HER2− tumors (p = 0.04). High PD-L1+ TILs score (> 50%) was independently associated with better outcome in TN tumors (HR = 0.27; 95%CI = 0.10-0.69). Within TN tumors, PD-L1 and TIL scores showed a modest but significant positive association with the number of silent mutations, but no association with BRCA1-like status. Multiplex image analyses indicated that PD-L1 is expressed on multiple immune cells (CD68+ macrophages, CD4+, FOXP3+, and CD8+ T cells) in the breast tumor microenvironment, independent of the PD-L1 status of the tumor cells. We found no evidence that levels of PD-L1+ TILs in TN breast cancer are driven by high mutation rate or BRCA1-like status.
Although different histologic subtypes of epithelial ovarian tumors have long been recognized, their molecular abnormalities have not been fully defined. We examined the prevalence of DNA mismatch ...repair (MMR) protein loss in these tumors. Tissue microarrays (TMA) of suspected ovarian carcinomas were stained for hMLH1, hMSH2, hMSH6, and hPMS2 and scored separately by 2 groups of investigators. Loss of staining (negative) or discrepant staining results on TMA were verified on whole-section slides. Intact (positive) staining results were also verified for an additional 25 randomly selected cases. Clinical data for cases demonstrating MMR protein loss were collected. A second set of TMA composed purely of mucinous tumors was also stained for antibodies to MMR proteins and scored by 1 group of investigators. TMA was an effective method for screening a large number of ovarian tumors for MMR protein expression, with a sensitivity of 100% for all 4 MMR proteins, and a specificity of 22.2%-53.8% for different MMR proteins. Of the primary epithelial tumors of the ovary, loss of expression of MMR proteins was significantly more common in the endometriosis-associated carcinomas (7/69; 10.1%) than in high-grade serous carcinomas (2/182; 1.1%): P=0.0021. The former group also showed more frequent loss of MMR proteins compared with mucinous intestinal-type carcinomas (0/32; P=0.0940). Cases within the group of endometriosis-associated carcinomas were endometrioid (2/29 cases), clear cell (1/27 cases), undifferentiated (1/8 cases), and mixed carcinomas with an endometrioid, clear cell, and/or undifferentiated component (3/5 cases). No loss of MMR protein expression was identified in epithelial tumors of other histologic subtypes. Our study demonstrated the loss of MMR protein expression in 10.1% of endometriosis-associated ovarian carcinomas. These results raise the possibility of selective screening for Lynch syndrome in patients with these types of ovarian carcinoma.
Ki67, a marker of cellular proliferation, is commonly assessed in surgical pathology laboratories. In breast cancer, Ki67 is an established prognostic factor with higher levels associated with worse ...long-term survival. However, Ki67 IHC is considered of limited clinical use in breast cancer management largely due to issues related to standardization and reproducibility of scoring across laboratories. Recently, both the American Food and Drug Administration (FDA) and Health Canada have approved the use of abemaciclib (CDK4/6 inhibitor) for patients with HR+/HER2: high-risk early breast cancers in the adjuvant setting. Health Canada and the FDA have included a Ki67 proliferation index of ≥20% in the drug monograph. The approval was based on the results from monarchE, a phase III clinical trial in early-stage chemotherapy-naïve, HR+, HER2 negative patients at high risk of early recurrence. The study has shown significant improvement in invasive disease-free survival (IDFS) with abemaciclib when combined with adjuvant endocrine therapy at two years. Therefore, there is an urgent need by the breast pathology and medical oncology community in Canada to establish national guideline recommendations for Ki67 testing as a predictive marker in the context of abemaciclib therapy consideration. The following recommendations are based on previous IKWG publications, available guidance from the monarchE trial and expert opinions. The current recommendations are by no means final or comprehensive, and their goal is to focus on its role in the selection of patients for abemaciclib therapy. The aim of this document is to guide Canadian pathologists on how to test and report Ki67 in invasive breast cancer. Testing should be performed upon a medical oncologist's request only. Testing must be performed on treatment-naïve tumor tissue. Testing on the core biopsy is preferred; however, a well-fixed resection specimen is an acceptable alternative. Adhering to ASCO/CAP fixation guidelines for breast biomarkers is advised. Readout training is strongly recommended. Visual counting methods, other than eyeballing, should be used, with global rather than hot spot assessment preferred. Counting 100 cells in at least four areas of the tumor is recommended. The Ki67 scoring app developed to assist pathologists with scoring Ki67 proposed by the IKWG, available for free download, may be used. Automated image analysis is very promising, and laboratories with such technology are encouraged to use it as an adjunct to visual counting. A score of <5 or >30 is more robust. The task force recommends that the results are best expressed as a continuous variable. The appropriate antibody clone and staining protocols to be used may take time to address. For the time being, the task force recommends having tonsils/+pancreas on-slide control and enrollment in at least one national/international EQA program. Analytical validation remains a pending goal. Until the data become available, using local ki67 protocols is acceptable. The task force recommends participation in upcoming calibration and technical validation initiatives.
The presence of atypical or usual epithelial proliferations within papillary breast lesions complicates their interpretation on core biopsy. We evaluated the combination of estrogen receptor (ER) and ...cytokeratin 5 (CK5) as an aid in the distinction of usual duct hyperplasia from atypical proliferations in this setting. Core biopsies from 185 papillary lesions were reviewed and of these, 82 cases were selected for immunohistochemical study based on the presence of an epithelial proliferation between the fibrovascular cores. Fifty-two cases were used as the test set and 30 cases, with subsequent surgical excision, were used as the validation set. The epithelial proliferation was evaluated for staining intensity and percentage of positive cells using CK5 and ER. Expression of both CK5 and ER was significantly different in nonatypical lesions when compared with atypical lesions (P<0.0001). Nonatypical lesions typically showed an ER-low/CK5-high profile and atypical lesions showed an ER-high/CK5-low profile with ER-high expression defined as diffuse strong staining in >90% of cells. CK5-high expression was defined as a mosaic pattern of staining in >20% of cells and CK5-low as absent or staining in <20% of cells. On the basis of their staining profile, 29 of the 30 validation cases were correctly classified using the excision specimen as the gold standard. Patterns and extent of ER and CK5 staining, when used together, are valuable adjunct stains to differentiate usual duct hyperplasia from atypical proliferations within papillary lesions on core biopsy.
Notch receptors regulate cell fate determination, stem cell self-renewal, proliferation and apoptosis. We previously reported that elevated mRNA expression of the Notch ligand JAG1 identifies breast ...cancer patients with a poor prognosis. Here we show through immunohistochemical analysis of the same breast cancer cases (N=127) that patients with tumors expressing high levels of JAG1 protein had a worse outcome than those with tumors expressing low levels (10-year survival 26 vs 48%, and median survival 63 vs 108 months, respectively; P=0.03). We also describe the novel application of the Allred score to quantify JAG1 mRNA and protein expression levels. Using the Allred score, patients with tumors expressing high levels of JAG1 mRNA had a worse outcome than those with tumors expressing low levels (10-year survival 16 vs 47%, and median survival 43 months vs 100 months, respectively; P<0.001). Interestingly, when tumors were classified as either high or low for JAG1 mRNA or protein expression, there was only 65% agreement (κ=0.08) between the two methods of expression analysis. When JAG1 mRNA and protein data were combined, patients with tumors expressing low levels of both had a 10-year survival of 53% and median survival of 131 months. In comparison, patients with tumors expressing either high levels of JAG1 protein, mRNA or both had reduced 10-year survival and median survival (31%, 19%, 11% and 77, 43, 23 months respectively; P<0.0001). There was marginal evidence of an interaction effect (P=0.055), which indicated that the prognostic value of JAG1 protein was limited to the JAG1 mRNA-low subgroup. These data show that the Allred score can be used to rapidly quantify JAG1 mRNA and protein levels in breast cancer to identify patients who have a significant survival disadvantage and who may benefit from therapies (such as γ-secretase inhibitors) that target signaling through the Notch pathway.
We previously observed that T-bet+ tumor-infiltrating T lymphocytes (T-bet+ TILs) in primary breast tumors were associated with adverse clinicopathological features, yet favorable clinical outcome. ...We identified BRD4 (Bromodomain-Containing Protein 4), a member of the Bromodomain and Extra Terminal domain (BET) family, as a gene that distinguished T-bet+/high and T-bet-/low tumors. In clinical studies, BET inhibitors have been shown to suppress inflammation in various cancers, suggesting a potential link between BRD4 and immune infiltration in cancer. Hence, we examined the BRD4 expression and clinicopathological features of breast cancer.
The cohort consisted of a prospectively ascertained consecutive series of women with axillary node-negative breast cancer with long follow-up. Gene expression microarray data were used to detect mRNAs differentially expressed between T-bet+/high (n = 6) and T-bet-/low (n = 41) tumors. Tissue microarrays (TMAs) constructed from tumors of 612 women were used to quantify expression of BRD4 by immunohistochemistry, which was analyzed for its association with T-bet+ TILs, Jagged1, clinicopathological features, and disease-free survival.
Microarray analysis indicated that BRD4 mRNA expression was up to 44-fold higher in T-bet+/high tumors compared to T-bet-/low tumors (p = 5.38E-05). Immunohistochemical expression of BRD4 in cancer cells was also shown to be associated with T-bet+ TILs (p = 0.0415) as well as with Jagged1 mRNA and protein expression (p = 0.0171, 0.0010 respectively). BRD4 expression correlated with larger tumor size (p = 0.0049), pre-menopausal status (p = 0.0018), and high Ki-67 proliferative index (p = 0.0009). Women with high tumoral BRD4 expression in the absence of T-bet+ TILs exhibited a significantly poorer outcome (log rank test p = 0.0165) relative to other subgroups.
The association of BRD4 expression with T-bet+ TILs, and T-bet+ TIL-dependent disease-free survival suggests a potential link between BRD4-mediated tumor development and tumor immune surveillance, possibly through BRD4's regulation of Jagged1 signaling pathways. Further understanding BRD4's role in different immune contexts may help to identify an appropriate subset of breast cancer patients who may benefit from BET inhibitors without the risk of diminishing the anti-tumoral immune activity.
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