Background: The HER2DX assay, a 27-gene test, was developed to provide prognostic information and predict treatment responses in patients with early-stage HER2+ breast cancer. This assay evaluates four gene expression signatures: immune/immunoglobulin (IGG), proliferation, luminal, and HER2, offering a comprehensive risk score, a likelihood estimates for pathological complete response (pCR) and ERBB2 expression levels. In this study, we evaluated the HER2DX assay in HER2+ DCIS, aiming to understand its biology and relationship with HER2+ invasive breast cancer. Methods: Standardized HER2DX genomic test was evaluated centrally on 28 formalin-fixed paraffin-embedded tumor samples of HER2+ DCIS across three hospitals in Spain. DCIS features such as nuclear grade, comedonecrosis, architectural pattern, tumor size and hormone receptor (HR) status were evaluated. Percentage (%) of stromal tumor infiltrating lymphocytes (TILs), their spatial distribution (i.e.: inflamed, desert or excluded), and presence of tertiary lymphoid structures (TLS, defined as spatially organized, non-encapsulated areas of immune cell aggregates with or without germinal center [GC]) were assessed on hematoxylin eosin slides. Descriptive statistics were used. Results: The HER2DX assay was evaluated in 28 cases of HER2+ DCIS. Most cases had a 3 nuclear grade (67.9%) and presented with comedonecrosis (60.7%). HR positivity (HR+) was found in 42.9% of cases, as determined by immunohistochemistry. The median tumor size was 30 mm, with a range from 5 mm to 90 mm. The median % of TILs was 20% (range 0.5-70%). TILs, as a continuous variable, showed a moderate correlation with the HER2DX IGG signature (Pearson correlation coefficient=0.43, p-value=0.023), and a tendency with the inflamed spatial distribution (p-value=0.063). TLS were identified in 82.1% of DCIS samples, and their presence was associated with higher expression of the IGG signature (52.2% TLS in IGG-high vs. 21.7% in IGG-low). Regarding the HER2DX luminal signature, 41.7% of cases were classified as luminal-high, associated with HR+ disease (p=0.025). For the HER2DX proliferation signature, 82.1% of cases were identified as proliferation-low. In HER2DX risk stratification, 100% of cases were categorized as low-risk, 46.4% were pCR-high, and 78.6% were ERBB2-high. Notably, only one (3.6%) HER2+ DCIS case was found to be ERBB2-low. Conclusions: The HER2DX assay revealed that all HER2+ DCIS cases were categorized as low-risk, with most cases showing high ERBB2 expression and a high predicted response to anti-HER2-based therapy. These findings underscore the underlying biology of HER2+ DCIS and its tumor immune microenvironment, indicating that HER2+ DCIS shares similar tumor biology with low-risk HER2+ breast cancer. Citation Format: Esther Sanfeliu, Anabel Martinez-Romero, Mercedes Marín-Aguilera, Vicente Marco, Felip Garcia, Vicente Peg, Blanca González-Farré, Ivonne Vazquez, Patricia Galván, Oleguer Castillo, Paula Blasco, Valeria Sirenko, Angela Aguirre, Laia Paré, Guillermo Villacampa, Antonio Martínez, Jesus Soberino, Aleix Prat, Fara Brasó-Maristany. HER2DX assay in HER2-positive (HER2+) breast ductal carcinoma in situ (DCIS) [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P5-07-18.

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.

Despite the remarkable clinical successes of chimeric antigen receptor (CAR) T-cell therapies in treating B-cell malignancies and multiple myeloma, similar outcomes have not been achieved in other indications. For patients with relapsed or refractory (R/R) acute myeloid leukemia (AML) or T-cell acute lymphoblastic leukemia (T-ALL), treatment options are limited, yet CART-cell therapies offer significant potential to address this unmet need. Here, we introduce a first-in-class CART-cell therapy targeting CD84, a novel antigen, for the treatment of R/R AML and T-ALL. CD84 is highly expressed on leukemic blasts, with limited expression on hematopoietic stem progenitor cells (HSPC), and is largely absent in healthy human tissues. Our second-generation CARTs targeting CD84 (CART84) demonstrate potent cytotoxicity against AML and T-ALL cells both in vitro and in vivo in patient-derived xenograft (PDX) models. Furthermore, CART84 eliminated primary leukemic blasts while exhibiting low cytotoxicity against CD34+ HSPC in vitro and in humanized mouse models in vivo, suggesting a low risk of myelotoxicity. These results support CD84 as a promising target for AML and T-ALL and provide the foundation for our upcoming first-in-human phase I/II clinical trial using CD84-directed CAR T cell therapy for patients with R/R AML and T-ALL (EudraCT 2024-519966-31-00).

BACKGROUND:An accurate assessment of tumor viability after first-line treatment is critical for predicting treatment failure in peripheral T-cell lymphomas (PTCLs). 18F-fluorodeoxyglucose (18F-FDG) positron emission tomography/computed tomography (PET/CT) has been adopted as the preferred assessment method in clinical trials, but its impact in clinical practice should be examined. This study aims to determine the prognostic significance of18F-FDG-PET/CT for survival following first-line treatment in PTCL patients. RESEARCH DESIGN AND METHODS:Retrospective observational study including 175 patients diagnosed with PTCL between 2008 and 2013 in 13 Spanish sites. RESULTS:Fifty patients were evaluated with18F-FDG-PET/CT following first-line therapy: 58% were18F-FDG-PET/CT-negative and 42% were18F-FDG-PET/CT-positive. Disease progression occurred in 37.9% of18F-FDG-PET/CT-negative patients and in 80.9% of18F-FDG-PET/CT-positive patients (p = 0.0037). Median progression-free survival and overall survival were 67 and 74 months for18F-FDG-PET/CT-negative patients, and 5 (p < 0.0001) and 10 months (p < 0.0001), respectively, in18F-FDG-PET/CT-positive patients. After multivariate analysis, only B symptoms emerged as a negative predictive factor of complete response (RR 7.08; 95% CI 1.60-31.31; p = 0.001). CONCLUSIONS:18F-FDG-PET/CT identifies high-risk PTCL patients who will have poor prognosis and survival following first-line treatment. However, more research is needed to confirm the best treatment options for PTCL patients.

BACKGROUND:Allergy to lipid transfer proteins (LPT) is common in Mediterranean Europe, and it causes severe reactions in patients and affects multiple foods, impairing the quality of life. OBJECTIVE:This study aimed to describe the clinical and sensitization profile of patients with LTP syndrome and to determine a clinical pattern of severity. Molecular diagnosis is shown in a broad population through microarrays. MATERIAL AND METHODS:This study was performed at the LTP Allergy Consultation of the Reina Sofia Hospital in Murcia, Spain. We analyzed the patients' characteristics, reactions, cofactors, food implicated, quality of life, skin prick test to food and aeroallergens, and serologic parameters, such as total immunoglobulin E, peach LTP (Pru p 3 IgE) and immunoglobulin G4, and microarray Immuno Solid-phase Allergen Chip (ISAC). We related the severity of the reactions with other variables. RESULTS:We presented a series of 236 patients diagnosed with LTP allergy, 54.66% suffering from anaphylaxis, 36.02% from urticaria angioedema, and 9.32% from oral allergy syndrome. The most frequently implicated food was peach, producing symptoms in 70% of patients, followed by walnut in 55%, peanut in 45%, hazelnut in 44%, and apple in 38% patients. Regarding the food that provoked anaphylaxis, walnut was the most frequent instigator, along with peach, peanut, hazelnut, almond, sunflower seed, and apple. According to the severity of LPT reaction, we did not discover significant differences in gender, age, food group involved, and serologic parameters. We found differences in the presence of cofactors, with 48.84% of cofactors in patients with anaphylaxis, compared to 27.1% in patients without anaphylaxis and in family allergy background (P < 0.0001). CONCLUSION:In our series of patients, 54% presented anaphylaxis, and the foods that most frequently produced symptoms were peaches, apples, and nuts. Cofactors and family allergy backgrounds were associated with the severity of LPT reaction.

Background: The HER2DX assay is a genomic test in early-stage HER2-positive (HER2+) breast cancer that provides prognostic and predictive information. HER2DX is a supervised learning algorithm incorporating tumor size, nodal staging, and 4 gene expression signature scores (immune/IGG, tumor cell proliferation, luminal differentiation and the expression of the HER2 amplicon). Among them, the IGG signature is associated with both overall survival and probability of achieving a pathologic complete response (pCR). Here, we studied the association of percentage (%) of tumor infiltrating lymphocytes (TILs) with HER2DX scores, immune genes and other breast cancer-related genes. Methods: HER2DX and %TILs were evaluated in 670 formalin-fixed paraffin-embedded (FFPE) samples of HER2+ breast cancer, including in 3 clinical studies SHORTHER (n=437), PAMELA (n=86), a cohort of patients treated with anti-HER2 therapy plus chemotherapy at Hospital Clínic of Barcelona (n=147). The %TILs were quantified by histological evaluation with hematoxylin eosin staining according to International TILs Working Group guidelines. The nCounter platform determined the expression of 192 genes and HER2DX scores. Pearson correlations (Cor) and Significance Analysis of Microarrays (SAM) with a false-discovery rate (FDR) < 5% assessed the association between %TILs and the expression of individual genes or HER2DX signature scores. Results: A moderate correlation was observed between %TILs and the immune IGG signature (Cor=0.56, p< 0.001). Of note, 171 (25.52%) cases had low %TILs (< 30%) and high IGG score, while 1 (0.15%) case had high %TILs (≥30%) and low IGG score. The %TILs were significantly associated with the expression of immune genes, ERBB2, IGG signature and HER2 amplicon score, and negatively associated with the expression of luminal genes (i.e., ESR1, PRG and BCL2). An unclear relationship between TILs and proliferation genes was observed. Finally, moderate correlations were observed between %TILs and HER2DX pCR score (Cor=0.48, p< 0.001) and between %TILs and HER2DX risk score (Cor=0.33, p< 0.001). Conclusions: Important differences exist between the %TILs and the HER2DX IGG signature in early-stage HER2+ breast cancer. The %TILs should not be used to predict the HER2DX scores. Biologically, a higher %TILs indirectly capture a higher ERBB2 expression and a lower expression of luminal genes, both associated with response to anti-HER2 treatment. Citation Format: Esther Sanfeliu, Fara Brasó-Maristany, Maria Vittoria Dieci, Mercedes Marín-Aguilera, Blanca González-Farré, Gaia Griguolo, Tomas Pascual, Patricia Galván, Laura Angelats, Oleguer Castillo, Paula Blasco, Valeria Sirenko, Pedro Jares, Joan Antón Puig-Butillé, Laia Paré, Antonio Martínez, Antonio Llombart-Cussac, Javier Cortés, Ana Vivancos, Patricia Villagrasa, Joel S Parker, Charles M Perou, Aleix Prat, PierFranco Conte, Valentina Guarneri. Association between tumor infiltrating lymphocytes (TILs) and the HER2DX assay in early-stage of HER2-positive (HER2+) breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-02-30.

AimsThe aim of this study is to extend the analysis of the Lung Cancer Biomarker Testing Registry (LungPath), by analysing the techniques used in the determination of epidermal growth factor receptor (EGFR), anaplastic lymphoma kinase (ALK), c-ros oncogene 1 (ROS1) and programmed death ligand-1 (PD-L1) for the diagnostic of patients with advanced non-small-cell lung cancer (NSCLC).MethodsInformation of the technique used for the determination of EGFR, ALK, ROS1 and PD-L1 was recorded from March 2018 to January 2019 from 44 centres, but only 34 centres matched with the 38 centres previously analysed, allowing to analyse the techniques used in 8970 matched determinations of EGFR, ALK, ROS1 and PD-L1. Therefore, a by-centre analysis studied the level of implementation of the techniques in the 44 centres, while a by-determination analysis made it possible to assess the overall frequency of the techniques used on the 9134 matched samples.ResultsBy-centre analysis showed that only 46.5% and 25.6% of the centres used reflex strategies for ALK and ROS1 determination, respectively. By-determination analysis showed that 94.4% of EGFR determinations were performed by PCR, 80.7% of ALK determinations were performed by IHC with clone D5F3, while 55.7% of ROS1 determinations were performed by IHC with clone D4D6. 22C3 were the PD-L1 clone more used (43.5%) followed by SP263 clone (31.1%).ConclusionsThe real-world evidence obtained from LungPath shows the effort of Spanish hospitals in performing biomarker determination in NSCLC with different methodologies despite that next-generation sequencing (NGS) utilisation in the year of the analysis was low. Biomarker determination results could be optimised with the incorporation of sequencing methods such as NGS in pathology departments.

On average, a case of 8– 10 fragments was processed in 120 min, from the time the sample was received in the laboratory until the pathologist performed the diagnosis. SOX10-stained frozen sections detected LM with a sensitivity of 94% and a specificity of 100% when compared with conventional analysis (paraffin-embedded sections) ( gold standard ) (Figure 1). The positive predictive value (PPV) was 100% and the negative predictive value (NPV) was 97.2%. False-negative results with SOX10-frozen sections were obtained in three patients (2.8% tissue specimens); a close follow-up was carried out and no recurrence was observed. SOX10 has shown a sensitivity approaching 100% for melanoma in formalin-fixed tissue. 5 Our study revealed that SOX10 reliably detects LM in FSMCT, allowing, in the majority of cases, the entire surgical procedure (FSCMT, debulking and reconstruction) to be carried out on the same day, avoiding multi-day procedures and potentially reducing costs. Our study has several limitations. It was performed in a single centre with a limited number of patients. On the other hand, the slides were not blindly assessed. In conclusion, SOX10 immunostaining in frozen tissue shows high sensitivity and specificity for the detection of LM, while reducing the inconveniences of multi-day procedures associated with conventional staged margin con-trol techniques

ABSTRACTBackgroundEx vivo confocal microscopy is a real-time technique that provides high-resolution images of fresh, non-fixed tissues, with an optical resolution comparable to conventional pathology. The objective of this study was to investigate the feasibility of using ex vivo confocal microscopy in fusion mode (FuCM) and the haematoxylin and eosin (H&E)-like digital staining that results for the analysis of basic patterns of lesion in nephropathology.MethodsForty-eight renal samples were scanned in a fourth-generation ex vivo confocal microscopy device. Samples were subjected to confocal microscopy imaging and were then processed using conventional pathology techniques. Concordance between the techniques was evaluated by means of the percentage of agreement and the κ index.ResultsAgreement between conventional microscopy and H&E-like digital staining was strong (κ = 0.88) in the evaluation of acute tubular damage and was substantial (κ = 0.79) in the evaluation of interstitial fibrosis, interstitial inflammation, arterial and arteriolar lesions. H&E-like digital staining also allows rapid identification of extracapillary proliferation (κ = 0.88), necrosis and segmental sclerosis (κ = .88) in the glomerular compartment, but the results reported here are limited because of the small number of cases with these glomerular findings.ConclusionsFuCM proved to be as effective as conventional techniques in evaluating the presence of acute tubular necrosis and interstitial fibrosis changes, but in fresh tissue. The ease of acquisition of ex vivo confocal microscopy images suggests that FuCM may be useful for rapid evaluation of kidney biopsies and to restructure the clinical workflow in renal histopathology.

PDF file - 6167K, (A) Flow cytometry analysis of cyclin D1 and p27KIP1 intracellular levels in a set of primary MCL samples (n=10, Table 1). In gray, isotypic control curves; in black, cyclin D1 and p27KIP1 curves. r indicates x-mean cyclin D1 or p27KIP1-Alexa-Fluor 647/xmean control ratio. (B) Semi-log curves time/f(p27KIP1 or cyclin D1/β-actin or β-tubulin) ratio deduced from densitometric analysis of cyclin D1 and p27KIP1 protein levels by Western blot in 4 MCL cell lines. (C) Time-dependent variation in cyclin D1 and p27KIP1 protein levels by SDS-PAGE in Ramos cells exposed for 3, 6 and 24 h to TAT-cyclin D1 fusion protein. β-tubulin was used as loading control.

1 - Serum and tumor cytokines according to MYD88 mutational status 2- Discrepancies in cell of origin distribution between gene expression and immunohistochemistry

p53 immunohistochemistry (IHC) has been proposed as a surrogate for TP53 mutations in penile squamous cell carcinomas (PSCC). We aimed to evaluate the performance of a pattern-based evaluation of p53 IHC in PSCC. Human papilloma virus (HPV) DNA testing, p16 and p53 IHC, and whole exome sequencing were performed in a series of 40 PSCC. p53 IHC was evaluated following a pattern-based framework and conventional p53 IHC evaluation. Out of 40 PSCC, 12 (30.0%) were HPV-associated, and 28 (70.0%) were HPV-independent. The agreement between the p53 IHC pattern-based evaluation and TP53 mutational status was almost perfect (k = 0.85). The sensitivity and accuracy of the pattern-based framework for identifying TP53 mutations were 95.5% and 92.5%, respectively, which were higher than the values of conventional p53 IHC interpretation (54.5% and 70.0%, respectively), whereas the specificity was the same (88.9%). In conclusions, the pattern-based framework improves the accuracy of detecting TP53 mutations in PSCC compared to the classical p53 IHC evaluation.

Topic: 24. Gene therapy, cellular immunotherapy and vaccination - Biology & Translational Research Background: Chimeric antigen receptor (CAR) T-cell therapies have achieved outstanding results in relapsed or refractory (R/R) B-cell malignancies, including multiple myeloma. However, none has been yet approved for other haematological malignancies. CD84 (SLAMF5) is a SLAM family immunoreceptor that is expressed in several immune cells and it has been reported to be overexpressed in chronic lymphocytic leukaemia (CLL). Aims: Validating CD84 as a target for haematological malignancies, in concrete, for acute myeloid leukaemia (AML) and T-cell acute lymphoblastic leukaemia (T-ALL). Engineer a first-in-class CD84-targeted CAR T-cell therapy (CART84) for the treatment of multiple haematological malignancies. Methods: CD84 expression was studied in immune cells (among them, in hematopoietic stem/progenitor cells (HSPC)) and in malignant cells by flow cytometry; and in human tissues by immunohistochemical staining. Second generation CART84 were designed with different murine or fully human anti-CD84 scFv and a 4-1BB as a costimulatory domain. CART84 in vitro efficacy was determined by cytotoxicity, cytokine production and proliferation assays. Potential myelotoxicity of CART84 was studied by cytotoxicity assays towards CD34+HSPC. CART84 in vivo efficacy was studied in immunocompromised NSG mice using AML and T-ALL cell lines or patient derived-xenografts (PDX). Results: We found that CD84 is highly expressed on the surface of primary patient malignant cells from CLL, AML and T-ALL, whereas its expression was low on CD34+ HSPC. Immunohistochemistry analysis of healthy human tissues revealed that CD84 is not expressed in lung, liver, kidney, myocardium, skin, or brain (cerebral cortex and cerebellum), although expression was detected in tissue macrophages. This expression pattern is similar to that of CD123, the target of several CAR T cells in clinical development. CART84 successfully expanded in vitro and exerted high cytotoxicity towards cell lines from different haematological malignancies including aggressive B-cell lymphoma (Ramos), AML (MOLM-13) and T-ALL (MOLT-4) (Figure 1.A). CART84 CD84.02, CD84.03, and CD84.05 showed specific proliferation and cytokine secretion when co-cultured with CD84-positive cells in vitro. The cytotoxic effect of CART84 against CD34+ HSPC was much lower than that against leukemic cells. Despite low cytotoxicity towards HSPC, we decided to focus on AML and T-ALL, since these patients can be rescued with an allogeneic hematopoietic stem cell transplant (allo-HSCT). In an immunodeficient NSG mouse model, CART84 CD84.02 and CD84.03 controlled both MOLM-13 (AML) and MOLT-4 (T-ALL) tumour progression and increased mice survival in comparison with the control group (Figure 1.B). Similarly, positive results were obtained in AML and T-ALL PDX models. Summary/Conclusion: CD84 is a promising CART target for the treatment of several haematological malignancies. CART84 displayed high cytotoxicity towards AML and T-ALL cells both in vitro and in vivo. Our data support the therapeutic use of CART84 for haematological malignancies. However, due to its potential myelotoxicity, we encourage it as a bridge therapy to allo-HSCT for patients with R/R AML and T-ALL.Keywords: CAR-T, T cell acute lymphoblastic leukemia, Acute myeloid leukemia

PDF file - 3780K, (A) Accumulation of p27KIP1 and activated capase-3 in cytosol-enriched protein extracts from Rec-1 cells cultured for 72 h with 1 μmol/L lenalidomide and subjected to a Human apoptosis antibody array (R&D Systems).