Primary testicular (PT) diffuse large B-cell lymphoma (DLBCL) is a rare and aggressive lymphoma with distinct clinical and molecular characteristics. To identify prognostic biomarkers in PT-DLBCL, in this study we analyzed DNA and RNA samples of PT-DLBCL tumors from 206 patients using next-generation sequencing platforms and assays. Genetic alteration analysis found that multiple chromosomal copy number variations (CNVs), TP53 transcript mutations with high variant allele frequency, and MCD subtype had significantly adverse prognostic effects, whereas elevated microsatellite instability had a significantly favorable prognostic effect in PT-DLBCL. Targeted RNA-seq analysis identified a PTL gene expression signature by comparing PT-DLBCL with systemic DLBCL and revealed the heterogeneity within PT-DLBCL by unsupervised clustering, which classified PT-DLBCLs into a testicular lymphoma tumor (TLT) subtype and a microenvironment (ME) subtype. The TLT subtype featured upregulation of genes functioning in DNA damage response, DNA repair, chromatin remodeling, the cell cycle, and the nucleus, and was associated with significantly poorer patient survival and higher frequencies of MYD88 mutations, multiple CNVs, MCD subtype, bulk tumors, and elderly patients in the PT-DLBCL cohort. In contrast, the ME subtype distinctively featured upregulation of various signaling pathway genes involving the tumor microenvironment and downregulation of BTK and B-cell receptor signaling genes, and was associated with significantly better clinical outcome than the TLT subtype of PT-DLBCL independently of CNVs, MCD and MYD88 mutation and than systemic DLBCL. Moreover, genomic microRNA profiling analysis identified a PTL microRNA signature significantly differentially expressed between PT-DLBCL and systemic DLBCL patients and within the PT-DLBCL cohort, and PT-DLBCL patients with higher expression of 16 PTL microRNAs (14 are testicular tissue-specific) had significantly better survival. In summary, this study revealed the molecular heterogeneity in genetic abnormalities and expression profiles of coding genes and microRNAs within the PT-DLBCL entity, and identified significant prognostic biomarkers and PTL signatures.

Self-supervised learning (SSL) automates the extraction and interpretation of histopathology features on unannotated hematoxylin-eosin-stained whole slide images (WSIs). We train an SSL Barlow Twins encoder on 435 colon adenocarcinoma WSIs from The Cancer Genome Atlas to extract features from small image patches (tiles). Leiden community detection groups tiles into histomorphological phenotype clusters (HPCs). HPC reproducibility and predictive ability for overall survival are confirmed in an independent clinical trial ( N = 1213 WSIs). This unbiased atlas results in 47 HPCs displaying unique and shared clinically significant histomorphological traits, highlighting tissue type, quantity, and architecture, especially in the context of tumor stroma. Through in-depth analyses of these HPCs, including immune landscape and gene set enrichment analyses, and associations to clinical outcomes, we shine light on the factors influencing survival and responses to treatments of standard adjuvant chemotherapy and experimental therapies. Further exploration of HPCs may unveil additional insights and aid decision-making and personalized treatments for colon cancer patients.

The benign neoplasm cardiac myxoma represents one of the hallmarks of Carney complex (CNC), a familial multiple neoplasia syndrome. About 80

Background: The TNM (tumor - node - metastasis) Evaluation Committee of Union for International Cancer Control (UICC) and College of American Pathologists (CAP) recommended to prospectively validate the cost-effective and robust tumor - stroma ratio (TSR) as an independent prognostic parameter, since high intratumor stromal percentages have previously predicted poor patient-related outcomes. Patients and methods: The ' Uniform Noting for International application of Tumor-stroma ratio as Easy Diagnostic tool ' (UNITED) study enrolled patients in 27 participating centers in 12 countries worldwide. The TSR, categorized as stromahigh ( > 50%) or stroma-low ( < 50%), was scored through standardized microscopic assessment by certi fi ed pathologists, and effect on disease-free survival (DFS) was evaluated with 3-year median follow-up. Secondary endpoints were bene fi t assessment of adjuvant chemotherapy (ACT) and overall survival (OS). Results: A total of 1537 patients were included, with 1388 eligible stage II/III patients curatively operated between 2015 and 2021. DFS was signi fi cantly shorter in stroma -high ( n = 428) than in stroma-low patients ( n = 960) (3-year rates 70% versus 83%; P < 0.001). In multivariate analysis, TSR remained an independent prognosticator for DFS ( P < 0.001, hazard ratio 1.49, 95% con fi dence interval 1.17-1.90). As secondary outcome, DFS was also worse in stage II and III stroma -high patients despite adjuvant treatment (3-year rates stage II 73% versus 92% and stage III 66% versus 80%; P = 0.008 and P = 0.011, respectively). In stage II patients not receiving ACT ( n = 322), the TSR outperformed the American Society of Clinical Oncology (ASCO) criteria in identifying patients at risk of events (event rate 21% versus 9%), with a higher discriminatory 3-year DFS rate (stroma -high 80% versus ASCO high risk 91%). A trend toward worse 5-year OS in stroma -high was noticeable (74% versus 83% stroma-low; P = 0.102). Conclusion: The multicenter UNITED study unequivocally validates the TSR as an independent prognosticator, con fi rming worse outcomes in stroma -high patients. The TSR improved current selection criteria for patients at risk of events, and stroma -high patients potentially experienced chemotherapy resistance. TSR implementation in pathology diagnostics and international guidelines is highly recommended as aid in personalized treatment.

Abstract Introduction: MYC+BCL2+ double expression (DE) is a biomarker for poor prognosis in diffuse large B-cell lymphoma (DLBCL), likely due to the cooperation between MYC and BCL2 oncoproteins in tumor cells. However, as generally MYC and BCL2 are separately assessed on two different slides by immunohistochemistry (IHC), MYC+ and BCL2+ results are from different cells. With the WHO-recommended cutoffs, ≥40% for MYC and ≥50% for BCL2 (sum < 100%), there is chance that the so-called DE comes from non-overlapping regions of the tumor (not single-cell DE). Moreover, tumor expression scores based on morphology assessment often vary among pathologists. In this study, we performed fluorescent multiplex IHC (fmIHC) using a MultiOmyxTM immunofluorescence platform (NeoGenomics Laboratories, Inc.) to determine single-cell MYC and BCL2 co-expression for prognostic analysis in DLBCL. Patients and Methods: Co-expression of MYC, BCL2, and 11 immunophenotypic markers by fmIHC was digitally quantified for 547 patients with DLBCL. Percentage of cells with MYC/BCL2 double or single expression was calculated for various cell types. Prognostic analysis was performed for de novo DLBCL cases, including 108 patients treated CHOP chemotherapy, and 356 patients treated with rituximab (R)-CHOP. Results: First, although MYC and BCL2 were expressed in multiple cell types, the vast majority (>90%) of MYC+BCL2+ DE cells were lymphoma cells. Frequencies of MYC and BCL2 co-expression were consistent with random distribution of independent MYC/BCL2 expression in positive cases. The activated B-cell-like subtype compared with the germinal center B-cell-like subtype had a significantly higher mean percentage of MYC+BCL2+ DE, but not single BCL2 or MYC expression, in PAX5+ cells. Second, in both the R-CHOP and CHOP cohorts, high percentages of DE in PAX5+ cells and in all DAPI+ cells were associated with significantly poorer survival of DLBCL patients. Very high percentage of single BCL2 expression in PAX5+ cells was also associated with poorer survival, however, only in a small number of patients in both cohorts. Only in the R-CHOP cohort, high single MYC expression in PAX5+ cells (in a significant proportion of the cohort) was associated with significantly poorer overall survival. In the R-CHOP cohort, eight cases had MYC/BCL2 double hit by FISH, including 3 cases with MYC/IGH fusions (DE) and 5 cases with MYC/non-IGH rearrangements (only one DE). The exclusion of these double hit cases did not impact the significance of DE and single MYC+ expression. Summary: True MYC+BCL2+ double protein expression at the single cell level is predominantly in lymphoma cells, and associated with significantly poor survival of DLBCL patients. Our results suggest high cutoffs for common MYC/BCL2 IHC to identify true double expressor lymphoma. Citation Format: Zijun Yidan Xu-Monette, Yong Li, Qingyan Au, Harry Nunns, Wenyu Shi, Alexandar Tzankov, Carlo Visco, Govind Bhagat, Eric Hsi, Xiaoxian X. Zhao, Karen Dybkaer, April Chiu, Wayne Tam, Youli Zu, Fredrick B. Hagemeister, Andrew Song, Michele Pellegrino, Heounjeong Go, Maurilio Ponzoni, Andrés Ferreri, Michael B. Møller, Benjamin Parsons, J. Han van Krieken, Miguel A. Piris, Jane N. Winter, Mingzhi Zhang, Bing Xu, Ken H. Young. Single-cell double expression of MYC and BCL2 proteins by fluorescent multiplex IHC is mainly in lymphoma cells with significant prognostic impact in large B-cell lymphoma while suggesting high cutoffs for routine IHC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6418.

The consensus molecular subtype (CMS) classification divides colon tumors into four subtypes holding promise as a predictive biomarker. However, the effect of adjuvant chemotherapy on recurrence free survival (RFS) per CMS in stage III patients remains inadequately explored. With this intention, we selected stage III colon cancer (CC) patients from the MATCH cohort (n = 575) and RadboudUMC (n = 276) diagnosed between 2005 and 2018. Patients treated with and without adjuvant chemotherapy were matched based on tumor location, T- and N-stage (n = 522). Tumor material was available for 464 patients, with successful RNA extraction and CMS subtyping achieved in 390 patients (surgery alone group: 192, adjuvant chemotherapy group: 198). In the overall cohort, CMS4 was associated with poorest prognosis (HR 1.55; p = .03). Multivariate analysis revealed favorable RFS for the adjuvant chemotherapy group in CMS1, CMS2, and CMS4 tumors (HR 0.19; p = .01, HR 0.27; p < .01, HR 0.19; p < .01, respectively), while no significant difference between treatment groups was observed within CMS3 (HR 0.68; p = .51). CMS subtyping in this non-randomized cohort identified patients with poor prognosis and patients who may not benefit significantly from adjuvant chemotherapy.

Despite high cure rates in classic Hodgkin lymphoma (cHL), relapses are observed. Whether relapsed cHL represents second primary lymphoma or an underlying T-cell lymphoma (TCL) mimicking cHL is underinvestigated. To analyze the nature of cHL recurrences, in-depth clonality testing of immunoglobulin (Ig) and T-cell receptor (TCR) rearrangements was performed in paired cHL diagnoses and recurrences among 60 patients, supported by targeted mutation analysis of lymphoma-associated genes. Clonal Ig rearrangements were detected by next-generation sequencing (NGS) in 69 of 120 (58%) diagnoses and recurrence samples. The clonal relationship could be established in 34 cases, identifying clonally related relapsed cHL in 24 of 34 patients (71%). Clonally unrelated cHL was observed in 10 of 34 patients (29%) as determined by IG-NGS clonality assessment and confirmed by the identification of predominantly mutually exclusive gene mutations in the paired cHL samples. In recurrences of >2 years, ∼60% of patients with cHL for whom the clonal relationship could be established showed a second primary cHL. Clonal TCR gene rearrangements were identified in 14 of 125 samples (11%), and TCL-associated gene mutations were detected in 7 of 14 samples. Retrospective pathology review with integration of the molecular findings were consistent with an underlying TCL in 5 patients aged >50 years. This study shows that cHL recurrences, especially after 2 years, sometimes represent a new primary cHL or TCL mimicking cHL, as uncovered by NGS-based Ig/TCR clonality testing and gene mutation analysis. Given the significant therapeutic consequences, molecular testing of a presumed relapse in cHL is crucial for subsequent appropriate treatment strategies adapted to the specific lymphoma presentation.

Supplementary Figures S1 to S3

Background:The amount of stroma within the primary tumor is a prognostic parameter for colon cancer patients. This phenomenon can be assessed using the tumor-stroma ratio (TSR), which classifies tumors in stroma-low (≤50% stroma) and stroma-high (>50% stroma). Although the reproducibility for TSR determination is good, improvement might be expected from automation. The aim of this study was to investigate whether the scoring of the TSR in a semi- and fully automated method using deep learning algorithms is feasible. Methods:A series of 75 colon cancer slides were selected from a trial series of the UNITED study. For the standard determination of the TSR, 3 observers scored the histological slides. Next, the slides were digitized, color normalized, and the stroma percentages were scored using semi- and fully automated deep learning algorithms. Correlations were determined using intraclass correlation coefficients (ICCs) and Spearman rank correlations. Results:37 (49%) cases were classified as stroma-low and 38 (51%) as stroma-high by visual estimation. A high level of concordance between the 3 observers was reached, with ICCs of 0.91, 0.89, and 0.94 (all P < .001). Between visual and semi-automated assessment the ICC was 0.78 (95% CI 0.23-0.91, P-value 0.005), with a Spearman correlation of 0.88 (P < .001). Spearman correlation coefficients above 0.70 (N=3) were observed for visual estimation versus the fully automated scoring procedures. Conclusion:Good correlations were observed between standard visual TSR determination and semi- and fully automated TSR scores. At this point, visual examination has the highest observer agreement, but semi-automated scoring could be helpful to support pathologists.

Supplementary Figure 1. Morphology and immunophenotype of pSTAT3+ DLBCL. Supplementary Figure 2. Relationship between STAT3 mRNA and pSTAT3 expression and survival analysis based on STAT3 mRNA level. Supplementary Figure 3. Survival analyses based on pSTAT3 expression in DLBCL with MYC/BCL2 double expression and DLBCL without MYC/BCL2 double expression after COO stratification. Supplementary Figure 4. Overall survival (OS) and progression-free survival (PFS) in all DLBCL cases, DLBCL with germinal center B-cell-like phenotype (GCB) and DLBCL with activated B-cell-like phenotype (ABC). Supplementary Figure 5. Survival analyses in 3 and 4 groups based on pSTAT3 expression in lymphoma cells. Supplementary Table 1. Number of cases based on pSTAT3 expression, MYC/BCL2 double expression and COO classification Supplementary Table 2. The numbers and percentages of pSTAT3 positive DLBCL with each cutoff Supplementary Table 3. Clinical characteristics and cell-of-origin differentiation with 30% cutoff for pSTAT3 expression Supplementary Table 4. Distribution of cell-of-origin classification based on 4 groups of pSTAT3 expression in lymphoma cells. Supplementary Table 5. Distribution of cell-of-origin classification based on 3 groups of pSTAT3 expression in lymphoma cells. Supplementary Table 6. Bivariate analyses of pSTAT3 with each variable and pSTAT3.

Supplementary Data from Identification of a Quantitative MINT Locus Methylation Profile Predicting Local Regional Recurrence of Rectal Cancer

Supplementary Table S1. Clinicopathologic characteristics of patients with de novo DLBCL with dual MYC/TP53 aberrations Supplementary Table S2. Frequency of single or dual abnormal Myc protein, p53 protein, BCL2 protein, MYC gene, and TP53 gene in the studied patients with DLBCL treated with R-CHOP Supplementary Table S3. Prognostic factors by univariate analysis and multivariate analysis in DLBCL Supplementary Table S4. Gene expression signatures identified by comparing DLBCL patients with concurrent MYC-R Mut-TP53 or Mychigh Mut-TP53 alterations (double-positive) with DLBCL patients withSupplementary Table S5. Molecular, genetic and phenotypic status of 8 DLBCL/HGBCL cell lines by targeted next-generation sequencing, fluorescence in situ hybridization (FISH), and immunohistochemistry analysis none or the alterations (double-negative) Supplementary Figure S1. Morphologic and immunophenotypic features of DLBCL with MYC rearrangement (MYC-R) and TP53 mutation (Mut-TP53) dual-alterations and highgrade B-cell lymphoma (HGBCL) with MYC/BCL2 double-hit (DH) in representative patients. Supplementary Figure S2. Prognostic impact of p53 and Myc protein overexpression as single or double abnormalities in overall DLBCL and GCB/ABC subtypes. Supplementary Figure S3. Heatmap for gene expression signatures of MYC/TP53 dual alterations. Supplementary Figure S4. Representative figures of flow cytometric analysis indicating G2/M phase-cell cycle arrest by NCB057643 treatment for 24 hours in cells with MYC-R or Myc overexpression with Wt-TP53 (OCI-LY19) or Mut-TP53 (GR and TMD8). Supplementary Figure S5. Heatmap for significantly up- or downregulated proteins after INCB057643 treatment (5µM, 24 hours) in OCI-LY19 cells. Supplementary Figure S6. A MDM2 inhibitor DS3032b shows cytotoxic effects selectively in high-grade B-cell lymphoma (HGBCL) with MYC/BCL2 double-hit (DH) and wild-type (Wt) TP53. Supplementary Figure S7. Combined DS3032b and INCB057643 treatment has no synergistic cytotoxicity in DLBCL cell lines with MYC aberrations and TP53 mutation (MutTP53). Supplementary Figure S8. INCB057643 treatment (1.25 µM) alone or in combination with ABT-199 (venetoclax, 6.25 mM) for 24 hours induced p21 expression in TMD8 cells, whereas had no effect on p53 (mutant) expression levels.

CCR Translation for this Article from Identification of a Quantitative MINT Locus Methylation Profile Predicting Local Regional Recurrence of Rectal Cancer

Approximately one-third of patients with diffuse large B-cell lymphoma (DLBCL) relapse and often require salvage chemotherapy followed by autologous stem cell transplantation. In most cases, the clonal relationship between the first diagnosis and subsequent relapse is not assessed, thereby potentially missing the identification of second primary lymphoma. In this study, the clonal relationship of 59 paired DLBCL diagnoses and recurrences was established by next-generation sequencing-based detection of immunoglobulin gene rearrangements. Among 50 patients with interpretable results, 43 patients (86%) developed clonally related relapsed disease. This was observed in 100% of early recurrences (<2 years), 80% of the recurrences with an interval between 2 and 5 years, and 73% of late recurrences (≥5 years). On the other hand, 7 (14%) out of 50 patients displayed different dominant clonotypes in primary DLBCL and clinical recurrences, confirming the occurrence of second primary DLBCL; 37% of DLBCL recurrences that occurred ≥4 years after diagnosis were shown to be second primary lymphomas. The clonally unrelated cases were Epstein-Barr virus positive in 43% of the cases, whereas this was only 5% in the relapsed DLBCL cases. In conclusion, next-generation sequencing-based clonality testing in late recurrences should be considered in routine diagnostics to distinguish relapse from second primary lymphoma, as this latter group of patients with DLBCL may benefit from less-intensive treatment strategies.

Supplementary Figure from Genetic Subtyping and Phenotypic Characterization of the Immune Microenvironment and MYC/BCL2 Double Expression Reveal Heterogeneity in Diffuse Large B-cell Lymphoma

The supplementary documents include: (1) Supplementary methods for detection of MYC mutations and rearrangements, assessment of Myc expression, and functional studies. (2) Supplementary Tables S1-S8. Supplementary Table S1. Numbers of MYC mutation present in the coding sequence and the untranslated regions. Supplementary Table S2. Clinical and molecular characteristics of the DLBCL cohort with wild-type or mutated Myc or N11S single nucleotide polymorphism. Supplementary Table S3. List of Myc mutations and corresponding patient survival. Supplementary Table S4. Multivariate survival analysis. Supplementary Table S5. Molecular characteristics of patients with wild-type or mutated MYC untranslated regions. Supplementary Table S6. List of MYC 3Ã,'UTR mutations and corresponding patient survival. Supplementary Table S7. Gene profiling comparisons between wild-type and mutated MYC. Supplementary Table S8. Brief summary of major findings by this study. (3) Legends for Supplementary Figures S1-S5. Supplementary Figure S1. Survival analysis for MYC mutations in DLBCL respective to GCB/ABC subtypes, homo/heterozygosity, and MYC translocations. Supplementary Figure S2. Comparison of Myc levels and gene expression profiles between DLBCL groups. Supplementary Figure S3. Differential expression of genes between the WT-Myc and MUT-Myc groups. Supplementary Figure S4. Comparison of gene expression between the WT-Myc and MUT-Myc groups. Supplementary Figure S5. Differential expression of proteins between the WT-Myc and MUT-Myc groups.

Supplementary Methods, Tables 1-11, and Figures 1-16

<p>Summary of all analyzed primary tumor slides used in the analysis, using the same format as in Figure 1B of the main manuscript to illustrate the T cell landscapes.</p>

List of top 10 sequences in the individual IGH repertoires of 138 DLBCL cases with a diagnostic IGH clonotype identified