Background:CD117 (c-KIT) is a tyrosine kinase receptor protein, mutations in which are important in the tumourigenesis of gastrointestinal stromal tumour (GIST). Immunohistochemical detection of CD117 is the primary identifying feature in its diagnosis. Introduction:UK NEQAS ICC & ISH conducts an EQA programme for CD117 expression in GIST. Between May-2020 and May-2025, 20 runs of this EQA were undertaken. The archived data was analysed to look for evidence regarding the quality of testing and associations between primary antibodies and stain quality. Discussion:Total submissions were 2,656 (mean per run = 132.8; range = 125-140). The number of submissions awarded a quality score indicating at least acceptable stain quality was 2,642 (99.5%). The total number of submissions that failed was 14 (mean proportion per run = 0.5%; range = 0.0%-2.4%); those obtaining a borderline score totalled 158 (mean proportion = 5.9%; range = 0.7%-10.9%); those obtaining an acceptable score totalled 394 (mean proportion = 14.8%; range = 4.3-24.4%); and those obtaining a good/excellent score totalled 2,090 (mean proportion = 78.7%; range = 57.7%-94.2%). There was a strong trend for the proportion of submissions obtaining a good/excellent quality score to increase over time. Four primary antibodies were used: Clone 9.7 (102 submissions, 3.9%), YR145 (263 submissions, 10.0%), EP10 supplied by Leica Biosystems (EP10(LB), 415 submissions, 15.8%), EP10 supplied by Roche Diagnostics (EP10(RD), 505 submissions, 19.2%), and a polyclonal antiserum supplied by Agilent Dako (1,263 submissions, 48.1%). A total of 76 submissions (2.9%) that used other primaries or suppliers were excluded from analysis. Conclusion:Demonstration of CD117 overexpression in GIST was undertaken to a very high quality standard in the majority of laboratories. For users of Dako Agilent supplied automation the highest quality staining was produced by using the polyclonal antiserum from the same supplier. Among users of Leica Biosystems and Roche Diagnostics automation the best staining was produced by using primary sourced from the same supplier that provided their automation. It is recommended that Clone 9.7 (Roche Diagnostics) should not be used as the results it produced were statistically significantly inferior to those of all other antibodies.

Aim Comparison of immunohistochemical detection of mismatch repair (MMR) proteins within tumours, as deficient MMR is important for (1) the detection of Lynch Syndrome, caused by inherited variants affecting the DNA MMR genes MLH1, MSH2, MSH6 and PMS2, (2) aiding MMR gene variant interpretation and (3) deciding on use of immune checkpoint blockade therapy. Methods This retrospective analysis compares the performance of different MMR immunohistochemistry (IHC) antibody clones, detection systems and automation IHC platforms using a decade of technical data submitted to the UK National External Quality Assessment Scheme (NEQAS) as part of its MMR EQA programme, with calculation of participants' final aggregated scores (FAS) for performance comparison. Results Between 2011 and 2022, there were 38 MMR assessment runs, with an average of 44.8 submissions per antigen per assessment run. MMR module participation greatly increased over this decade. Average FAS showed a small non-significant upward trend with the lowest scores (14.6) observed in the first half of the decade, with greater concordance between FAS scores (15.1) in the second half of the decade. For MMR IHC, the antibody clones most frequently used were M1 and ES05 (FAS 15.7 and 15.1) for MLH1, G219-1129 and FE11 (FAS 14.8 and 14.1) for MSH2, EP51 and A16-4 (FAS 15.2 and 15.2) for PMS2 and SP93 and EP49 (FAS 16.1 and 15.6) for MSH6. The most common detection systems were Ventana Optiview, Leica BondMax, Leica Bond Refine, Dako FLEX+ and Ventana UltraView. Conclusions The quality of submitted MMR IHC sections has risen, with recent assessment scores showing lower variability, indicating better antibody clone performance, improved detection systems and IHC automation platform technology, allied to increasing technical competence among participants. UK NEQAS provides insight and feedback relating to MMR IHC protocols that most reliably produce high-quality MMR IHC staining to facilitate accurate reporting of the increasingly important tumour MMR status.

Ki-67 is a well-established marker of tumour proliferation and an important prognostic and predictive biomarker in breast cancer, particularly in hormone receptor-positive (HR-positive), HER2-negative disease. Despite its biological relevance, clinical implementation has been limited by the reported interobserver and interlaboratory variability. Recent therapeutic advances have created an increased need for accurate and reproducible Ki-67 assessment in clinical practice. This review summarizes the biological basis for the use of Ki-67 as a marker of proliferation, technical requirements for reliable immunohistochemistry and the influence of pre-analytical and analytical variables on staining performance. We evaluate established and emerging scoring approaches and provide scoring recommendations for practising pathologists. A simplified calibrated global assessment method is presented as an alternative to exhaustive visual quantification that preserves its accuracy while substantially reducing scoring time and avoiding the variability of estimated methods. Image analysis/artificial intelligence (AI) using validated algorithms is recommended where available. We also review the role of Ki-67 in predicting response to neoadjuvant endocrine and chemotherapy, its integration into prognostic models such as the PEPI score, and its utility in selecting patients for adjuvant CDK4/6 inhibition. The limitations of fixed cut-off values are discussed, together with the potential advantages of tiered classification and continuous modelling. Finally, we outline the growing role of digital pathology and AI, which have demonstrated improved reproducibility, reduced turnaround time, and prognostic performance superior to manual scoring. Ki-67 is a clinically meaningful biomarker, the value of which can only be fully realized through rigorous standardization, validated scoring approaches and close communication between pathologists and oncologists. This guidance provides a practical framework for high-quality Ki-67 assessment and supports its safe and effective integration into contemporary breast cancer management.

Inter-observer concordance data for the HER2 category as assessed by a group of 16 specialist breast pathologists on 50 diagnostic core biopsies was compared with that produced by digital image analysis (DIA) using the HER2 APP, CE2797 (VP APP; Visiopharm, Hoersholm, Denmark). Comparing pathologists' consensus scores and DIA scores, 36 cases (73.5%) agreed. Fleiss' kappa statistic was 0.433 (indicative of moderate agreement). Cohen's weighted kappa was used to compare the scores of individual raters to consensus scores; for all 50 cases the kappa scores had a range between 0.412 and 0.854; the VP APP was ranked 12th of 17 raters (kappa score 0.638 indicating substantial agreement). Results for HER2-low cases (N = 44) showed a kappa score range of 0.295 to 0.823; the VP APP ranked 12th of 17 (score 0.535 indicating moderate agreement). For high agreement cases the kappa score range was 0.664 to 1.000 for all HER2 scores (N = 24) and the VP APP scored 0.916 (indicating almost perfect agreement). For the HER2-low scores (N = 20), the kappa score range was 0.506-1.000 and the VP APP scored 0.860 (almost perfect agreement). DIA of the proportions of tumour cells showing expression within each of the HER2 categories demonstrated that the majority of cases showing a low level of agreement between pathologists showed heterogeneity and/or a level of expression close to a cut-point for decision making. This study demonstrates that the VP APP produces results that are extremely well-aligned to those of expert pathologists in cases with good overall agreement, and in difficult cases its reproducibility will outperform that of the visual scorer. The results also suggest that use of the VP APP has the potential to reduce the proportion of cases referred for gene amplification testing by reducing the number of cases incorrectly classified as HER2 2+.

Validation of biomarker assays is mandatory not only for their applications in clinical trials but also for their subsequent transfer to clinical laboratories in routine clinical care. There are two critical components relevant to their transfer to clinical practice: regulatory oversight and methodology transfer. Both aspects are simplified where companion diagnostic (CDx) assays relevant to a given indication are being implemented in clinical laboratories. However, when laboratory developed tests (LDTs) are being used either because CDx is not available or because LDT is preferred, both aspects need special consideration from regulatory agencies as well as clinical laboratories. The key component that links these two aspects is evidence of validation of the new LDTs. For predictive and prognostic biomarkers in oncology, clinical validation is feasible only in clinical trials. This approach is not available or feasible to clinical laboratories that develop LDTs. While clinical laboratories routinely perform technical/analytical validation, depending on the type of biomarker, this may not be sufficient to provide evidence of the LDT's clinical relevance. Laboratories must perform and document their assessment for the need for indirect clinical validation. When indirect clinical validation is required, it must be performed according to existing guidelines for this purpose. This paper provides expert consensus guidance and recommendations on how to assess for the need for indirect clinical validation and how to perform indirect clinical validation where required. This paper also provides a conceptual framework to regulatory agencies for determining requirements for validation of predictive and prognostic biomarkers in oncology.

An EQA specifically designed for the assessment of immunohistochemical stain quality in the area of HER2-low testing was established by UK National Quality Assessment Scheme for Immunocytochemistry and In-Situ Hybridsiation (UK NEQAS ICC & ISH) at the beginning of 2023. We report on the results produced during the first 18-months of its operation (7 assessment runs, 527 submissions). At the first run 54.5% of participants’ submissions failed. The principal reason for failure was weak staining at this and at all subsequent assessments where submissions failed. The fail-rate continued to be above 50% for the first three assessment runs; beginning at run 4 and continuing in all following runs, the fail-rate declined such that it was around 20% at run 7. This change was not associated with any methodological parameter on which data was available (primary antibody, antigen retrieval, detection system or automation platform). Overall, 2258 core samples were available, 99.2% of all samples expected to be negative (HER2 0) showed staining concordant with that category; 71.% of those expected to stain as HER2 1+ and 60.5% of those expected to stain as HER2 2+ were assessed as showing the correct level of staining. Core samples showing weaker than expected staining totalled 531 (23.5%) while 52 (2.3%) showed stronger staining than expected. The 4B5 clone (Ventana) was the most commonly used primary antibody with 391 (87.3%) of submissions using this clone; of which, 275 (70.3%) passed. In contrast none of the 25 (5.6%) submissions that used the CB11 clone (Oracle, Leica) achieved a pass.

To the Editor.—We read with great interest and a growing sense of excitement the recently published editorial by Miller,1 and the accompanying editorial by Magnani and Taylor.2As we are, respectively, the director and manager of the United Kingdom (UK) National External Quality Assessment Scheme for Immunocytochemistry and In-Situ Hybridisation (UK NEQAS ICC & ISH; also herein referred to as the “Scheme”), the subject of these editorials is obviously one which is of great professional interest to us.The College of American Pathologists (CAP) immunohistochemistry (IHC) proficiency testing program is one of the largest, longest-running, and well-respected external quality assessment (EQA) programs in the world, as well as being one of the most influential. It was heartening, therefore, to read of its plans to disengage the interpretive element of immunohistochemical testing from its underlying technical aspects. And in doing so, to begin the process of putting the science, or, as Miller1 terms it, the “chemistry,” back into IHC.Why are we excited by this? Well, it is mainly because, like CAP’s committee review procedures, UK NEQAS ICC & ISH has been examining the methodologic aspects of IHC and their effects on quality since its inception, in our case, 1984. That was when the founding group of biomedical scientists and pathologists that latterly became UK NEQAS ICC & ISH first began to critically assess the quality of κ light chain demonstration in formalin-fixed tissues. This was the birth of EQA for immunohistochemical testing, and indeed the first time EQA was introduced into the discipline of cellular pathology as a whole, certainly within the UK.Since then, UK NEQAS ICC & ISH has continued to extend its repertoire of EQA programs in an effort to keep pace with the ever-increasing use of immunohistochemical markers across the breadth of cellular pathology. We no longer offer a stand-alone program that solely requires its participants to demonstrate κ light chains. Some would argue that this is a shame, as success with the method required a masterful understanding of the science behind each step in the immunohistochemical methodology, such that it could be successfully manipulated to reliably demonstrate a protein that is present at both huge concentrations (in κ plasma cells) and at vanishingly small amounts (on the cell membranes of a subpopulation of B cells in the mantle zone).But times have moved on, and being at the forefront of delivery of technical EQAs for IHC (and latterly also for in situ hybridization testing), the Scheme has needed to move on also and to explore and introduce innovative methods. Examples of which include the following: The requirement for tissue controls that express the target protein at a range of concentrations around the critical clinical cutpoint. Important for diagnostic predictive markers. This was first introduced by UK NEQAS ICC & ISH into its estrogen receptors in breast cancer module, which started in early 1998 and continues to this day—more than 100 runs and 25 years later. (In passing, it should be noted that we are the only Scheme as far as we know that also assesses its participants’ in-house controls for adequacy in addition to the assessment of distributed materials.)The utilization of multiple cell-line controls in which each cell line expresses the chosen protein at a clinically relevant level as a set of reference standards for use in EQA was first reported by the Scheme in its breast cancer human epidermal growth factor 2 (HER2) IHC module, which started in 2002,3 marking a further sea change by introducing the use of IHC as a predictive test for a single drug—trastuzumab (Herceptin)—and so beginning the era of companion diagnostics.This was quickly followed by another first, with the introduction in 2004 of the ISH module for cellular pathology laboratories performing this technique to demonstrate HER2 gene copy number and its amplification status, once again as a predictive marker of response to trastuzumab in breast cancer. And our venture into the brave new world (for us) of interpretive as well as technical EQA.We can skip forward nearly 20 years to the present day, missing as we do so the burgeoning growth in the use of IHC and in situ hybridization–based companion diagnostics in all areas of personalized medicine (not least in immuno-oncology, which didn’t even exist 20 years ago). And here once again we see UK NEQAS ICC & ISH responding to the need for a novel approach, this time in the introduction of a truly quantitative assessment method that uses digital image analysis to assess the quality of results produced by Ki-67 to measure proliferation,4 together with the use of purpose-designed cell-line arrays (Array Sciences LLC).Which brings us right up to date with our most recently introduced module for low-level HER2 expression in breast cancer. Here we are addressing a critical educational need by combining expert peer-led visual assessment with a specially developed digital algorithm to give individually tailored feedback on technical quality and interpretive proficiency to our participants.5Away from the focus on external quality, there have been significant advances made in addressing day-to-day internal quality control in the IHC laboratory, and in perhaps one area, the statement by Magnani and Taylor2 that “Of necessity, every IHC laboratory uses a different (nonstandardized) control tissue,” need no longer be true thanks to advances in the development of systems such as Qualitopix (Visiopharm A/S, Denmark) that monitor and audit day-to-day immunohistochemical staining consistency on standardized, validated test materials in real time.We recognize that we are not the only EQA program that examines IHC at the methodologic level, and indeed we actively collaborate with many of our fellow schemes across the world, including CAP, through our association with the International Quality Network for Pathology (https://www.iqnpath.org/) organization. And we acknowledge that they, like us, have grown and developed novel tools and ways of assessing the quality of IHC.By highlighting all these advances and developments, we are not intending to say to the CAP IHC committee “we got there first, now look how much catching up you need to do.” But rather, yes, you are right to do this now when we are at last beginning to have at our disposal all of the tools we need to objectively assess the effects that methodology has on immunohistochemical stain quality, which is the bedrock on which correct interpretation has its foundations.We welcome this new development from the CAP IHC committee and are sure that the knowledge it will produce will greatly strengthen the science of IHC and the quality of its results, to the immeasurable benefit of the patients it serves.It’s good to have you on board.

Abstract Background and Aims Ki-67 is a well-established biomarker of proliferation in breast cancer (BC). However, its value in treatment decision making is hampered by a lack of analytical reproducibility. Regular participation in external quality assessment (EQA) substantially improves inter-laboratory concordance. Pre-requisites in establishing a fit-for-purpose EQA for Ki-67 are a well-validated testing substrate and a reproducible method of assessing Ki-67 scores in participants’ submitted materials. We report here on the results of work using cell line controls analysed by digital image analysis (DIA) as a first step towards providing such an EQA. Materials and Methods A formalin-fixed paraffin embedded (FFPE) cell line microarray (CLMA) was designed and produced in conjunction with Array Sciences LLC (Sausalito, USA). It was comprised of cores taken from a pure population of Sf9 caterpillar cells, which have been shown to be completely unreactive with most commercial antibodies to human Ki-67, together with cores of Sf9 cells mixed with four different human BC cell lines. These were BT-20 (85% BC cells), ZR-75-1 (75%), BT-474 (65%) and BT-483 (55%). Sections from the CLMA were mounted onto glass microscope slides together with sections from a FFPE tonsil sample and two BC samples; one BC showed high (~30%, BC-high) and the second, low proliferation (~5%, BC-low). In both BC samples proliferating cancer cells were distributed homogeneously throughout the the block. Unstained sections were distributed to laboratories participating in the Scheme’s Ki-67 BC programme; after routine IHC-staining for Ki-67 returned slides were centrally visually assessed for stain quality and subjected to DIA using an in-house application developed on Visiopharm software (Visiopharm A/S, Hoersholm, Denmark). Results Slides were returned by 37 laboratories. Analysis of Ki-67 scores obtained on the Sf9 core identified two distinct groups. The first (n = 27) were negative or showed low Ki-67 scores (mean = 1.1%, 95% CIs: 0.2-1.9%), the second (n = 10) displayed a step-change in scores (mean = 49.9%, 95% CIs: 33.2-66.7%); the means of the two groups were significantly different (P< 0.0001). When Ki-67 scores for each of the tissue samples were dichotomized into similar groups, the means of those groups differed significantly for the two BC samples (P< 0.001), but not for tonsil. Quality scores generated by visual assessment did not differ significantly between the two groups. However, when slides bearing Sf9 cores demonstrating aberrant Ki-67 scores were visually examined nuclear staining was clearly visible, and non-specific nuclear staining could also be identified in the matched BC tissue samples, but not in the tonsil sections. Correlation of Ki-67 scores between the four BC cell line cores and each of the BC tissue samples was examined using Pearson’s correlation statistics. The r statistic range was 0.57-0.71 in comparisons between BC cell line cores and the BC-high sample, and 0.53-0.70 for those with BC-low; in each case BT-483 showed the highest correlation score and BT-20 the lowest. A similar analysis was undertaken between the tonsil and the two BC tissues. The r statistic for correlation between tonsil and BC-high scores was 0.72; it was 0.64 for those between tonsil and BC-low. Conclusions By using a pure population of Sf9 cells we have developed a sensitive indicator of non-specific nuclear staining in IHC preparations stained for Ki-67 which identifies the presence of the artefact quantifiably. Cores made from Sf9/BC cell line mixtures (especially BT-483) produce Ki-67 scores which correlate with those obtained in breast cancer samples at a similar level to those achieved between tonsil and BC samples; this is true for both high and the low proliferation ranges. Cell line mixtures can be adjusted to show Ki-67 scores in the clinically relevant ranges, and they do not show the inherent biological variations seen in tissue controls such as tonsil. Citation Format: Andrew Dodson, Fitim Berisha, Dawn Wilkingson, Lila Zabaglo, Suzanne Parry. Digital image analysis and a novel set of cell line samples as aids in the development of a quantitative external quality assessment programme for Ki-67 [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-26-09.

Claudin 18.2 (CLDN18.2), the dominant isoform of CLDN18 in gastric tissues, is a highly specific tight junction protein of the gastric mucosa with variably retained expressions in gastric and gastroesophageal junction cancers. Additionally, CLDN18.2-targeted treatment with zolbetuximab, in combination with chemotherapy, has recently been assessed in 2 phase-III studies of patients with HER2-negative, locally advanced, unresectable, or metastatic gastric or gastroesophageal junction adenocarcinoma. These trials used the investigational VENTANA CLDN18 (43-14A) RxDx immunohistochemistry (IHC) assay on the Ventana BenchMark platform to identify patients eligible for CLDN18.2-targeted treatment. We report the findings of a global ring study evaluating the analytical comparability of concordance of the results of 3 CLDN18 antibodies (Ventana, LSBio, and Novus) stained on 3 IHC-staining platforms (Ventana, Dako, and Leica). A tissue microarray (TMA), comprising 15 gastric cancer cases, was stained by 27 laboratories across 11 countries. Each laboratory stained the TMAs using at least 2 of the 3 evaluated CLDN18 antibodies. Stained TMAs were assessed and scored using an agreed IHC-scoring algorithm, and the results were collated for statistical analysis. The data confirmed a high level of concordance for the VENTANA CLDN18 (43-14A; Ventana platform only) and LSBio antibodies on both the Dako and Leica platforms, with accuracy, precision, sensitivity, and specificity rates all reaching a minimum acceptable ≥85% threshold and good-to-excellent levels of concordance as measured by Cohen's kappa coefficient. The Novus antibody showed the highest level of variability against the reference central laboratory results for the same antibody/platform combinations. It also failed to meet the threshold for accuracy and sensitivity when used on either the Dako or Leica platform. These results demonstrated the reliability of IHC testing for CLDN18 expression in gastric tumor samples when using commercially available platforms with an appropriate methodology and primary antibody selection.

Abstract Background: Mechanisms of resistance to endocrine therapy are not well understood within ER+HER2+ breast cancer (BC). Our prior work suggested that intrinsic HER2-Enriched (HER2E) molecular subtype predicts early resistance to aromatase inhibitors (AI) (Bergamino eBioMedicine 2022) and high on-treatment (on-Txt) Ki67 levels predict poor survival (Smith Lancet Oncol 2020). Improved early detection of persistent proliferating tumor cells with endocrine resistance pathways could be targeted by pre-emptive personalized therapy and reduction in recurrence. In this study, we proposed to further identify additional alterations/features from genomic and spatial data to provide unprecedented new insight into intrinsic and adaptive resistant pathways in tumor cells that may assist to identify molecular targets for treatment. Materials: POETIC was a phase III trial of post-menopausal patients with ER/PR+ invasive BC (n = 4480) randomized 2:1 to 2 weeks of peri-operative AI (POAI) vs control, followed by standard-of-care treatment. Ki67 was assessed by IHC and intra-tumor heterogeneity was evaluated (5-15 regions) for all the POETIC POAI samples (N = 2487). ER+HER2+ samples were classified as good responders (GR) or poor responders (PR) based on a reduction in Ki67 between pre-treatment (pre-Txt) and 2-week on-Txt samples. Tumor-infiltrating lymphocytes were assessed; multiplex Immunofluorescence (mIF) was performed to measure immune cell densities in tumor and stroma compartments (CD3, CD20, CD68, FOXP3, and CD3 FOXP3 co-expression). Gene expression profiles by BC360™ (Nanostring) on all 210 pairs of POAI treated ER+/HER2+; whole exome sequencing (WES, 100X) were performed on pre-Txt tumor and blood samples from 13 GR, 17 PR, and 9 HER2E GR. We performed GeoMx Whole Transcriptome on 4 pairs (pre-Txt and on-Txt) of GR and GeoMx Proteins (77 including IO proteins) on 6 pairs of GRs and 6 pairs of PRs. Results: The most frequently mutated genes were TP53, PIK3CA, GATA3, and CHD4. Only TP53 was associated with PR (Fisher’s exact p=0.01). TP53 mutated cases had higher expression of TP53 mutant-like gene expression signature compared to wild-type cases (Wilcoxon test p=0.001), mIF FOXP3 (Wilcoxon test p = 0.0005), and CD68 (Wilcoxon test p = 0.019) density score. However, within the HER2-E subset, we found that TP53 mutations were associated with GR (Fisher’s exact p=0.02). We found spatial heterogeneity of Ki67 IHC levels across POAI samples. Examining IHC, while there was higher heterogeneity of Ki67 in the ER+HER2- samples (n = 2264) with 3% of pre-Txt and 9% on-Txt, 6% of ER+HER2+ samples (13/223, 6 LumA, 5 LumB, and 2 HER2E) showed heterogeneity of Ki67 exclusively on-Txt. Even in GR tumors with Ki67 < 10% on-Txt, we identified hotspots with retained proliferating Ki67+ cells after 2 weeks of POAI. The lobular tumors were GR and had characteristic CDH1 mutations. Importantly, cases with persistent areas of Ki67+ cells, regardless of Her2 status, were associated with late relapse. To further explore intratumoral heterogeneity, we performed spatial whole transcriptomics profiling on 95 regions from 4 pairs of GR samples (Ki67 > 10% at baseline and Ki67 < 10% on-Txt) and found low intratumoral heterogeneity in the pre-Txt samples that increased at 2 weeks on-Txt. In a larger set of samples including both GR and PR with the GeoMx protein method, we found increased intratumoral heterogeneity in the PR vs GR. Conclusion: While TP53 mutation was generally a predictor of poor response; in HER2-E it paradoxically was associated with a good early response to aromatase inhibitor which warrants further investigation. Ki67 levels in ER+HER2+ showed higher intratumoral heterogeneity in a subset of patients on treatment suggesting the potential of persistent, proliferating cells leading to later recurrence. Our spatial RNA and protein data further observe the intratumoral heterogeneity that identifies pathways for use as potential spatial biomarkers. Citation Format: Maggie Chon U Cheang, Xixuan Zhu, Orsolya Sipos, Anastasia Alataki, Mikayla Feldbauer, Elena López-Knowles, Holly Tovey, Lucy Kilburn, Milana Bergamino Sirvén, Dhrusti Patel, Hui Xiao, Perry Maxwell, Anthony Skene, Chris Holcombe, Manuel Salto-Tellez, Nicholas Turner, Andrew Dodson, Ian Smith, John Robertson, Judith Bliss, Gene Schuster, Roberto Salgado, Mitch Dowsett, Katherine A Hoadley. Genomic characterization of endocrine resistance in ER+HER2+ breast cancers in the POETIC Trial [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PS09-08.

With interest, we read the article titled "Quantitative comparison of PD-L1 IHC assays against NIST standard reference material 1934" by Sompuram et al, 1 Sompuram S.R. Torlakovic E.E. 't Hart N.A. Vani K. Bogen S.A. Quantitative comparison of PD-L1 IHC assays against NIST standard reference material 1934. Mod Pathol. 2022; 35: 326-332 Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar and we compliment them on the methodology and their aim of standardizing PD-L1 immunohistochemistry (IHC). In Reply: Programmed Death-Ligand 1 (PD-L1) Immunohistochemistry CalibrationModern PathologyVol. 36Issue 2PreviewThe authors welcome the opportunity to discuss the commutability of programmed death-ligand 1 (PD-L1) calibrators. To summarize, calibration is a foundational principle for accurate and reproducible clinical laboratory testing. It is the process that links signal strength (eg, stain intensity) with the analyte concentration. Our article described the first application of quantitative calibration to PD-L1 immunohistochemistry (IHC) testing.1 Haragan et al2 raise the concern that one of our findings does not match their previously published conclusion. Full-Text PDF

E2F activation metagene & Fluorescence activated cell sorting

Supplementary Data from Biomarkers of Response and Resistance to Palbociclib Plus Letrozole in Patients With ER+/HER2− Breast Cancer

Purpose Ki67 assessed at diagnosis (Ki67 baseline ) is an important prognostic factor in primary oestrogen receptor-positive (ER +) breast cancer. Proportional change in Ki67 after 2 weeks (∆Ki67 2week ) is associated with clinical benefit from endocrine therapies and residual Ki67 (Ki67 2week ) with recurrence-free survival. The aim was to define the association between Ki67 baseline and after aromatase inhibitor (AI) exposure ∆Ki67 2week and Ki67 2week with key prognostic and biologic factors utilising data from the POETIC study. Patients and methods In POETIC 4480 postmenopausal patients with primary ER and/or PgR + breast cancer were randomised 2:1 to 2 weeks’ presurgical AI (anastrozole or letrozole) or no presurgical treatment (control). Ki67 was measured centrally in core-cut biopsies taken prior to AI and in core-cuts or the excision biopsy at surgery. Relationships between the Ki67 and biologic factors were explored using linear regression. Results Established associations of Ki67 baseline with biologic factors including PgR status, tumour grade, tumour size, histological subtype, nodal status, and vascular invasion were confirmed in the HER2- subpopulation. In the HER2 + subpopulation only grade and tumour size were significantly associated with Ki67 baseline . In control group Ki67 2week was 18% lower than Ki67 baseline (p < 0.001) when Ki67 2week was measured in excision biopsies but not when measured in core-cuts. Median suppression by AIs (∆Ki67 2week ) was 79.3% (IQR: −89.9 to −54.6) and 53.7% (IQR: −78.9 to −21.1) for HER2-negative and HER2-positive cases, respectively. Significantly less suppression occurred in PgR- vs PgR + and HER2 + vs HER2- tumours which remained apparent after adjustment for 2-week sample type. Conclusions The magnitude of this study allowed characterisation of relationships between Ki67 baseline , ∆Ki67 2week and Ki67 2week with high degrees of confidence providing a reference source for other studies. Lower values of Ki67 occur when measured on excision biopsies and could lead to apparent but artefactual decreases in Ki67: this should be considered when either ∆Ki67 2week or Ki67 2week is used in routine clinical practice to aid treatment decisions or in clinical trials assessing new drug therapies .

Ki67 has potential clinical importance in breast cancer but has yet to see broad acceptance due to inter-laboratory variability. Here we tested an open source and calibrated automated digital image analysis (DIA) platform to: (i) investigate the comparability of Ki67 measurement across corresponding core biopsy and resection specimen cases, and (ii) assess section to section differences in Ki67 scoring. Two sets of 60 previously stained slides containing 30 core-cut biopsy and 30 corresponding resection specimens from 30 estrogen receptor-positive breast cancer patients were sent to 17 participating labs for automated assessment of average Ki67 expression. The blocks were centrally cut and immunohistochemically (IHC) stained for Ki67 (MIB-1 antibody). The QuPath platform was used to evaluate tumoral Ki67 expression. Calibration of the DIA method was performed as in published studies. A guideline for building an automated Ki67 scoring algorithm was sent to participating labs. Very high correlation and no systematic error ( p = 0.08) was found between consecutive Ki67 IHC sections. Ki67 scores were higher for core biopsy slides compared to paired whole sections from resections ( p ≤ 0.001; median difference: 5.31%). The systematic discrepancy between core biopsy and corresponding whole sections was likely due to pre-analytical factors (tissue handling, fixation). Therefore, Ki67 IHC should be tested on core biopsy samples to best reflect the biological status of the tumor.

Accurate PD-L1 testing for non-small cell lung cancer (NSCLC) maximizes the benefits of immune checkpoint inhibitor (ICI) drugs like pembrolizumab. False negative test results deny ICI treatments to eligible patients, worsening clinical and economic outcomes, while false positives increase costs by using ICI treatments without their benefits. This study evaluates the cost-effectiveness of PD-L1 testing with an in vitro diagnostic (IVD) compared to a laboratory-developed test (LDT) for allocating patients with NSCLC to treatment with either pembrolizumab or chemotherapy using the German healthcare system as a model. We developed a decision analytical model to evaluate the cost-effectiveness of PD-L1 testing with a regulatory body approved IVD compared to an LDT from the national German healthcare payer (statutory health insurance system) perspective. Accuracy of PD-L1 testing was based on data from two independent proficiency testing programs. The 1-year model was based on outcomes data from the KEYNOTE-024 clinical trial and treatment patterns reflecting current German practices. IVDs produced accurate PD-L1 testing results in 93