Johan Hjm van Krieken

Aims: Tumour–stroma ratio (TSR) scores of biopsy material in rectal carcinoma (RC) could aid a biomarker‐based, upfront and personalised treatment strategy selection for RC patients. In a large retrospective, multicentre cohort, we aimed to validate the predictive value of biopsy‐scored TSR on neoadjuvant therapy response, and secondarily, disease‐free and overall survival (DFS, OS).Methods and results: Scanned haematoxylin and eosin‐stained RC biopsy slides were collected from Leiden University Medical Center (N = 116) and from the clinical PROCTOR‐SCRIPT (N = 142) and RAPIDO (N = 271) trials. TSR was scored per protocol and categorised as stroma‐low (≤ 50%) or stroma‐high (> 50%). Major response was defined as tumour regression grade (TRG) 1 + 2 by Mandard, including pathological complete response. Ultimately, a large and varied cohort with 373 RC patients was established. Locally advanced RC was more often stroma‐high (P < 0.001). We subsequently observed significantly lower major response rates in the stroma‐high RC after a neoadjuvant treatment approach (hazard ratio = 0.63, 95% confidence interval = 0.41–0.99; P = 0.044). Despite correction for well‐known risk factors in Cox hazard regression analysis, such as (y)pTNM substages or residual tumour status, the TSR had no singular significant influence on DFS nor OS in multivariate analysis (P = 0.438; P = 0.934, respectively).Conclusions: Biopsy‐scored TSR can predict neoadjuvant therapy efficacy, as RC patients with stroma‐high biopsies show less major response. However, patient survival is multifactorial, although response is an important predictor, influenced by TSR. Scoring TSR on RC biopsy material is a reliable histological parameter, implementation of which in treatment guidelines could improve upfront selection for a watch‐and‐wait strategy.

The benign neoplasm cardiac myxoma represents one of the hallmarks of Carney complex (CNC), a familial multiple neoplasia syndrome. About 80% of the index cases have germline mutations in PRKAR1A encoding the RIα regulatory subunit of cAMP-dependent protein kinase A (PKA). However, the role of PRKAR1A gene mutations in the pathogenesis of non-CNC-associated sporadic cardiac myxoma is less well established. Here, we investigated the presence of PRKAR1A gene variants in a cohort of 24 sporadic cardiac myxomas using targeted next-generation sequencing. Our study shows that 14 out of 24 cases (58%) harbor PRKAR1A gene mutations, represented mostly by frameshift, nonsense, and splice site mutations (together 84%), leading to a premature stop codon predicted to be degraded via non-sense mediated mRNA decay. The other 16% of PRKAR1A genetic alterations involved missense mutations, often located in important functional domains of the regulatory subunit RIα. Notably, 64% (n = 9/14) of the cases harbored more than one PRKAR1A gene variant, suggesting compound heterozygous mutations either in cis or trans. In conclusion, PRKAR1A gene mutations associated with loss of RIα function leading to increased PKA activity were observed in ~ 60% of sporadic cardiac myxomas, strongly supporting an essential role for PKA in mediating formation of cardiac myxoma.

Purpose: Tumor-infiltrating B lymphocytes (TIL-B) have demonstrated prognostic and predictive significance in solid cancers. In this study, we aimed to distinguish TIL-Bs from malignant B-cells in diffuse large B-cell lymphoma (DLBCL) and determine the clinical and biological significance. Experimental Design: A total of 269 patients with de novo DLBCL from the International DLBCL R-CHOP Consortium Program were studied. Ultra-deep sequencing of the immunoglobulin genes was performed to determine B-cell clonotypes. The frequencies and numbers of TIL-B clonotypes in individual repertoires were correlated with patient survival, gene expression profiling (GEP) data, and frequencies of DLBCL-infiltrating immune cells quantified by fluorescent multiplex IHC at single-cell resolution. Results: TIL-B abundance, evaluated by frequencies of normal B-cell clonotypes in the immunoglobulin repertoires, remarkably showed positive associations with significantly better survival of patients in our sequenced cohorts. DLBCLs with high versus low TIL-B abundance displayed distinct GEP signatures, increased pre-memory B-cell state and naïve CD4 T-cell state fractions, and higher CD4+ T-cell infiltration. TIL-B frequency, as a new biomarker in DLBCL, outperformed the germinal center (GC) B-cell–like/activated B-cell–like classification and TIL-T frequency. The identified TIL-B–high GEP signature, including genes upregulated during T-dependent B-cell activation and those highly expressed in normal GC B cells and T cells, showed significant favorable prognostic effects in several external validation cohorts. Conclusions: TIL-B frequency is a significant prognostic factor in DLBCL and plays a crucial role in antitumor immune responses. This study provides novel insights into the prognostic determinants in DLBCL and TIL-B functions with important therapeutic implications.

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.

Diffuse large B-cell lymphoma (DLBCL) is the major type of aggressive B-cell lymphoma. High-grade B-cell lymphoma (HGBCL) with MYC/BCL2 double-hit (DH) represents a distinct entity with dismal prognosis after standard immunochemotherapy in the current WHO lymphoma classification. However, whether TP53 mutation synergizes with MYC abnormalities (MYC rearrangement and/or Myc protein overexpression) contributing to HGBCL-like biology and prognosis is not well investigated. In this study, patients with DLBCL with MYC/TP53 abnormalities demonstrated poor clinical outcome, high-grade morphology, and distinct gene expression signatures. To identify more effective therapies for this distinctive DLBCL subset, novel MYC/TP53/BCL-2–targeted agents were investigated in DLBCL cells with MYC/TP53 dual alterations or HGBCL-MYC/BCL2-DH. A BET inhibitor INCB057643 effectively inhibited cell viability and induced apoptosis in DLBCL/HGBCL cells regardless of MYC/BCL2/TP53 status. Combining INCB057643 with a MDM2-p53 inhibitor DS3032b significantly enhanced the cytotoxic effects in HGBCL-DH without TP53 mutation, while combining with the BCL-2 inhibitor venetoclax displayed potent therapeutic synergy in DLBCL/HGBCL cells with and without concurrent TP53 mutation. Reverse-phase protein arrays revealed the synergistic molecular actions by INCB057643, DS3032b and venetoclax to induce cell-cycle arrest and apoptosis and to inhibit AKT/MEK/ERK/mTOR pathways, as well as potential drug resistance mechanisms mediated by upregulation of Mcl-1 and RAS/RAF/MEK/ERK pathways. In summary, these findings support subclassification of DLBCL/HGBCL with dual MYC/TP53 alterations, which demonstrates distinct pathobiologic features and dismal survival with standard therapy, therefore requiring additional targeted therapies.Implications:The clinical and pharmacologic studies suggest recognizing DLBCL with concomitant TP53 mutation and MYC abnormalities as a distinctive entity necessary for precision oncology practice.Visual Overview:http://mcr.aacrjournals.org/content/molcanres/19/2/249/F1.large.jpg.

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.

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

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

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