The Genetic Landscape of Primary Breast Marginal Zone Lymphoma Identifies a Mutational-driven Disease With Similarities to Ocular Adnexal Lymphoma.

Extranodal marginal zone lymphomas (eMZL) can occur in any organ and site of the body. Recent research has shown that they differ from organ to organ in terms of their mutational profile. In this study, we investigated a cohort of primary breast marginal zone lymphomas (PBMZL) to get a better insight into their morphologic and molecular profile. A cohort of 15 cases (14 female and 1 male) was characterized by immunohistochemistry (IHC) for 19 markers, fluorescence in situ hybridization (FISH), and high throughput sequencing (HTS) using a lymphoma panel comprising 172 genes. In addition, PCR for the specific detection of Borrelia spp. and metagenomics whole genome sequencing were performed for infectious agent profiling. Follicular colonization was observed in most cases, while lymphoepithelial lesions, though seen in many cases, were not striking. All 15 cases were negative for CD5, CD11c, and CD21 and positive for BCL2 and pan B-cell markers. There were no cases with BCL2, BCL10, IRF4, MALT1, or MYC translocation; only 1 had a BCL6 rearrangement. HTS highlighted TNFAIP3 (n=4), KMT2D (n=2), and SPEN (n=2) as the most frequently mutated genes. There were no Borrelia spp., and no other pathogens detected in our cohort. One patient had a clinical history of erythema chronicum migrans affecting the same breast. PBMZL is a mutation-driven disease rather than fusion-driven. It exhibits mutations in genes encoding components affecting the NF-κB pathway, chromatin modifier-encoding genes, and NOTCH pathway-related genes. Its mutational profile shares similarities with ocular adnexal and nodal MZL.

Primary bone diffuse large B-cell lymphoma (PB-DLBCL): a distinct extranodal lymphoma of germinal centre origin, with a common EZB-like mutational profile and good prognosis.

AIMS: Primary bone diffuse large B-cell lymphoma (PB-DLBCL) is not recognized as a separate entity by the current classification systems. Here we define and highlight its distinctive clinical presentation, morphology, phenotype, gene expression profile (GEP), and molecular genetics. METHODS: We collected 27 respective cases and investigated their phenotype, performed gDNA panel sequencing covering 172 genes, and carried out fluorescence in situ hybridization to evaluate MYC, BCL2, and BCL6 translocations. We attempted to genetically subclassify cases using the Two-step classifier and performed GEP for cell-of-origin subtyping and in silico comparison to uncover up- and downregulated genes as opposed to other DLBCL. RESULTS: Most cases (n = 22) were germinal centre B-cell-like (GCB) by immunohistochemistry and all by GEP. Additionally, PB-DLBCL had a mutational profile similar to follicular lymphoma and nodal GCB-DLBCL, with the exception of more frequent TP53 and B2M mutations. The GEP of PB-DLBCL was unique, and the frequency of BCL2 rearrangements was lower compared to nodal GCB-DLBCL. The Two-step classifier categorized eight of the cases as EZB, three as ST2, and one as MCD. CONCLUSION: This study comprehensively characterizes PB-DLBCL as a separate entity with distinct clinical and morpho-molecular features. These insights may aid in developing tailored therapeutic strategies and shed light on its pathogenesis.

Molecular characterization and genetic subclassification comparison of diffuse large B-cell lymphoma (DLBCL) - real-life experience with 74 cases.

INTRODUCTION: Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous entity. Lately, several algorithms achieving therapeutically and prognostically relevant DLBCL subclassification have been published. METHODS: A cohort of 74 routine DLBCL cases was broadly characterized by immunohistochemistry (IHC), fluorescence in situ hybridization (FISH) of the BCL2, BCL6 and MYC loci, and comprehensive high throughput sequencing (HTS). Based on the genetic alterations found, cases were reclassified using two probabilistic tools - LymphGen and Two-step classifier, allowing for comparison of the two models. RESULTS: Hans and Tally's overall IHC-based subclassification success rate was 96% and 82%, respectively. HTS and FISH data allowed the LymphGen algorithm to successfully classify 11/55 cases, (1 - BN2, 7 - EZB, 1 - MCD, and 2 - genetically composite EZB/N1). The total subclassification rate was 20%. On the other hand, the Two-step classifier categorized 36/55 cases, with 65.5% success (9 - BN2, 12 - EZB, 9 - MCD, 2 - N1, and 4 - ST2). Clinical correlations highlighted MCD as an aggressive subtype associated with higher relapse and mortality. CONCLUSIONS: The Two-step algorithm has a better success rate at subclassifying DLBCL cases based on genetic differences. Further improvement of the classifiers is required to increase the number of classifiable cases and thus prove their applicability in routine diagnostics.

Combined therapy with ibrutinib and bortezomib followed by ibrutinib maintenance in relapsed or refractory mantle cell lymphoma and high-risk features: a phase 1/2 trial of the European MCL network (SAKK 36/13).

Background: The Bruton's tyrosine kinase inhibitor ibrutinib and the proteasome inhibitor bortezomib have single-agent activity, non-overlapping toxicities, and regulatory approval in mantle cell lymphoma (MCL). In vitro, their combination provides synergistic cytotoxicity. In this investigator-initiated phase 1/2 trial, we established the recommended phase 2 dose of ibrutinib in combination with bortezomib, and assessed its efficacy in patients with relapsed or refractory MCL. Methods: In this phase 1/2 study open in 15 sites in Switzerland, Germany and Italy, patients with relapsed or refractory MCL after ≤2 lines of chemotherapy and both ibrutinib-naïve and bortezomib-naïve received six cycles of ibrutinibb and bortezomib, followed by ibrutinib maintenance. For the phase 1 study, a standard 3 + 3 dose escalation design was used to determine the recommended phase 2 dose of ibrutinib in combination with bortezomib. The primary endpoint in phase 1 was the dose limiting toxicities in cycle 1. The phase 2 study was an open-label, single-arm trial with a Simon's two-stage min-max design, with a primary endpoint of overall response rate (ORR) assessed by CT/MRI. This study was registered with ClinicalTrials.gov, NCT02356458. Findings: Between August 2015 and September 2016, nine patients were treated in the phase 1 study, and 49 patients were treated between November 2016 and March 2020 in the phase 2 of the trial. The ORR was 81.8% (90% CI 71.1, 89.8%, CR(u) 21.8%) which increased with continued ibrutinib (median 10.6 months) to 87.3%, (CR(u) 41.8%). 75.6% of patients had at least one high-risk feature (Ki-67 > 30%, blastoid or pleomorphic variant, p53 overexpression, TP53 mutations and/or deletions). In these patients, ibrutinib and bortezomib were also effective with an ORR of 74%, increasing to 82% during maintenance. With a median follow-up of 25.4 months, the median duration of response was 22.7, and the median PFS was 18.6 months. PFS reached 30.8 and 32.9 months for patients with a CR or Cru, respectively. Interpretation: The combination of ibrutinib and bortezomib shows durable efficacy in patients with relapsed or refractory MCL, also in the presence of high-risk features. Funding: SAKK (Hubacher Fund), Swiss State Secretariat for Education, Research and Innovation, Swiss Cancer Research Foundation, and Janssen.

Interplay between the tyrosine kinases Chk and Csk and phosphatase PTPRJ is critical for regulating platelets in mice.

The Src family kinases (SFKs) Src, Lyn, and Fyn are essential for platelet activation and also involved in megakaryocyte (MK) development and platelet production. Platelet SFKs are inhibited by C-terminal Src kinase (Csk), which phosphorylates a conserved tyrosine in their C-terminal tail, and are activated by the receptor-type tyrosine phosphatase PTPRJ (CD148, DEP-1), which dephosphorylates the same residue. Deletion of Csk and PTPRJ in the MK lineage in mice results in increased SFK activity, but paradoxically hypoactive platelets resulting from negative feedback mechanisms, including upregulation of Csk homologous kinase (Chk) expression. Here, we investigate the role of Chk in platelets, functional redundancy with Csk, and the physiological consequences of ablating Chk, Csk, and PTPRJ in mice. Platelet count was normal in Chk knockout (KO) mice, reduced by 92% in Chk;Csk double KO (DKO) mice, and partially rescued in Chk;Csk;Ptprj triple KO (TKO) mice. Megakaryocyte numbers were significantly increased in both DKO and TKO mice. Phosphorylation of the inhibitory tyrosine of SFKs was almost completely abolished in DKO platelets, which was partially rescued in Src and Fyn in TKO platelets. This residual phosphorylation was abolished by Src inhibitors, revealing an unexpected mechanism in which SFKs autoinhibit their activity by phosphorylating their C-terminal tyrosine residues. We demonstrate that reduced inhibitory phosphorylation of SFKs leads to thrombocytopenia, with Csk being the dominant inhibitor in platelets and Chk having an auxiliary role. PTPRJ deletion in addition to Chk and Csk ameliorates the extent of thrombocytopenia, suggesting targeting it may have therapeutic benefits in such conditions.