Fit-for-Purpose Ki-67 Immunohistochemistry Assays for Breast Cancer.

New therapies are being developed for breast cancer, and in this process, some "old" biomarkers are reutilized and given a new purpose. It is not always recognized that by changing a biomarker's intended use, a new biomarker assay is created. The Ki-67 biomarker is typically assessed by immunohistochemistry (IHC) to provide a proliferative index in breast cancer. Canadian laboratories assessed the analytical performance and diagnostic accuracy of their Ki-67 IHC laboratory-developed tests (LDTs) of relevance for the LDTs' clinical utility. Canadian clinical IHC laboratories enrolled in the Canadian Biomarker Quality Assurance Pilot Run for Ki-67 in breast cancer by invitation. The Dako Ki-67 IHC pharmDx assay was employed as a study reference assay. The Dako central laboratory was the reference laboratory. Participants received unstained slides of breast cancer tissue microarrays with 32 cases and performed their in-house Ki-67 assays. The results were assessed using QuPath, an open-source software application for bioimage analysis. Positive percent agreement (PPA, sensitivity) and negative percent agreement (NPA, specificity) were calculated against the Dako Ki-67 IHC pharmDx assay for 5%, 10%, 20%, and 30% cutoffs. Overall, PPA and NPA varied depending on the selected cutoff; participants were more successful with 5% and 10%, than with 20% and 30% cutoffs. Only 4 of 16 laboratories had robust IHC protocols with acceptable PPA for all cutoffs. The lowest PPA for the 5% cutoff was 85%, for 10% was 63%, for 20% was 14%, and for 30% was 13%. The lowest NPA for the 5% cutoff was 50%, for 10% was 33%, for 20% was 50%, and for 30% was 57%. Despite many years of international efforts to standardize IHC testing for Ki-67 in breast cancer, our results indicate that Canadian clinical LDTs have a wide analytical sensitivity range and poor agreement for 20% and 30% cutoffs. The poor agreement was not due to the readout but rather due to IHC protocol conditions. International Ki-67 in Breast Cancer Working Group (IKWG) recommendations related to Ki-67 IHC standardization cannot take full effect without reliable fit-for-purpose reference materials that are required for the initial assay calibration, assay performance monitoring, and proficiency testing.

Reinventing Nuclear Histo-score Utilizing Inherent Morphologic Cutoffs: Blue-brown Color H-score (BBC-HS).

Immunohistochemistry (IHC) is a testing methodology that is widely used for large number of diagnostic, prognostic, and predictive biomarkers. Although IHC is a qualitative methodology, in addition to threshold-based stratification (positive vs. negative), the increasing levels of expression of some of these biomarkers often lead to more intense staining, which published evidence linked to specific diagnosis, prognosis, and responses to therapy. It is essential that the descriptive thresholds between positive and negative staining, as well as between frequently used graded categories of staining intensity (eg, 1+, 2+, 3+) are standardized and reproducible. Histo-score (H-score) is a frequently used scoring system that utilizes these categories. Our study introduces categorization of the cutoff points between positive and negative results and graded categories of staining intensity for nuclear IHC biomarker assays based on color interaction between hematoxylin and diaminobenzidine (DAB); the Blue-brown Color H-score (BBC-HS). Six cases of diffuse large B-cell lymphoma were stained for a nuclear marker MUM1. The staining was assessed by H-score by 12 readers. Short tutorial and illustrated instructions were provided to readers. The novel scoring system in this study uses the interaction between DAB (DAB, brown stain) and hematoxylin (blue counterstain) to set thresholds between "0" (negative nuclei), "1+" (weakly positive nuclei), "2+" (moderately positive nuclei), and "3+" (strongly positive nuclei). The readers recorded scores for 300 cells. Krippendorff alpha (K-alpha) and intraclass correlation coefficient (ICC) were calculated. We have also assessed if reliability improved when counting the first 100 cells, first 200 cells, and for the total 300 cells using K-alpha and ICC. To assess the performance of each individual reader, the mean H-score and percent positive score (PPS) for each case was calculated, and the bias was calculated between each reader's score and the mean. K-alpha was 0.86 for H-score and 0.76 for PPS. ICC was 0.96 for H-score and 0.92 for PPS. The biases for H-score ranged from -58 to 41, whereas for PPS it ranged from -27% to 33%. Overall, most readers showed very low bias. Two readers were consistently underscoring and 2 were consistently overscoring compared with the mean. For nuclear IHC biomarker assays, our newly proposed cutoffs provide highly reliable/reproducible results between readers for positive and negative results and graded categories of staining intensity using existing morphologic parameters. BBC-HS is easy to teach and is applicable to both human eye and image analysis. BBC-HS application should facilitate the development of new reliable/reproducible scoring schemes for IHC biomarkers.

Human epidermal growth factor receptor 2 testing in primary breast cancer in the era of standardized testing: a Canadian prospective study.

PURPOSE: Therapies that target overexpression of human epidermal growth factor receptor 2 (HER2) rely on accurate and timely assessment of all patients with new diagnoses. This study examines HER2 testing of primary breast cancer tissue when performed with immunohistochemistry (IHC) and additional in situ hybridization (ISH) for negative cases (IHC 0/1+). The analysis focuses on the rate of false-negative HER2 tests, defined as IHC 0/1+ with an ISH ratio ≥ 2.0, in eight pathology centers across Canada. PATIENTS AND METHODS: Whole sections of surgical resections or tissue microarrays (TMAs) from invasive breast carcinoma tissue were tested by both IHC and ISH using standardized local methods. Samples were scored by the local breast pathologist, and consecutive HER2-negative IHC results (IHC 0/1+) were compared with the corresponding fluorescence or silver ISH result. RESULTS: Overall, 711 surgical excisions of primary breast cancer were analyzed by IHC and ISH; HER2 and chromosome 17 centromere (CEP17) counts were available in all cases. The overall rate of false-negative samples was 0.84% (six of 711 samples). Interpretable IHC and ISH scores were available in 1,212 cases from TMAs, and the overall rate of false-negative cases was 1.6% (16 of 978 cases). CONCLUSION: Our observation confirms that IHC is an adequate test to predict negative HER2 status in primary breast cancer in surgical excision specimens, even when different antibodies and IHC platforms are used. The study supports the American Society of Clinical Oncology/College of American Pathologists and Canadian testing algorithms of using IHC followed by ISH for equivocal cases.

Predicting local recurrence following breast-conserving therapy for early stage breast cancer: the significance of a narrow (≤ 2 mm) surgical resection margin.

BACKGROUND AND OBJECTIVES: Controversy continues over the extent of surgical resection margin required to minimize the risk of local recurrence (LR) in breast-conserving therapy (BCT) for early stage breast cancer. This study explores whether or not a narrow (≤ 2 mm) but negative resection margin affects LR. METHODS: All patients registered at the Saskatoon Cancer Center between January 1, 1991 and December 31, 2000 with a diagnosis of early stage invasive duct carcinoma treated with BCT were examined. All charts and pathology reports were reviewed with a review of the pathology for all cases where the resection margin was unclear in the original report. Other factors known or thought to effect LR (age, radiation boost, grade, extensive DCIS, ER/PR receptor status) were considered in the statistical analysis. RESULTS: Amongst the 200 narrow margin cases 19 LR were detected (19/201 = 9.5%) while 52 LR were detected in the 491 wide margin cases (52/491 =10.6%). This difference was not statistically significant. CONCLUSIONS: A narrow (≤ 2 mm) surgical resection margin does not result in an increase in LR compared to a surgical resection margin 2 mm in BCT for early stage duct carcinoma and does not warrant re-excision.

Periodic acid-schiff is superior to hematoxylin and eosin for screening prophylactic gastrectomies from CDH1 mutation carriers.

Hereditary diffuse gastric cancer (HDGC), an autosomal dominant cancer susceptibility syndrome, is largely attributable to germline mutations and deletions in the gene encoding E-cadherin, CDH1. Asymptomatic, mutation-positive individuals often choose prophylactic gastrectomy for cancer risk reduction. Examination of the entire mucosa of prophylactic gastrectomy specimens is essential and shows occult gastric cancers in most cases. We hypothesized that primary screening entire cases stained with periodic acid-Schiff (PAS) instead of hematoxylin and eosin (H&E) could improve diagnostic accuracy and speed of detecting invasive signet-ring adenocarcinoma. Serial sections from 6 prophylactic gastrectomy specimens with molecularly confirmed CDH1 mutations were stained with PAS and H&E, respectively (108 to 164 blocks per case). PAS-stained and H&E-stained slides were randomized for each case and examined microscopically for the presence of invasive signet-ring cells. The time to examine each slide was recorded. Our results showed that significantly fewer lesions were missed (ie, the lesion was initially identified on only 1 section, but present on both sections) on PAS-stained slides (6 missed lesions) than on H&E-stained slides (23 missed lesions); (P<0.05). Furthermore, it took significantly less time to screen a PAS-stained case (3 h 05+/-41 min) than an H&E-stained case (4 h 59+/-1 h 2 min) (P<0.05). Selected lesions were confirmed as epithelial by pan-keratin-positive immunohistochemistry. Thus, doing PAS staining instead of H&E on CDH1 mutation-positive prophylactic gastrectomy specimens may increase the detection rate of adenocarcinoma while reducing screening time.

Long term survival in a patient with pulmonary lymphangioleiomyomatosis.

A patient with pulmonary lymphangioleiomyomatosis was diagnosed more than 22 years after the onset of symptoms by a thoracoscopic lung biopsy, after a high resolution computerized tomogram of the chest was highly suggestive of the disease. After nearly 30 years since the onset of her symptoms, the patient leads a relatively normal life with only mildly abnormal lung function and has minimal reduction in her exercise tolerance. There have been few reports of patients surviving for such a long time after the onset of this disease; the literature suggests that most patients die within 15 years of symptom onset.