Testing for t(3;8) in MYC/BCL6 re-arranged large B cell lymphoma identifies a high risk subgroup with inferior survival.

A reciprocal t(3;8) BCL6::MYC fusion is common in large B cell lymphoma (LBCL) with MYC and BCL6 disruption. These pseudo-double hit cases are not adverse, whereas t(3;8) negative MYC/BCL6 lymphoma has an inferior prognosis relative to other MYC-rearranged LBCL.

Inter- and Intra-Observer Agreement of PD-L1 SP142 Scoring in Breast Carcinoma-A Large Multi-Institutional International Study.

The assessment of PD-L1 expression in TNBC is a prerequisite for selecting patients for immunotherapy. The accurate assessment of PD-L1 is pivotal, but the data suggest poor reproducibility. A total of 100 core biopsies were stained using the VENTANA Roche SP142 assay, scanned and scored by 12 pathologists. Absolute agreement, consensus scoring, Cohen's Kappa and intraclass correlation coefficient (ICC) were assessed. A second scoring round after a washout period to assess intra-observer agreement was carried out. Absolute agreement occurred in 52% and 60% of cases in the first and second round, respectively. Overall agreement was substantial (Kappa 0.654-0.655) and higher for expert pathologists, particularly on scoring TNBC (6.00 vs. 0.568 in the second round). The intra-observer agreement was substantial to almost perfect (Kappa: 0.667-0.956), regardless of PD-L1 scoring experience. The expert scorers were more concordant in evaluating staining percentage compared with the non-experienced scorers (R2 = 0.920 vs. 0.890). Discordance predominantly occurred in low-expressing cases around the 1% value. Some technical reasons contributed to the discordance. The study shows reassuringly strong inter- and intra-observer concordance among pathologists in PD-L1 scoring. A proportion of low-expressors remain challenging to assess, and these would benefit from addressing the technical issues, testing a different sample and/or referring for expert opinions.

UK recommendations for HER2 assessment in breast cancer: an update.

The last UK breast cancer (BC) human epidermal growth factor receptor 2 (HER2) testing guideline recommendations were published in 2015. Since then, new data and therapeutic strategies have emerged. The American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) published a focused update in 2018 that reclassified in situ hybridisation (ISH) Group 2 (immunohistochemistry (IHC) score 2+and HER2/chromosome enumeration probe 17 (CEP17) ratio ≥2.0 and HER2 copy number <4.0 signals/cell), as well as addressed other concerns raised by previous guidelines. The present article further refines UK guidelines, with specific attention to definitions of HER2 status focusing on eight key areas: (1) HER2 equivocal (IHC 2+) and assignment of the ASCO/CAP ISH group 2 tumours; (2) the definition of the group of BCs with low IHC scores for HER2 with emphasis on the distinction between IHC score 1+ (HER2-Low) from HER2 IHC score 0 (HER2 negative); (3) reporting cases showing HER2 heterogeneity; (4) HER2 testing in specific settings, including on cytological material; (5) repeat HER2 testing, (6) HER2 testing turnaround time targets; (7) the potential role of next generation sequencing and other diagnostic molecular assays for routine testing of HER2 status in BC and (8) use of image analysis to score HER2 IHC. The two tiered system of HER2 assessment remains unchanged, with first line IHC and then ISH limited to IHC equivocal cases (IHC score 2+) but emerging data on the relationship between IHC scores and levels of response to anti-HER2 therapy are considered. Here, we present the latest UK recommendations for HER2 status evaluation in BC, and where relevant, the differences from other published guidelines.

The frequency and clinical significance of centromere enumeration probe 17 alterations in human epidermal growth factor receptor 2 immunohistochemistry-equivocal invasive breast cancer.

BACKGROUND AND AIMS: Chromosome 17 alterations affect the assessment of HER2 gene amplification in breast cancer (BC), but its clinical significance remains unclear. This study aimed to identify the prevalence of centromere enumeration probe 17 (CEP17) alterations, and its correlation with response to neoadjuvant therapy (NAT) in BC patients with human epidermal growth factor receptor 2 (HER2) immunohistochemistry-equivocal score. METHODS AND RESULTS: A large BC cohort (n = 6049) with HER2 immunohistochemistry score 2+ and florescent in-situ hybridisation (FISH) results was included to assess the prevalence of CEP17 alterations. Another cohort (n = 885) with available clinicopathological data was used to evaluate the effect of CEP17 in the setting of NAT. HER2-amplified tumours with monosomy 17 (CEP17 copy number < 1.5 per nucleus), normal 17 (CEP17 1.5-< 3.0) and polysomy 17 (CEP17 ≥ 3.0) were observed in 16, 59 and 25%, respectively, compared with 3, 74 and 23%, respectively, in HER2-non-amplified tumours. There was no significant relationship between CEP17 alterations and pathological complete response (pCR) rate in both HER2-amplified and HER2-non-amplified tumours. The independent predictors of pCR were oestrogen (ER) negativity in HER2-amplified tumours [ER negative versus positive; odds ratio (OR) = 11.80; 95% confidence interval (CI) = 1.37-102.00; P = 0.02], and histological grade 3 in HER2 non-amplified tumours (3 versus 1, 2; OR = 5.54; 95% CI = 1.61-19.00; P = 0.007). CONCLUSION: The impacts of CEP17 alterations are not as strong as those of HER2/CEP17 ratio and HER2 copy number. The hormonal receptors status and tumour histological grade are more useful to identify BC patients with a HER2 immunohistochemistry-equivocal score who would benefit from NAT.

Analytical validation of a standardised scoring protocol for Ki67 immunohistochemistry on breast cancer excision whole sections: an international multicentre collaboration.

AIMS: The nuclear proliferation marker Ki67 assayed by immunohistochemistry has multiple potential uses in breast cancer, but an unacceptable level of interlaboratory variability has hampered its clinical utility. The International Ki67 in Breast Cancer Working Group has undertaken a systematic programme to determine whether Ki67 measurement can be analytically validated and standardised among laboratories. This study addresses whether acceptable scoring reproducibility can be achieved on excision whole sections. METHODS AND RESULTS: Adjacent sections from 30 primary ER+ breast cancers were centrally stained for Ki67 and sections were circulated among 23 pathologists in 12 countries. All pathologists scored Ki67 by two methods: (i) global: four fields of 100 tumour cells each were selected to reflect observed heterogeneity in nuclear staining; (ii) hot-spot: the field with highest apparent Ki67 index was selected and up to 500 cells scored. The intraclass correlation coefficient (ICC) for the global method [confidence interval (CI) = 0.87; 95% CI = 0.799-0.93] marginally met the prespecified success criterion (lower 95% CI ≥ 0.8), while the ICC for the hot-spot method (0.83; 95% CI = 0.74-0.90) did not. Visually, interobserver concordance in location of selected hot-spots varies between cases. The median times for scoring were 9 and 6 min for global and hot-spot methods, respectively. CONCLUSIONS: The global scoring method demonstrates adequate reproducibility to warrant next steps towards evaluation for technical and clinical validity in appropriate cohorts of cases. The time taken for scoring by either method is practical using counting software we are making publicly available. Establishment of external quality assessment schemes is likely to improve the reproducibility between laboratories further.

ALK Immunohistochemistry in NSCLC: Discordant Staining Can Impact Patient Treatment Regimen.

INTRODUCTION: Diagnostic immunohistochemistry (IHC) is increasingly accepted as a screening method for anaplastic lymphoma receptor tyrosine kinase gene (ALK) rearrangements in NSCLC. We have sought to establish an ongoing robust external quality assessment process to gauge quality of anaplastic lymphoma kinase (ALK) IHC, which can have an impact on interpretation of patient samples. METHODS: Unstained tissue and cell line samples were distributed on a quarterly basis to participating laboratories from 30 countries. Participants stained the slide using their routine diagnostic ALK IHC method and returned the slide along with their in-house control and methodology details. Slides were assessed by a team of pathologists and scientists. RESULTS: Overall, there was a mean pass rate of 83% (range 71%-98%), with 38 variations in staining protocol. Methods included the following: the Roche D5F3 assay (65% of users, pass rate 93%); Novocastra 5A4 (15% of users, pass rate 65%); Cell Signaling Technology D5F3 (7% of users, pass rate 91%), and Dako ALK1 (5% of users, pass rate 50%). Choice of methodology directly affected final interpretation of distributed ALK-positive and ALK-negative NSCLC cases, which were correctly identified by 89% and 88% of participants, respectively. Antibody detection method was a contributing factor in false-negative staining results. The choice of laboratory controls was found to be unsuitable, and as such, in-house control recommendations are also provided. CONCLUSIONS: ALK IHC is a robust screening technique, but there is concern that some diagnostic laboratories are using inadequate staining methods, which has a direct impact on final interpretation. External assessment helps provide laboratories with continued confidence in their ALK IHC testing.

Analytical validation of a standardized scoring protocol for Ki67: phase 3 of an international multicenter collaboration.

Pathological analysis of the nuclear proliferation biomarker Ki67 has multiple potential roles in breast and other cancers. However, clinical utility of the immunohistochemical (IHC) assay for Ki67 immunohistochemistry has been hampered by unacceptable between-laboratory analytical variability. The International Ki67 Working Group has conducted a series of studies aiming to decrease this variability and improve the evaluation of Ki67. This study tries to assess whether acceptable performance can be achieved on prestained core-cut biopsies using a standardized scoring method. Sections from 30 primary ER+ breast cancer core biopsies were centrally stained for Ki67 and circulated among 22 laboratories in 11 countries. Each laboratory scored Ki67 using three methods: (1) global (4 fields of 100 cells each); (2) weighted global (same as global but weighted by estimated percentages of total area); and (3) hot-spot (single field of 500 cells). The intraclass correlation coefficient (ICC), a measure of interlaboratory agreement, for the unweighted global method (0.87; 95% credible interval (CI): 0.81-0.93) met the prespecified success criterion for scoring reproducibility, whereas that for the weighted global (0.87; 95% CI: 0.7999-0.93) and hot-spot methods (0.84; 95% CI: 0.77-0.92) marginally failed to do so. The unweighted global assessment of Ki67 IHC analysis on core biopsies met the prespecified criterion of success for scoring reproducibility. A few cases still showed large scoring discrepancies. Establishment of external quality assessment schemes is likely to improve the agreement between laboratories further. Additional evaluations are needed to assess staining variability and clinical validity in appropriate cohorts of samples.

FKBPL: a marker of good prognosis in breast cancer.

FK506-binding protein-like (FKBPL) has established roles as an anti-tumor protein, with a therapeutic peptide based on this protein, ALM201, shortly entering phase I/II clinical trials. Here, we evaluated FKBPL's prognostic ability in primary breast cancer tissue, represented on tissue microarrays (TMA) from 3277 women recruited into five independent retrospective studies, using immunohistochemistry (IHC). In a meta-analysis, FKBPL levels were a significant predictor of BCSS; low FKBPL levels indicated poorer breast cancer specific survival (BCSS) (hazard ratio (HR) = 1.30, 95% confidence interval (CI) 1.14-1.49, p < 0.001). The prognostic impact of FKBPL remained significant after adjusting for other known prognostic factors (HR = 1.25, 95% CI 1.07-1.45, p = 0.004). For the sub-groups of 2365 estrogen receptor (ER) positive patients and 1649 tamoxifen treated patients, FKBPL was significantly associated with BCSS (HR = 1.34, 95% CI 1.13-1.58, p < 0.001, and HR = 1.25, 95% CI 1.04-1.49, p = 0.02, respectively). A univariate analysis revealed that FKBPL was also a significant predictor of relapse free interval (RFI) within the ER positive patient group, but it was only borderline significant within the smaller tamoxifen treated patient group (HR = 1.32 95% CI 1.05-1.65, p = 0.02 and HR = 1.23 95% CI 0.99-1.54, p = 0.06, respectively). The data suggests a role for FKBPL as a prognostic factor for BCSS, with the potential to be routinely evaluated within the clinic.

An international study to increase concordance in Ki67 scoring.

Although an important biomarker in breast cancer, Ki67 lacks scoring standardization, which has limited its clinical use. Our previous study found variability when laboratories used their own scoring methods on centrally stained tissue microarray slides. In this current study, 16 laboratories from eight countries calibrated to a specific Ki67 scoring method and then scored 50 centrally MIB-1 stained tissue microarray cases. Simple instructions prescribed scoring pattern and staining thresholds for determination of the percentage of stained tumor cells. To calibrate, laboratories scored 18 'training' and 'test' web-based images. Software tracked object selection and scoring. Success for the calibration was prespecified as Root Mean Square Error of scores compared with reference <0.6 and Maximum Absolute Deviation from reference <1.0 (log2-transformed data). Prespecified success criteria for tissue microarray scoring required intraclass correlation significantly >0.70 but aiming for observed intraclass correlation ≥0.90. Laboratory performance showed non-significant but promising trends of improvement through the calibration exercise (mean Root Mean Square Error decreased from 0.6 to 0.4, Maximum Absolute Deviation from 1.6 to 0.9; paired t-test: P=0.07 for Root Mean Square Error, 0.06 for Maximum Absolute Deviation). For tissue microarray scoring, the intraclass correlation estimate was 0.94 (95% credible interval: 0.90-0.97), markedly and significantly >0.70, the prespecified minimum target for success. Some discrepancies persisted, including around clinically relevant cutoffs. After calibrating to a common scoring method via a web-based tool, laboratories can achieve high inter-laboratory reproducibility in Ki67 scoring on centrally stained tissue microarray slides. Although these data are potentially encouraging, suggesting that it may be possible to standardize scoring of Ki67 among pathology laboratories, clinically important discrepancies persist. Before this biomarker could be recommended for clinical use, future research will need to extend this approach to biopsies and whole sections, account for staining variability, and link to outcomes.

Updated UK Recommendations for HER2 assessment in breast cancer.

Human epidermal growth factor receptor 2 (HER2) overexpression is present in approximately 15% of early invasive breast cancers, and is an important predictive and prognostic marker. The substantial benefits achieved with anti-HER2 targeted therapies in patients with HER2-positive breast cancer have emphasised the need for accurate assessment of HER2 status. Current data indicate that HER2 test accuracy improved following previous publication of guidelines and the implementation of an external quality assessment scheme with a decline in false-positive and false-negative rates. This paper provides an update of the guidelines for HER2 testing in the UK. The aim is to further improve the analytical validity and clinical utility of HER2 testing by providing guidelines of test performance parameters, and recommendations on the postanalytical interpretation of test results. HER2 status should be determined in all newly diagnosed and recurrent breast cancers. Testing involves immunohistochemistry with >10% complete strong membrane staining defining a positive status. In situ hybridisation, either fluorescent or bright field chromogenic, is used either upfront or in immunohistochemistry borderline cases to detect the presence of HER2 gene amplification. Situations where repeat HER2 testing is advised are outlined and the impact of genetic heterogeneity is discussed. Strict quality control and external quality assurance of validated assays are essential. Testing laboratories should perform ongoing competency assessment and proficiency tests and ensure the reliability and accuracy of the assay. Pathologists, oncologists and surgeons involved in test interpretation and clinical use should adhere to published guidelines and maintain accurate performance and consistent interpretation of test results.

The updated ASCO/CAP guideline recommendations for HER2 testing in the management of invasive breast cancer: a critical review of their implications for routine practice.

The American Society of Clinical Oncology and the College of American Pathologists have issued joint updated comprehensive guideline recommendations for human epidermal growth factor receptor 2 (HER2) testing in breast cancer. The update not only provides guidelines for the test performance parameters, with the aim of improving test accuracy, reproducibility, and precision, but also provides comprehensive recommendations on the post-analytical interpretation of the results, and requires improved communication among healthcare providers. The updated guidelines are targeted at testing laboratories, pathologists, oncologists, surgeons, and, indirectly, other healthcare providers. Although the guidelines contribute to the improved analytical validity and clinical utility of laboratory assays required for successful molecularly targeted therapy in the era of personalized medicine, the implications of such recommendations have to be acknowledged. Certain recommendations, particularly those related to repeating the test and pathological concordance, have lower levels of supportive evidence than existing key recommendations, and the associated workload implications will be challenging to support in most healthcare systems. In this commentary, we critically address the key updated recommendations and their impact on service provision and patient care.

Virtual tissue microarrays: a novel and viable approach to optimizing tissue microarrays for biomarker research applied to ductal carcinoma in situ.

AIMS: Tissue microarrays (TMAs) are effective tools for performing high-throughput standardization analyses of biomarkers, but evidence indicating the core number required to be representative of the whole tumour is lacking. Ductal carcinoma in situ (DCIS) is a non-obligate precursor of invasive breast cancer. The number and size of cores that can best represent a DCIS lesion are unknown. Rather than performing extensive experiments using several variants of physical TMAs, the aim of this study was to develop a 'virtual TMA' approach that is effective at optimizing biomarker discovery and validation. METHODS AND RESULTS: Whole DCIS sections from 95 patients were evaluated by immunohistochemistry for oestrogen receptor (ER), progesterone receptor (PgR), HER2, and Ki67. Histoscores were generated manually for ER, PgR, and HER2, as well as percentage positivity for Ki67. Slides were scanned using the FDA-approved Ariol SL50 Image Analysis system, and the virtual array (V-Array) module was used. Virtual cores created virtual TMAs, and our validated scoring classifiers were applied. Automated histoscores and percentage positivity were determined, and compared against increasing numbers of cores. The optimal number of cores was based on concordant results between virtual TMAs and corresponding whole sections. CONCLUSIONS: We have shown that virtual arrays constitute an important tool in digital pathology in both research and clinical settings.

HER2 gene amplification in breast cancer: a rogues' gallery of challenging diagnostic cases: UKNEQAS interpretation guidelines and research recommendations.

International and national guidelines highlight the importance of accuracy, reproducibility, and quality control of in situ hybridization (ISH) methods for testing breast carcinomas. However, few guidelines cover the reporting of ISH cases with "unusual" signal patterns, including, eg, heterogeneity and loss of chromosome enumeration probe or gene signals. These cases are, in fact, relatively frequent, and there is a need for developing evidence- or consensus-based reporting guidelines to ensure consistency of treatment. Following an audit of cases from a single center (including >1,700 cases) we show that approximately 10% of ISH results reflect unusual signal patterns. We illustrate the most common of these patterns and provide reporting guidelines for diagnosticians and recommendations for future research. Our goal is to ensure that in the future such "rogues" are reported in a consistent manner that, ultimately, will be supported by molecular and biochemical evidence.

A UK NEQAS ISH multicenter ring study using the Ventana HER2 dual-color ISH assay.

We performed a multicenter assessment of a new HER2 dual-color chromogenic in situ hybridization (CISH) test and herein report on concordance of CISH data with fluorescence in situ hybridization (FISH) data and intraobserver and interlaboratory scoring consistency. HER2 results were evaluated using duplicate cores from 30 breast cancers in 5 laboratories using the Ventana HER2 dual-color ISH assay (Ventana Medical Systems, Cambridgeshire, England) and in 1 central laboratory using a standard FISH assay. Overall 93.3% of cases were successfully analyzed by CISH across the 5 participating laboratories. There was excellent concordance (98.0% overall) for diagnosis of HER2 amplification by CISH compared with FISH. Intraobserver variability (7.7%) and intersite variability (9.1%) of absolute HER2/chromosome enumeration probe 17 ratios were tightly controlled across all participating laboratories. The Ventana HER2 dual-color ISH assay is robust and reproducible, shows good concordance with a standard FISH assay, and complies with requirements in national and international guidelines for performance of ISH-based diagnostic tests.

A UK NEQAS ICC and ISH multicentre study using the Kreatech Poseidon HER2 FISH probe: intersite variation can be rigorously controlled using FISH.

AIM: To assess a new HER2 fluorescence in situ hybridization (FISH) test and report on multicentre intrasite and intersite variation. METHODS AND RESULTS: HER2 results were scored from 45 breast cancers in eight laboratories using the Kreatech Poseidon HER2 FISH probe (Kreatech Diagnostics, Amsterdam, the Netherlands). Overall, 80.9% of cores were successfully analysed. Mean intrasite variation for HER2 ratio assessment was low (4.74%). Intersite variation in ratio was in line with previous reports (11.9+/-0.8%) for both reference and non-reference laboratories; only one laboratory displayed significantly higher intersite variation (P=0.009) than the remaining seven laboratories. The overall incidence of misclassification of cores was <1.3%, demonstrating an excellent level of concordance (>98.7%) across all eight laboratories, irrespective of whether they were 'reference' or 'routine diagnostic' laboratories. CONCLUSIONS: The Kreatech Poseidon HER2 FISH test is robust and reproducible. Highly quantitatively reproducible FISH results were obtained from eight 'diagnostic' and 'reference' laboratories; however, continued quality assessments are essential to good performance.

Chromogenic in situ hybridization: a multicenter study comparing silver in situ hybridization with FISH.

Our purposes were to perform a robust assessment of a new HER2 chromogenic in situ hybridization test and report on concordance of silver in situ hybridization (SISH) data with fluorescence in situ hybridization (FISH) data and on intraobserver and interlaboratory scoring consistency. HER2 results were scored from 45 breast cancers in 7 laboratories using the Ventana (Tucson, AZ) INFORM HER-2 SISH assay and in 1 central laboratory using a standard FISH assay. Overall, 94.8% of cases were successfully analyzed by SISH across the 6 participating laboratories that reported data. Concordance for diagnosis of HER2 amplification by SISH compared with FISH was high (96.0% overall). Intraobserver variability (8.0%) and intersite variability (12.66%) of absolute HER2/chromosome 17 ratios appear to be tightly controlled across all 6 participating laboratories. The Ventana INFORM HER-2 SISH assay is robust and reproducible, shows good concordance with a standard FISH assay, and complies with requirements in national guidelines for performance of diagnostic tests.

Mcl-1 expression has in vitro and in vivo significance in chronic lymphocytic leukemia and is associated with other poor prognostic markers.

Bcl-2 family proteins play a critical role in the regulation of apoptosis in chronic lymphocytic leukemia (CLL). However, their association with established prognostic markers is unknown. In this study, we analyzed the expression of Bcl-2, Bax, and Mcl-1 in 185 CLL patients and evaluated their relationship with other prognostic markers, in vitro sensitivity to fludarabine, and clinical outcome. Mcl-1 expression was significantly correlated with stage of disease (P < .001), lymphocyte doubling time (P = .01), V(H) gene mutation status (P < .001), CD38 expression (P < .001), and ZAP-70 expression (P = .003). In addition, Mcl-1 and Mcl-1/Bax ratios showed strong correlations with in vitro resistance to fludarabine (P = .005 and P < .001, respectively). Furthermore, elevated Mcl-1 expression and Mcl-1/Bax ratios were predictive of time to first treatment in the whole cohort (P < .001 and P < .001, respectively) and in stage A patients only (P = .002 and P = .001, respectively). Taken together, our data show that Mcl-1 is a key controller of in vitro drug resistance and is an important regulator of disease progression and outcome in CLL. It therefore represents a promising therapeutic target in this incurable condition. The close correlation between Mcl-1 expression and V(H) gene mutation status, CD38 expression, and ZAP-70 expression offers a biologic explanation for their association with adverse prognosis.