FFPE Samples Research Articles

Benefits of FAIMS to Improve the Proteome Coverage of Deteriorated and/or Cross-Linked TMT 10-Plex FFPE Tissue and Plasma-Derived Exosomes Samples.

The proteome characterization of complex, deteriorated, or cross-linked protein mixtures as paired clinical FFPE or exosome samples isolated from low plasma volumes (250 µL) might be a challenge. In this work, we aimed at investigating the benefits of FAIMS technology coupled to the Orbitrap Exploris 480 mass spectrometer for the TMT quantitative proteomics analyses of these complex samples in comparison to the analysis of protein extracts from cells, frozen tissue, and exosomes isolated from large volume plasma samples (3 mL). TMT experiments were performed using a two-hour gradient LC-MS/MS with or without FAIMS and two compensation voltages (CV = -45 and CV = -60). In the TMT experiments of cells, frozen tissue, or exosomes isolated from large plasma volumes (3 mL) with FAIMS, a limited increase in the number of identified and quantified proteins accompanied by a decrease in the number of peptides identified and quantified was observed. However, we demonstrated here a noticeable improvement (>100%) in the number of peptide and protein identifications and quantifications for the plasma exosomes isolated from low plasma volumes (250 µL) and FFPE tissue samples in TMT experiments with FAIMS in comparison to the LC-MS/MS analysis without FAIMS. Our results highlight the potential of mass spectrometry analyses with FAIMS to increase the depth into the proteome of complex samples derived from deteriorated, cross-linked samples and/or those where the material was scarce, such as FFPE and plasma-derived exosomes from low plasma volumes (250 µL), which might aid in the characterization of their proteome and proteoforms and in the identification of dysregulated proteins that could be used as biomarkers.

160P CICLADES-CE study: Genomic signatures detected in DNA from FFPE samples of patients with advanced or metastatic breast cancers treated with anti-aromatase and CDK4/6 inhibitors

160P CICLADES-CE study: Genomic signatures detected in DNA from FFPE samples of patients with advanced or metastatic breast cancers treated with anti-aromatase and CDK4/6 inhibitors

Two Oncoviruses of HPV and EBV in Breast Cancer: An Iran-based Study

OBJECTIVE: To answer the question of whether viral infections such as Human papillomavirus (HPV) and Epstein-Barr virus (EBV) are involved in the occurrence of breast cancer or not. METHODOLOGY: In this case-control study, 50 paraffin-embedded breast samples (FFPE) and 50 non- cancerous FFPE samples were prepared from patients referred to Abadan hospitals from 2018 to 2021 to identify the genome of HPV and EBV by polymerase chain reaction (PCR). Data were analyzed using SPSS 15 with P-values of 0.05. Confirmed breast cancer biopsy samples with no medical history of chemotherapy and/or radiotherapy, the patient not being pregnant at the time of sampling, not using anti-cancer treatments, and not suffering from systemic inflammatory diseases such as rheumatoid arthritis. Patients of any age, with any breast cancer, and any size or stage of tumor were included without restrictions. RESULTS: Using PCR, HPV DNA was detected in 7/50 (14%) and 2/50(4%) of breast cancers and normal control, respectively. Moreover, 71.42% and 28.57% of HPV-infected persons in the patient's group were genotypes 16 and 18, respectively. All HPV-infected people in the control group were low-risk genotypes 26 and 53. Also, EBV DNA was found in 2/50 (4%) and 6/50 (12%) of breast cancers and control samples, respectively. Co-infection was not detected in samples. CONCLUSION: Although our study does not provide substantial Evidence about the role of viral infection in the progressiveness of breast tumors, significant studies have shown the critical role of these infections in the progression of breast tumors. KEYWORDS: Human Papillomavirus, HPV, Epstein–Barr virus, EBV, breast cancer

Validation of the Xpert Breast Cancer STRAT 4 Assay on the GeneXpert instrument to Assess Hormone Receptor, Ki67, and HER2 Gene Expression Status in Breast Cancer Tissue Samples.

Breast cancer is the commonest cause of cancer-related mortality in African females where patients often present later and with advanced disease. Causes for delayed diagnosis include restricted diagnostic access and international controversy on interpretation of ancillary tests like immunohistochemistry (IHC). Fine needle aspirates (FNAC) are an attractive alternative although may have reduced sensitivity. The Xpert Breast Cancer STRAT4 (STRAT4) (CE-IVD*) assay (Cepheid, Sunnyvale) is a semi-quantitative reverse-transcription polymerase chain reaction assay which detects messenger RNA (mRNA) expression in breast samples for estrogen receptor ( ESR1 ), progesterone receptor ( PGR1 ), human epidermal growth factor receptor/Erb-B2 receptor tyrosine kinase 2 (HER2/ ERBB2 ) and the proliferation marker, MKi67 . We assessed the performance of this assay on both formalin-fixed paraffin-embedded (FFPE, n=31) and matched FNAC (n=20) samples from patients presenting with breast cancer to the Johannesburg academic hospitals. IHC and Fluorescent in situ hybridization analysis (performed on HER2-indeterminate samples) was compared with the mRNA expression of the corresponding target genes in FFPE samples, and mRNA expression on FNAC samples was compared with the FFPE results for both mRNA expression and IHC. Concordance between IHC/FISH and Xpert Breast Cancer STRAT4 in FFPE and FNAC samples using the Quick lysis (Q) method (a research-use-only modification of the validated FFPE-lysis method), showed an overall percentage agreement for ESR1 expression of 90.3% and 81.3%, and for PGR1 expression at 86.7% and 81.3% respectively in FFPE and FNAC samples. Concordance was lowest for Ki67 expression, using a binary IHC cutoff for Ki67 positivity at ≥20% staining) at 83.9% and 62.5%, for FFPE and FNAC samples, respectively. This suggests that the STRAT4 assay may be a useful ancillary test in determining HR and Ki67 status in FFPE samples and that use on FNAC samples may be feasible. Future studies should expand the sample numbers and establish locally relevant cutoffs.

OS10.8.A APPLICATIONS OF NOVEL FFPE BASED TECHNOLOGIES FOR THE DIAGNOSTICS OF GLIOMAS

Abstract BACKGROUND Due to the lack of consistent tumour-cell specific markers, the diffuse brain invasion of glioblastoma (GB) presents a significant diagnostic challenge, especially for specimens with a low tumour cell fraction or scarce tissue. The only common alteration found in most GB is the gain of chromosome 7 and loss of chromosome 10. The emergence of new technologies such as spatial and single nucleus transcriptomics that allow for detection of copy number alterations (CNVs) may allow us to push the diagnostic boundaries. MATERIAL AND METHODS We performed 10x Visium spatial transcriptomic analysis of FFPE tissue from bulk resections of 14 FFPE GB and stereotactic biopsies of 6 GB, 7 IDH mutant astrocytomas, 4 oligodendrogliomas and 1 diffuse glioma NOS. Visium tissue spots were then deconvolved using data from single nucleus transcriptomic data from nuclei isolated from FFPE tissue. Fresh frozen and FFPE single nucleus transcriptomic libraries were also generated from 4 GB with matching fresh-frozen and FFPE tissue available. RESULTS The infiltration zone, the tumour core and healthy brain tissue could be differentiated using the inferred CNVs of chromosome 7 and chromosome 10. Additionally, inferred CNVs of tissue fragments smaller than 1 mm2 were in accordance with Infinium MethylationEPIC array analysis (a well-established method for CNV detection) from the same tumour tissue. Using the spatial transcriptomics approach, the loss of chromosome 1p and 19q, which is characteristic for oligodendroglioma was also detected. To overcome the low resolution of Visium, deconvolution approaches using a single cell dataset can be used to determine the tumour content of a given tissue more precisely. To test if the single nuclei data from FFPE samples is suitable for deconvolution of spatial transcriptomics, we compared fresh-frozen and FFPE single nuclei data from matching tumours. As expected, FFPE and fresh frozen nuclei clustered together according to cell types and tumours in the UMAP space. CONCLUSION Spatial transcriptomics can be useful for brain tumour diagnostic workup, especially in cases where a limited amount of tissue prevents analysis by standard molecular diagnostic METHODS . Additionally, CNVs inferred from the spatial data as well as the spatial expression of marker genes help to better distinguish tumour infiltration zones from the core tumour and healthy brain tissue in the case of GB. This analysis can be further supported by deconvolution with a reference set generated from single nucleus transcriptomic analysis of FFPE tissue.

JS04.7.A ANALYSIS OF RNA CLASSIFIES NEWLY DIAGNOSED GLIOBLASTOMA PATIENTS AND IDENTIFIES PATIENTS VULNERABLE TO TARGETED METABOLIC THERAPIES

Abstract BACKGROUND Recent computational analysis of glioblastoma (GBM) single-cell RNA-sequencing data and bulk tumors identified four stable cellular states that embody metabolic (mitochondrial and glycolytic/plurimetabolic) and developmental (neuronal and proliferative/progenitor) attributes and generated a new GBM classification. Interestingly, the mitochondrial subtype depends on oxidative phosphorylation (OXPHOS), stratifies patients with specific metabolic pharmacologic vulnerabilities. Here, we set up and optimized RNA Sequencing of frozen and FFPE samples of GBM to apply the new proposed classification to a retrospective cohort of IDH wild type GBM patients. METHODS Tumor samples from IDH wild-type (wt) GBM patients diagnosed in Pitiè-Salpétrière Hospital from 2007 and 2019 and treated by first line standard radio-chemotherapy were analyzed by 3’ mRNA NGS. RESULTS Two hundreds thirty-two newly diagnosed (232) GBM IDH patients were analyzed. Median age was 60 years old (yo) (range 31-87 yo), median KPS at diagnosis was 80, Male/female ratio 1.5, pMGMT resulted hypermethylated in 48 out of 115 tested samples (42%). RNA analysis classified patients into molecular subgroups as it follows: 60 OXPHOS+, 48 Neuronal, 55 Proliferative and 58 Glycolytic; 11 unclassifiable. A subgroup of 28 patients was analyzed by RNA Sequencing from both FFPE and frozen tumors with concordant results in 25 patients (89%). Since F3T3 gene fusions correlate with hyperexpression of mitochondrial markers, we separately analyzed a subgroup of 20 IDHwt F3T3+ GBM patients. We found that F3T3+ are most frequently OXPHOS+ (50%) but not exclusively. Survival analysis shows significant longer overall survival (OS) of OXPHOS+F3T3- patients (22.9 months) compared to OXPHOS-F3T3-(18.4 months), (p=0.045) [including Neuronal patients (OS 18.4 months), Glycolytic (18.2 m), Proliferative 18.6 m)]. PFS after first line radio-chemotherapy did not differ significantly (p=0.11) between subgroups. Among F3T3+ patients, OXPHOS+ were long survivors with a median OS of 40.1 months while Glycolytic, Neuronal and Proliferative F3T3+ patients showed a worse prognosis similar to F3T3- patients (median OS 17.3 months; p=0.01). CONCLUSIONS In conclusions the RNA expression analysis classifies GBM patients from frozen and FFPE samples in metabolic subgroups. We identified OXPHOS+ GBM patients with more indolent clinical behavior compared to Glycolytic and other subgroups and with specific vulnerabilities to metabolic manipulations, as in the case of OXPHOS+ patients that could benefit from targeted metabolic therapies. RNA sequencing of FFPE is feasible in the setting of a prospectively clinical trial, aiming to select OXPHOS+ patients among newly diagnosed GBM patients, eligible for mitochondrial inhibitors add-on to first-line standard radio-chemotherapy (Optimum trial, NCT04945148).

P10.12.B MOLECULAR MECHANISMS INVOLVED IN THE DEVELOPMENT OF BRAIN METASTASIS FROM THE MATCHED PAIRED LUNG ADENOCARCINOMA AND BREAST CANCER

Abstract BACKGROUND Up to 50% of patients with common cancers will develop brain metastases and incidences of this devastating complication are still rising. In our recent work, we found that the immune response in the brain is significantly stronger when the metastasis develops from lung adenocarcinoma (LUAD) compared to breast cancer (BC). Importantly, we discovered the higher expression of the drug target, VISTA, only in the brain metastasis of the LUAD. The aim of the current study was to investigate the molecular mechanisms driving the formation of brain metastasis from the two different lineages of cancer. In addition, we aimed to examine the expression level of the drugable molecules between primary tumors and their brain metastasis. MATERIAL AND METHODS Primary tumor and their matched brain metastasis samples of 11 LUAD and 11 breast cancer (total = 44 samples) were included in this study. RNA was extracted from the FFPE samples and the targeted gene expression profiles were measured using the PanCancer IO360™ Panel that includes 770 cancer-related genes (NanoString technology). Data were analysed using the nSolver software. RESULTS A first comparison was made between the primary tumors with their brain metastases for LUAD and BC separately. The second comparison was made between the common genes of both lineages. Pathway analysis indicated that there is higher metabolic stress in breast cancer-brain metastasis compared to that in the lung. In addition, we identified 12 common up-regulated genes in brain metastasis, despite the origin of the primary tumor. Importantly, by comparing the primary tumors of BC and LUAD with their brain metastasis, we found that VISTA is predominantly highly expressed in the primary tumors of LUAD and their matched brain metastasis. CONCLUSION Although the general mechanisms of metastasis have been described, our study demonstrates that different cancer lineages utilize various genes/pathways to metastasize to the brain. Moreover, primary LUAD and their brain metastasis express high levels of VISTA highlighting the possibilities for targeted therapy.

A qPCR technology for direct quantification of methylation in untreated DNA

DNA methylation is important for gene expression and alterations in DNA methylation are involved in the development and progression of cancer and other major diseases. Analysis of DNA methylation patterns has until now been dependent on either a chemical or an enzymatic pre-treatment, which are both time consuming procedures and potentially biased due to incomplete treatment. We present a qPCR technology, EpiDirect®, that allows for direct PCR quantification of DNA methylations using untreated DNA. EpiDirect® is based on the ability of Intercalating Nucleic Acids (INA®) to differentiate between methylated and unmethylated cytosines in a special primer design. With this technology, we develop an assay to analyze the methylation status of a region of the MGMT promoter used in treatment selection and prognosis of glioblastoma patients. We compare the assay to two bisulfite-relying, methyl-specific PCR assays in a study involving 42 brain tumor FFPE samples, revealing high sensitivity, specificity, and the clinical utility of the method.

Clinical importance of the range of detectable variants between the Oncomine Dx target test and a conventional single-gene test for EGFR mutation

Although we have experienced some cases with discordant results between the Oncomine Dx target test (ODxTT) and conventional single gene tests for detecting EGFR alterations, the clinical efficacy of EGFR-TKIs in these discordant cases remains little known. We retrospectively reviewed consecutive patients with non-small-cell lung cancer whose FFPE samples were simultaneously submitted for the ODxTT, and a PNA-LNA PCR clamp test. We evaluated the clinical efficacy of EGFR-TKIs in patients with discordant results between the two tests, focusing on the common EGFR mutations. Among 444 successful results, 10 patients had discordant results for common EGFR mutations (9 Ex 19 deletion and 1 Ex 21 L858R mutation), and all of these were detected only by the PNA-LNA PCR clamp test. Among six discordant cases treated with EGFR-TKI, the mutations detected in 3 patients were not included in the list of detectable variants that are reportable by the ODxTT, while the mutations detected in the other 3 patients were included in the list. For all three discordant cases harboring the mutations not reportable by the ODxTT, good clinical responses were demonstrated. However, among the other three discordant cases harboring the mutations reportable by the ODxTT, only one patient had a clinical response with short duration. Among the discordant cases for common EGFR mutations between the ODxTT and the conventional single gene test, there are a certain number of suitable patients responsive to EGFR-TKIs, especially when the cause of the discordant results comes from the difference in the range of detectable variants that are reportable between the tests.

Effect of spatial transcriptomics on tumor immune microenvironment resistant to immune checkpoint inhibitors in metastatic urothelial carcinoma.

68 Background: Immune checkpoint inhibitors (ICBs) for metastatic urothelial carcinoma (mUC) are widely used, but their response rates are inadequate, around 20%. Currently, analyzing samples prior to ICB administration has accumulated so far; thereby predicting response to ICBs by high tumor mutation burden levels and PD-L1 positivity. However, little is known about what kind of tumor microenvironment is formed in recurrent or residual tumors post ICBs, and how it contributes to ICB resistance. Therefore, we applied large datasets of spatial transcriptome analysis to mUC samples post ICBs in order to reveal the diversity of the tumor microenvironment after ICB administration. Methods: The Visium spatial transcriptome analysis was performed on FFPE samples from 12 mUC patients with viable tumor resection after ICB administration. Further, 4 FFPE samples of non-metastatic UC treated-surgically were subjected to spatial transcriptome analysis and were used as ICB-naïve tumor controls. First, the tumor and stromal regions were pathologically annotated. We then identified CD4-positive, CD8A-positive, and FOXP3-positive spots in both tumor and stromal regions, respectively, and analyzed changes in differentially expressed genes (DEGs) and pathways in the tumor space where cancer cells survive after ICB treatment. Results: For DEGs and pathway analysis, 8082 spots in the tumor region and 10964 spots in the stromal region were used. In the tumor and stromal areas, 15.4% and 15.1% were CD4 positive, 6.5% and 6.3% were CD8A positive, and 2.9% and 2.0% were FOXP3 positive, respectively. In tumor regions of ICB-treated specimens, the expression of SULF1, an extracellular matrix-related gene, was increased in all CD4-, CD8A-, and FOXP3-positive spots compared to negative spots. In addition, the expression of complement-related genes such as C1QB, C1QC, and C1R was upregulated in CD8A-positive spots in the tumor regions after ICB. Pathway analysis revealed that oxidative phosphorylation was enriched in CD8A-positive spots in tumor regions prior to ICB, while angiogenesis was enriched in CD8A-positive spots after ICB. Conclusions: Spatial transcriptome analysis of a large dataset of UC samples showed distinct features of immune infiltration involved in therapeutic resistance in the tumor microenvironment under immunotherapy. Notably, T-cells may be exhausted by metabolic changes in CD8A-positive spots in ICB-refractory tumor environment spaces.

Formalin-Fixed, Paraffin-Embedded–Targeted Locus Capture: A Next-Generation Sequencing Technology for Accurate DNA-Based Gene Fusion Detection in Bone and Soft Tissue Tumors

Chromosomal rearrangements are important drivers in cancer and their robust detection is essential for diagnosis, prognosis and treatment selection, particularly for bone and soft tissue tumors. Current diagnostic methods are hindered by limitations including difficulties with multiplexing targets and poor quality of RNA. A novel targeted DNA-based NGS method 'FFPE-TLC' has shown advantages over current diagnostic methods when applied on FFPE lymphomas, including the ability to detect novel rearrangements. Here, the utility of FFPE-TLC in bone and soft tissue tumor diagnostics was evaluated. FFPE-TLC sequencing was successfully applied on non- and decalcified FFPE samples (n=44) and control samples (n=19). In total, 58 rearrangements were identified in 40 FFPE tumor samples, including three previously negative samples and none in the FFPE control samples. In all five discordant cases, FFPE-TLC could identify gene fusions where other methods had failed either due to detection limits or poor sample quality. FFPE-TLC achieved a high specificity and sensitivity (no false positives and negatives). These results indicate that FFPE-TLC is applicable in cancer diagnostics to simultaneously analyze many genes for their involvement in gene fusions. Similar to the observation in lymphomas, FFPE-TLC is a good DNA-based alternative to the conventional methods for detection of rearrangements in bone and soft tissue tumors.

Development of the NOGGO GIS v1 Assay, a Comprehensive Hybrid-Capture-Based NGS Assay for Therapeutic Stratification of Homologous Repair Deficiency Driven Tumors and Clinical Validation.

The worldwide approval of the combination maintenance therapy of olaparib and bevacizumab in advanced high-grade serous ovarian cancer requires complex molecular diagnostic assays that are sufficiently robust for the routine detection of driver mutations in homologous recombination repair (HRR) genes and genomic instability (GI), employing formalin-fixed (FFPE) paraffin-embedded tumor samples without matched normal tissue. We therefore established a DNA-based hybrid capture NGS assay and an associated bioinformatic pipeline that fulfils our institution's specific needs. The assay´s target regions cover the full exonic territory of relevant cancer-related genes and HRR genes and more than 20,000 evenly distributed single nucleotide polymorphism (SNP) loci to allow for the detection of genome-wide allele specific copy number alterations (CNA). To determine GI status, we implemented an %CNA score that is robust across a broad range of tumor cell content (25-85%) often found in routine FFPE samples. The assay was established using high-grade serous ovarian cancer samples for which BRCA1 and BRCA2 mutation status as well as Myriad MyChoice homologous repair deficiency (HRD) status was known. The NOGGO (Northeastern German Society for Gynecologic Oncology) GIS (GI-Score) v1 assay was clinically validated on more than 400 samples of the ENGOT PAOLA-1 clinical trial as part of the European Network for Gynaecological Oncological Trial groups (ENGOT) HRD European Initiative. The "NOGGO GIS v1 assay" performed using highly robust hazard ratios for progression-free survival (PFS) and overall survival (OS), as well a significantly lower dropout rate than the Myriad MyChoice clinical trial assay supporting the clinical utility of the assay. We also provide proof of a modular and scalable routine diagnostic method, that can be flexibly adapted and adjusted to meet future clinical needs, emerging biomarkers, and further tumor entities.

Multi-dimensional characterization of immunological profiles in small cell lung cancer uncovers clinically relevant immune subtypes with distinct prognoses and therapeutic vulnerabilities

Small-cell lung cancer (SCLC) is generally considered a 'homogenous' disease, with little documented inter-tumor heterogeneity in treatment guidance or prognosis evaluation. The precise identification of clinically relevant molecular subtypes remains incomplete and their translation into clinical practice is limited. In this retrospective cohort study, we comprehensively characterized the immune microenvironment in SCLC by integrating transcriptional and protein profiling of formalin-fixation-and-paraffin-embedded (FFPE) samples from 29 patients. We identified two distinct disease subtypes: immune-enriched (IE-subtype) and immune-deprived (ID-subtype), displaying heterogeneity in immunological, biological, and clinical features. The IE-subtype was characterized by abundant immune infiltrate and elevated levels of interferon-alpha/gamma (IFNα/IFNγ) and inflammatory response, while the ID-subtype featured a complete lack of immune infiltration and a more proliferative phenotype. These two immune subtypes are associated with clinical benefits in SCLC patients treated with adjuvant therapy, with the IE-subtype exhibiting a more favorable response leading to improved survival and reduced disease recurrence risk. Additionally, we identified and validated a personalized prognosticator of immunophenotyping, the CCL5/CXCL9 chemokine index (CCI), using machine learning. The CCI demonstrated superior predictive abilities for prognosis and clinical benefits in SCLC patients, validated in our institute immunohistochemistry cohort and multicenter bulk transcriptomic data cohorts. In conclusion, our study provides a comprehensive and multi-dimensional characterization of the immune architecture of SCLC using clinical FFPE samples and proposes a new immune subtyping conceptual framework enabling risk stratification and the appropriate selection of individualized therapy.

A Comparison of Two Different FFPE Tissue Dissection Methods for Routine Diagnostics in Molecular Pathology: Manual Macrodissection versus Automated Microdissection Using the Roche "AVENIO Millisect" System.

Currently, in routine diagnostics, most molecular testing is performed on formalin-fixed, paraffin-embedded tissue after a histomorphological assessment. In order to find the best possible and targeted individual therapy, knowing the mutational status of the tumour is crucial. The "AVENIO Millisect" system Roche introduced an automation solution for the dissection of tissue on slides. This technology allows the precise and fully automated dissection of the tumour area without wasting limited and valuable patient material. In this study, the digitally guided microdissection was directly compared to the manual macrodissection regarding the precision and duration of the procedure, their DNA concentrations as well as DNA qualities, and the overall costs in 24 FFPE samples. In 21 of 24 cases (87.5%), the DNA yields of the manually dissected samples were higher in comparison to the automatically dissected samples. Shorter execution times and lower costs were also benefits of the manual scraping process. Nevertheless, the DNA quality achieved with both methods was comparable, which is essential for further molecular testing. Therefore, it could be used as an additional tool for precise tumour enrichment.

MDB-25. LARGE CELL/ANAPLASTIC MEDULLOBLASTOMA REPRESENTS MORE THAN ONE DISEASE ENTITY

Abstract Medulloblastoma (MB) with predominant large cell or anaplastic cytological features have been identified to be associated to an aggressive clinical course and poor survival. Because cases can show mixed cytologies, these histologically defined MB types were merged in the recent WHO classification of tumors of the CNS to large cell/anaplastic (LC/A) MB. In this study, we collected FFPE samples of 137 LC/A MB of all age groups (0-39 years) diagnosed at the DGNN Brain Tumor Reference Center in Bonn, Germany, and performed a comprehensive histological, immunophenotypical, genetic and epigenetic analysis by next-generation panel sequencing (NGS), 450k/850k methylation bead-array hybridization and molecular inversion probe (MIP) technology. 73% of cases showed predominant anaplastic histology, 27% predominant large cell histology. Methylation-based subtyping and sequencing showed that the cohort of LC/A MB represented different biological entities: 6% WNT-MB, 7% SHH-MB-TP53-wildtype, 10% SHH-MB-TP53-mutant, and 77% non-WNT/non-SHH MB (with predominant epigenetic subtype II). High-resolution copy number profiling by MIP demonstrated MYC amplification in 40% of non-WNT/non-SHH MB. MYC amplification was highly associated to infant disease and predominant large cell histology. On the contrary, predominant anaplastic histology was highly associated to non-infant disease, MYCN amplification and SHH activation. By NGS we detected recurrent mutations in 37 MB-related genes; the frequency of mutations of individual MB-related genes differed significantly between the histologically and genetically defined MB types. Our data clearly demonstrate that large cell MB and anaplastic MB represent different biological entities. Therefore, and also in respect to the future development of targeted treatment approaches to these highly malignant MB types, they should be precisely diagnosed as separate MB entities.

Optimizing identification of consensus molecular subtypes in muscle-invasive bladder cancer: a comparison of two sequencing methods and gene sets using FFPE specimens

BackgroundMolecular subtypes predict prognosis in muscle-invasive bladder cancer (MIBC) and are explored as predictive markers. To provide a common base for molecular subtyping and facilitate clinical applications, a consensus classification has been developed. However, methods to determine consensus molecular subtypes require validation, particularly when FFPE specimens are used. Here, we aimed to evaluate two gene expression analysis methods on FFPE samples and to compare reduced gene sets to classify tumors into molecular subtypes.MethodsRNA was isolated from FFPE blocks of 15 MIBC patients. Massive analysis of 3’ cDNA ends (MACE) and the HTG transcriptome panel (HTP) were used to retrieve gene expression. We used normalized, log2-transformed data to call consensus and TCGA subtypes with the consensusMIBC package for R using all available genes, a 68-gene panel (ESSEN1), and a 48-gene panel (ESSEN2).ResultsFifteen MACE-samples and 14 HTP-samples were available for molecular subtyping. The 14 samples were classified as Ba/Sq in 7 (50%), LumP in 2 (14.3%), LumU in 1 (7.1%), LumNS in 1 (7.1%), stroma-rich in 2 (14.3%) and NE-like in 1 (7.1%) case based on MACE- or HTP-derived transcriptome data. Consensus subtypes were concordant in 71% (10/14) of cases when comparing MACE with HTP data. Four cases with aberrant subtypes had a stroma-rich molecular subtype with either method. The overlap of the molecular consensus subtypes with the reduced ESSEN1 and ESSEN2 panels were 86% and 100%, respectively, with HTP data and 86% with MACE data.ConclusionDetermination of consensus molecular subtypes of MIBC from FFPE samples is feasible using various RNA sequencing methods. Inconsistent classification mainly involves the stroma-rich molecular subtype, which may be the consequence of sample heterogeneity with (stroma)-cell sampling bias and highlights the limitations of bulk RNA-based subclassification. Classification is still reliable when analysis is reduced to selected genes.

Novel LTK fusions based on NGS sequencing data in Chinese patients with solid tumors.

3144 Background: Leukocyte receptor tyrosine kinase ( LTK) gene has the same kinase domain (PTKc_ALK_LTK) as ALK, and could functionally response to ALK inhibitors such as lorlatinib. Previous study reported that CLIP1-LTK fusion was a novel oncogenic driver mutation in lung cancer (occur in 0.4% of non-small cell lung cancer), which was sensitive to the ALK tyrosine kinase inhibitor lorlatinib. So far, LTK fusion has been rarely reported. Methods: A total of 11 Chinese solid tumor patients were reviewed, including 6 cases of LTK positive rearrangements in DNA and 5 cases of LTK positive fusion in RNA. FFPE samples were collected for NGS-based 450 cancer gene panel detection in both DNA and RNA level, and matched blood samples were used as normal control to filter out germline mutations. LTK fusions were analyzed by OrigiFus algorithm. Results: We found 6 patients with LTK rearrangements by DNA-based NGS detection, including lung adenocarcinoma, colorectal cancer, stomach cancer, spindle cell sarcoma of small intestine, adenocarcinoma of the pancreas, and cancer of unknown primary, and 5 soft tissue sarcoma patients with RPAP1 E25- LTK E2 fusions by RNA-based NGS detection. We analyzed the patterns of partner genes and breakpoints of the 6 LTK rearrangements. The partner genes included NUSAP1 (n=2), NDUFAF1 (n=1), LOC105370802 (n=1), EHD4 (n=1), and AGBL1 (n=1). All these partner genes were located on the same chromosome as LTK, rather than CLIP1, which was identified as a translocation. The breakpoints on LTK included exon20 (n=2), intron1 (n=2), exon6 (n=1), and exon10 (n=1). Five of the 6 fusions consisted of a complete PTKc_ALK_LTK domain. Among the 6 LTK rearrangements, LTK E20- NUSAP1 E2 was predicted as a fusion. Interestingly, this clinical real-world sample also harbored a classic NTRK1 fusion, LMNA E2- NTRK1 E11. The other NUSAP1- LTK was predicted to have a transcript of 3’-3’, but one of the breakpoints was observed at LTK exon6, which could be located at the kinase domain, so the possibility of eventual fusion cannot be excluded. The two NUSAP1-LTK fusions were found in a stomach tumor and a soft tissue sarcoma, respectively. One non-small-cell lung cancer (NSCLC) patient had a breakpoint detected on LTK exon10, which is the same as the exon of CLIP1- LTK reported. Conclusions: This study revealed novel LTK fusions/rearrangements and their cancer distribution in Chinese patients with solid tumors. We gave the details of these fusions/rearrangements which enrich our understanding of the LTK fusion. Although the small number of cases, our analysis promoted the attention on LTK and its potential value of drug research and clinical treatment in pan-cancer.

17th International Conference on Malignant Lymphoma, Palazzo dei Congressi, Lugano, Switzerland, 13 - 17 June, 2023.

Introduction: DLBCL patients treated with R-CHOP regimen have prolonged survival, but 20%–40% of patients survive less than 5 years. Autoantibodies (AAbs) have the advantages of easy specimen acquisition and dynamic real-time monitoring. Blood-based autoantibodies have stratified lymphoma patients outcomes in previous studies, such as anti-ALK autoantibody titers were found to predict anaplastic large B-cell lymphoma (ALCL) recurrence risk stratification. This study was designed to find prognostic AAbs biomarkers for early progression, PFS, and OS in DLBCL patients treated with R-CHOP by utilizing a proteomic test based on autoantibodies. Patients and methods: Plasma samples from 325 DLBCL patients and FFPE samples from 37 DLBCL patients with biopsy confirmation were retrospectively collected between 2006 and 2020. All patients were previously untreated and collected before first-line R-CHOP and had complete clinical data. Patients were divided into relapse and non-relapse groups based on EFS24. There were three cohorts in this study. In the discovery phase, 20 samples were conducted comprehensively high-throughput protein microarray (∼20 K proteins, HuProtTM). In the verification phase, 181 samples were conducted low-density focused array (200 proteins). And in the validation phase, 135 samples were conducted for ELISA detection. Besides, exploratory analyses revealed the expression difference of prognostic AAbs target proteins in DLBCL FFPE samples. Results: Global autoantibody profiling of DLBCL patients were observed. Four autoantibodies were significantly different in relapse and non-relapse patients (p < 0.05) and associated with superior survival outcomes (PFS and OS) both in the verification phase (n = 181) and validation phase (n = 135). Based on the AUC for EFS24, 2 AAbs were chosen for the riskscore model conduction. 2 AAbs signature riskscore model was predictive of PFS with AUC of 0.72, 0.76 and 0.82 at 1, 3 and 5 years independent of IPI score in validation phase. Combining with the IPI score, 2 AAbs signature riskscore identified a more poor prognosis group (5-year PFS rate: 17.86%) versus IPI score high group (33.3%), a mediate group(56.49%) versus IPI score high_inter/low_inter group (46.3% and 48.2%) and a more favorable group (89%) versus IPI score low group (85.78%). Furthermore, 2 AAbs target proteins were also higher in DLBCL patients with superior outcomes (p < 0.05). Conclusions: We have demonstrated the extensive profiling of autoantibodies in DLBCL patients, and identified a novel prognostic AAbs panel and it can also contribute to improving the prognostic stratification ability of IPI score. Keywords: autoantibody, biomarker, diffuse large b-cell lymphoma, protein microarray Encore Abstract - previously submitted to regional or national meetings (up to <1000 attendees), EBMT 2023, AACR 2023, ASCO 2023, EHA 2023 The research was funded by: China National Major Project for New Drug Innovation (2017ZX09304015, 2019ZX09201-002) Keywords: aggressive B-cell non-Hodgkin lymphoma, diagnostic and prognostic biomarkers, risk models No conflicts of interests pertinent to the abstract.

Tumor immune environment in multiple primary malignancies involving colorectal cancers: Comparison with single primary colorectal cancers.

e15536 Background: Multiple primary malignancies (MPM) are characterized by two or more primary tumors in an individual patient, excluding relapse or subordinate relationship. Because the therapeutic options usually become limited, the diagnosis of MPM cause a lot of troubles to both clinicians and patients. A comprehensive investigation in comparing the tumor immune environment in MPM involving colorectal cancers (CRC) with single primary CRC will provide clues for the etiology and the management of MPM. Methods: Three patients with MPM involving CRC and twenty four patients with single primary CRC from our hospital were enrolled in the study. FFPE samples from colorectal lesions were analyzed for the tumor infiltrating lymphocytes [PD-L1+, PD-1+, CD3+, CD4+, CD8+, CD20+, FoxP3+, CD56+, CD163+, CD68+] by multiplex immunofluorescence technique. The tumor-enriched segments and stroma areas annotated by pathologists on images. Then artificial intelligence (AI) employing algorithms of image processing, machine learning, and deep learning techniques distinguished the core and peritumor areas, and calculated the density (counts per mm2) of each marker in two regions. A nonparametric approach (Kruskal–Wallis) was used to compare the density of immune cells between MPM involving CRC and single primary CRC groups. Results: The density of CD4+, CD56dim, FoxP3+ cells in patients with single primary CRC were significantly higher than those in MPM both in the stroma and tumor-enriched segments (all, p &lt; 0.05). In the tumor-enriched segments, the density of PD-1+ in patients with single primary CRC was also significantly more than that in MPM. Moreover, the density of PD-1+CD8+ cells in patients with single primary CRC showed a trend of higher density than those in MPM patients in the stroma area (p = 0.06). However, the density of PD-1+ and PD-1+CD8+ cells did not show significant differences related to cancer types (MPM and single primary CRC) in the stroma. Other indicators such as CD3+, CD20+, CD8+, CD68+CD163+, CD68+CD163-, CD56bright and PD-L1+, had no significant difference related to cancer types (MPM and single primary CRC) regardless of the area (tumor-enriched segments and stroma area). Conclusions: We identified that the tumor infiltrating lymphocytes such as CD4+ cells infiltrated more in single primary CRC than those in MPM involving CRC, suggesting that MPM involving CRC may unresponsive to immunotherapy monotherapy. With preclinical studies suggesting potential for synergistic effects, combining immunotherapies may be a promising approach to treat MPM, which requires a further validation with a larger sample size.

Tumor immune environment in colitis-associated colorectal cancers: Comparison with sporadic colorectal cancers.

e15531 Background: Persistent inflammatory diseases such as ulcerative colitis (UC) and Crohn’s disease (CD) contribute to the tumor initiation and progression in colitis-associated cancers (CAC). Intestinal damage caused by persistent inflammation promotes clonal expansion of somatic and epithelial cells and alters the immune microenvironment. A comprehensive investigation in comparing the tumor immune environment in CAC with sporadic colorectal cancers (CRC) will provide more clinical usefulness of immunoprofiling in CRC. Methods: Thirteen patients with CAC (6 with UC and 7 with CD) and 10 patients with sporadic colorectal cancer from our hospital were enrolled in the study. FFPE samples from patients were analyzed for the tumor infiltrating lymphocytes [PD-L1+, CD3+, CD4+, CD8+, CD20+, CD68+, CD163+] by multiplex immunofluorescence technique. The core and peritumor areas annotated by pathologists on images. Then artificial intelligence (AI) algorithms delineated the tumor-enriched segments and stroma, and calculated the density and percentage of each marker in two regions. A nonparametric approach (Kruskal–Wallis) was used to compare the density and percentage of immune cells between CAC and sporadic colorectal cancer groups. Results: In the stroma area, the density (counts per mm2) of PD-L1+CD68+ cells in patients with CAC were significantly higher than those in sporadic cancer patients (p &lt; 0.05). However, the density of PD-L1+CD68+ did not show significant differences related to disease types (CAC or sporadic CRC) in the tumor-enriched segments. Significantly, the density of CD3+CD4+ cells and the percentage of CD68+CD163- cells in patients with CAC showed a trend of higher density than those in sporadic cancer patients in the stroma area (all, 0.05 &lt; p &lt; 0.1). The density and the percentage of CD20+ cells in CAC patients had a higher density tendency than those in sporadic cancer patients both in the tumor-enriched segments and stroma (all, 0.05 &lt; p &lt; 0.1). Other indicators in tumor-enriched segments and stroma, such as CD3+, CD4+, CD8+, CD68+CD163+ and PD-L1+, had no significant difference between CAC and sporadic CRC patients, regardless of the percentage or the density. Conclusions: We identified that the tumor immune environment of CAC was different from that of sporadic CRC. Some tumor infiltrating lymphocytes such as PD-L1+CD68+ cells infiltrated more in CAC than those in sporadic cancer, suggesting that CAC may respond better to immunotherapy than sporadic CRC. However, this hypothesis requires a further validation with a larger sample size.