Recent advances in genomics and therapeutics in mantle cell lymphoma.

Over the past decades, significant strides have been made in understanding the pathobiology, prognosis, and treatment options for mantle cell lymphoma (MCL). The heterogeneity observed in MCL's biology, genomics, and clinical manifestations, including indolent and aggressive forms, is intricately linked to factors such as the mutational status of the variable region of the immunoglobulin heavy chain gene, epigenetic profiling, and Sox11 expression. Several intriguing subtypes of MCL, such as Cyclin D1-negative MCL, in situ mantle cell neoplasm, CCND1/IGH FISH-negative MCL, and the impact of karyotypic complexity on prognosis, have been explored. Notably, recent immunochemotherapy regimens have yielded long-lasting remissions in select patients. The therapeutic landscape for MCL is continuously evolving, with a shift towards nonchemotherapeutic agents like ibrutinib, acalabrutinib, and venetoclax. The introduction of BTK inhibitors has brought about a transformative change in MCL treatment. Nevertheless, the challenge of resistance to BTK inhibitors persists, prompting ongoing efforts to discover strategies for overcoming this resistance. These strategies encompass non-covalent BTK inhibitors, immunomodulatory agents, BCL2 inhibitors, and CAR-T cell therapy, either as standalone treatments or in combination regimens. Furthermore, developing novel drugs holds promise for further improving the survival of patients with relapsed or refractory MCL. In this comprehensive review, we methodically encapsulate MCL's clinical and pathological attributes and the factors influencing prognosis. We also undertake an in-depth examination of stratified treatment alternatives. We investigate conceivable resistance mechanisms in MCL from a genetic standpoint and offer precise insights into various therapeutic approaches for relapsed or refractory MCL.

Chronic Lymphocytic Leukemia With Two B-Cell Populations of Discordant Light Chain Restrictions in Individual Patients: Parallel Development of Biclonal B-Cell Neoplasms or Clonal Evolution With Isotype Switch?

OBJECTIVES: To evaluate clinicopathologic characteristics of biclonal chronic lymphocytic leukemia (CLL). METHODS: Retrospectively analyze clinical data and pathologic features. RESULTS: Ten cases were identified in which flow cytometry demonstrated an abnormal B-cell population with a CLL-like immunophenotype but showed no definitive light chain restriction. All had cytogenetic abnormalities detected, including seven with two CLL-related abnormalities. Four of these showed features suggestive of clonal evolution, all having del(13q) as a "stem-line" abnormality and three showing del(11q) as a "side-line" abnormality. Five (50%) cases demonstrated deleterious NOTCH1 mutations, in contrast to 11.8% in a control group of monoclonal CLL (P < .05). Of the 10 patients, 5 received treatment, with good/partial response in three cases and therapeutic resistance in one case. The median treatment-free survival was estimated at 68 months. CONCLUSIONS: Despite a polytypic pattern of light chain expression, the neoplastic nature of biclonal CLL is suggested by a characteristic CLL phenotype and can be confirmed by cytogenetic and genomic analyses. The two clones with discordant light chain isotypes may share a "stem-line" cytogenetic abnormality, suggesting possible clonal evolution. Biclonal CLL is associated with NOTCH1 mutations, which may occur in a small subclone and gradually evolve in clonal size. Genomic analysis on light chain-sorted and/or chronologically collected samples may provide insight into clonal evolution in CLL.

Human herpesvirus 8-negative effusion-based large B-cell lymphoma: a distinct entity with unique clinicopathologic characteristics.

Rare cases of human herpesvirus 8 (HHV8)-negative effusion-based large B-cell lymphoma (EB-LBCL) occur in body cavities without antecedent or concurrent solid mass formation. In contrast to HHV8 + primary effusion lymphoma (PEL), EB-LBCL has no known association with HIV or HHV8 infection. However, the small sample sizes of case reports and series worldwide, especially from non-Japanese regions, have precluded diagnostic uniformity. Therefore, we conducted a retrospective, multi-institutional study of 55 cases of EB-LBCL and performed a comprehensive review of an additional 147 cases from the literature to identify distinct clinicopathologic characteristics. In our study, EB-LBCL primarily affected elderly (median age 80 years), immunocompetent patients and manifested as lymphomatous effusion without a solid component. The lymphomatous effusions mostly occurred in the pleural cavity (40/55, 73%), followed by the pericardial cavity (17/55, 31%). EB-LBCL expressed CD20 (53/54, 98%) and PAX5 (23/23, 100%). Most cases (30/36, 83%) were of non-germinal center B-cell subtype per the Hans algorithm. HHV8 infection was absent (0/55, 0%), while Epstein-Barr virus was detected in 6% (3/47). Clinically, some patients were managed with drainage alone (15/34, 44%), while others received rituximab alone (4/34, 12%) or chemotherapy (15/34, 44%). Eventually, 56% (22/39) died with a median overall survival (OS) of 14.9 months. Our findings were similar to those from the literature; however, compared to the non-Japanese cases, the Japanese cases had a significantly higher incidence of pericardial involvement, a higher rate of chemotherapy administration, and longer median OS. Particularly, we have found that Japanese residence, presence of pericardial effusion, and absence of MYC rearrangement are all favorable prognostic factors. Our data suggest that EB-LBCL portends a worse prognosis than previously reported, although select patients may be managed conservatively. Overall, EB-LBCL has distinct clinicopathologic characteristics, necessitating the establishment of separate diagnostic criteria and consensus nomenclature.

Sequential Development of JAK2V617F Mutation and BCR-ABL1 Fusion in Individual Patients With Myeloproliferative Neoplasms.

CONTEXT.­: Concomitant BCR-ABL1 and JAK2V617F in myeloproliferative neoplasms (MPNs) is rare, and its pathogenesis and clinical significance are unclear. OBJECTIVE.­: To investigate the clonal relationship between the 2 genomic alterations, as well as the clinicopathologic impact. DESIGN.­: Retrospective analysis of MPNs with sequential development of BCR-ABL1 and JAK2V617F. RESULTS.­: Of 6 cases, 5 had JAK2V617F-positive MPN diagnosed before acquiring BCR-ABL1 years later, and 1 had BCR-ABL1+ chronic myeloid leukemia before JAK2V617F-positive myelofibrosis completely replaced the BCR-ABL1+ clone 1 year after tyrosine kinase inhibitor therapy. Among the former group, treatment for the initial MPN involved hydroxyurea, ruxolitinib, and/or supportive care, and the latency to the development of JAK2V617F ranged from 4 to 13 years (median of 9 years). Four cases showed retention of JAK2V617F, whereas BCR-ABL1 emerged as the major clone, including 2 cases that exhibited parallel increases in JAK2V617F and BCR-ABL1 burdens, with both genomic markers exceeding 50%. Three patients received stem cell transplants and demonstrated sustained engraftment, with the genomic markers below detectable levels. CONCLUSIONS.­: Most MPNs with concomitant JAK2V617F and BCR-ABL1 are actually composite MPNs with a "second hit" residing on a different clone. Rare cases demonstrate a subclone harboring a "double-hit" in a background of a JAK2V617F-positive stem line clone. The probability of a "double-hit" with a BCR-ABL1+ stem line clone is probably reduced by effective tyrosine kinase inhibitor treatment. The treatment often involves combined kinase inhibitors and/or hydroxyurea, but the outcome is unpredictable; hematopoietic stem cell transplantation may be the ultimate therapeutic option for this complicated disease.

Myeloid neoplasms in the setting of chronic lymphocytic leukaemia/chronic lymphocytic leukaemia-like disease: a clinicopathological study of 66 cases comparing cases with prior history of treatment to those without.

AIMS: Myeloid neoplasms occur in the setting of chronic lymphocytic leukaemia (CLL)/CLL-like disease. The underlying pathogenesis has not been elucidated. METHODS: Retrospectively analysed 66 cases of myeloid neoplasms in patients with CLL/CLL-like disease. RESULTS: Of these, 33 patients (group 1) had received treatment for CLL/CLL-like disease, while the other 33 patients (group 2) had either concurrent diagnoses or untreated CLL/CLL-like disease before identifying myeloid neoplasms. The two categories had distinct features in clinical presentation, spectrum of myeloid neoplasm, morphology, cytogenetic profile and clinical outcome. Compared with group 2, group 1 demonstrated a younger age at the diagnosis of myeloid neoplasm (median, 65 vs 71 years), a higher fraction of myelodysplastic syndrome (64% vs 36%; OR: 3.1; p<0.05), a higher rate of adverse unbalanced cytogenetic abnormalities, including complex changes, -5/5q- and/or -7/7q- (83% vs 28%; OR: 13.1; p<0.001) and a shorter overall survival (median, 12 vs 44 months; p<0.05). CONCLUSIONS: Myeloid neoplasm in the setting of CLL/CLL-like disease can be divided into two categories, one with prior treatment for CLL/CLL-like disease and the other without. CLL-type treatment may accelerate myeloid leukaemogenesis. The risk is estimated to be 13-fold higher in patients with treatment than those without. The causative agent could be attributed to fludarabine in combination with alkylators, based on the latency of myeloid leukaemogenesis and the cytogenetic profile.

Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma With Secondary Acquisition of t(11;14)(q13;q32)/CCND1-IGH: A Rare Variant Of Richter Transformation to Mantle Cell Lymphoma.

INTRODUCTION: Chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) occasionally undergoes Richter transformation, mostly to diffuse large B-cell lymphoma, but its evolution to other types of B-cell lymphoma is rare. We report a CLL evolved to mantle cell lymphoma by acquiring t(11;14)(q13;q32); CCND1-IGH. METHOD: A Retrospective review of clinical and laboratory data. RESULTS: A 39-year-old male patient was diagnosed with CLL/SLL, and was initially followed without specific treatment, but subsequently received chlorambucil/fludarabine/rituximab due to exacerbated lymphocytosis. While his CLL/SLL waned and waxed, the immunophenotype and genotype of neoplastic B-cells remained unchanged, without cyclin D1 expression and CCND1-IGH fusion. Eleven years after the diagnosis, the patient's disease showed evidence of progression. Bone marrow examination demonstrated "CLL" with the morphology and immunophenotype similar to those seen in the previous biopsies. Unexpectedly, the neoplastic B-cells demonstrated cyclin D1 expression and harbored t(11;14)(q13;q32); CCND1-IGH, suggesting a clonal evolution to mantle cell lymphoma. He subsequently received cytoreductive chemotherapy followed by allogenic bone marrow transplant and remained in remission since then. CONCLUSION: The retention of immunophenotype suggests a clonal relationship between CLL/SLL and mantle cell lymphoma. While the acquisition of t(11;14)(q13;q32); CCND1-IGH likely alters the disease course, the pathogenesis of this illegitimate translocation in CLL remains to be studied.

Incidental finding of abdominal splenosis with mononucleated cell infiltration leading to a diagnosis of acute myeloid leukemia.

Splenosis refers to ectopic splenic implants that are often found incidentally years after splenic rupture/splenectomy, and the nodules of splenosis are usually small, less than 3 cm for the majority. We report a case of splenosis with a 5-centimeter large mass in the anterior abdomen in a 79 year-old male with a remote history of splenic rupture/splenectomy. Unexpectedly, needle core biopsy of the abdominal mass demonstrated splenic tissue with a mononucleated cell infiltrate blurring the splenic architecture that was highlighted only by CD8 stain. This finding prompted a bone marrow examination resulting in the diagnosis of acute myeloid leukemia in the patient. Retrospectively, enlargement of this ectopic spleen may have been caused by this leukemic infiltrate. This case underscores the importance of being aware of this rare pathological condition and its retained vulnerability for involvement by hematolymphoid neoplasms, as well as significance of identifying splenic architecture highlighted by CD8 stain to reach a correct diagnosis.

The practice of universal screening for Lynch syndrome in newly diagnosed endometrial carcinoma.

BACKGROUND: Lynch syndrome (LS) accounts for 5% of all endometrial cancer (EC) cases and 4% of all lifetime risk of developing colorectal cancer. While current guidelines recommend LS screening for all patients with newly diagnosed colorectal cancer, there is no such guideline for screening patients with EC. DISCUSSION: This review addresses LS screening and discusses algorithms for testing patients in the setting of newly diagnosed EC. CONCLUSION: The successful diagnosis of LS has important implications, including prevention of LS-associated cancers among relatives and immunotherapy recommendations for patients with advanced EC and loss of expression of mismatch repair immunohistochemistry and microsatellite instability positive following failure of traditional treatment.

Bacterial lipoproteins and other factors released by Francisella tularensis modulate human neutrophil lifespan: Effects of a TLR1 SNP on apoptosis inhibition.

Francisella tularensis infects several cell types including neutrophils, and aberrant neutrophil accumulation contributes to tissue destruction during tularaemia. We demonstrated previously that F. tularensis strains Schu S4 and live vaccine strain markedly delay human neutrophil apoptosis and thereby prolong cell lifespan, but the bacterial factors that mediate this aspect of virulence are undefined. Herein, we demonstrate that bacterial conditioned medium (CM) can delay apoptosis in the absence of direct infection. Biochemical analyses show that CM contained F. tularensis surface factors as well as outer membrane components. Our previous studies excluded roles for lipopolysaccharide and capsule in apoptosis inhibition, and current studies of [14 C] acetate-labelled bacteria argue against a role for other bacterial lipids in this process. At the same time, studies of isogenic mutants indicate that TolC and virulence factors whose expression requires FevR or MglA were also dispensable, demonstrating that apoptosis inhibition does not require Type I or Type VI secretion. Instead, we identified bacterial lipoproteins (BLPs) as active factors in CM. Additional studies of isolated BLPs demonstrated dose-dependent neutrophil apoptosis inhibition via a TLR2-dependent mechanism that is significantly influenced by a common polymorphism, rs5743618, in human TLR1. These data provide fundamental new insight into pathogen manipulation of neutrophil lifespan and BLP function.

Francisella tularensis Modulates a Distinct Subset of Regulatory Factors and Sustains Mitochondrial Integrity to Impair Human Neutrophil Apoptosis.

Tularemia is a disease characterized by profound neutrophil accumulation and tissue destruction. The causative organism, Francisella tularensis, is a facultative intracellular bacterium that replicates in neutrophil cytosol, inhibits caspase activation and profoundly prolongs cell lifespan. Here, we identify unique features of this infection and provide fundamental insight into the mechanisms of apoptosis inhibition. Mitochondria are critical regulators of neutrophil apoptosis. We demonstrate that F. tularensis significantly inhibits Bax translocation and Bid processing during 24-48 h of infection, and in this manner sustains mitochondrial integrity. Downstream of mitochondria, X-linked inhibitor of apoptosis protein (XIAP) and proliferating cell nuclear antigen (PCNA) inhibit caspase-9 and caspase-3 by direct binding. Notably, we find that PCNA disappeared rapidly and selectively from infected cells, thereby demonstrating that it is not essential for neutrophil survival, whereas upregulation of calpastatin correlated with diminished calpain activity and reduced XIAP degradation. In addition, R-roscovitine is a cyclin-dependent kinase inhibitor developed for the treatment of cancer; it also induces neutrophil apoptosis and can promote the resolution of several infectious and inflammatory disorders. We confirm the ability of R-roscovitine to induce neutrophil apoptosis, but also demonstrate that its efficacy is significantly impaired by F. tularensis. Collectively, our findings advance the understanding of neutrophil apoptosis and its capacity to be manipulated by pathogenic bacteria.

Regulation of human neutrophil apoptosis and lifespan in health and disease.

Neutrophils (also called polymorphonuclear leukocytes, PMNs) are the most abundant white blood cells in humans and play a central role in innate host defense. Another distinguishing feature of PMNs is their short lifespan. Specifically, these cells survive for less than 24 hours in the bloodstream and are inherently pre-programed to die by constitutive apoptosis. Recent data indicate that this process is regulated by intracellular signaling and changes in gene expression that define an "apoptosis differentiation program." Infection typically accelerates neutrophil turnover, and as such, phagocytosis-induced cell death (PICD) and subsequent clearance of the corpses by macrophages are essential for control of infection and resolution of the inflammatory response. Herein we reprise recent advances in our understanding of the molecular mechanisms of neutrophil apoptosis with a focus on regulatory factors and pathway intermediates that are specific to this cell type. In addition, we summarize mechanisms whereby perturbation of PMN death contributes directly to the pathogenesis of many infectious and inflammatory disease states.

Francisella tularensis alters human neutrophil gene expression: insights into the molecular basis of delayed neutrophil apoptosis.

We demonstrated recently that Francisella tularensis profoundly impairs human neutrophil apoptosis, but how this is achieved is largely unknown. Herein we used human oligonucleotide microarrays to test the hypothesis that changes in neutrophil gene expression contribute to this phenotype, and now demonstrate that F. tularensis live vaccine strain (LVS) caused significant changes in neutrophil gene expression over a 24-hour time period relative to the uninfected controls. Of approximately 47,000 genes analyzed, 3,435 were significantly up- or downregulated by LVS, including 365 unique genes associated with apoptosis and cell survival. Specific targets in this category included genes asso-ciated with the intrinsic and extrinsic apoptotic pathways (CFLAR, TNFAIP3, TNFRSF10D, SOD2, BCL2A1, BIRC4, PIM2, TNFSF10, TNFRSF10C, CASP2 and CASP8) and genes that act via the NFĸB pathway and other mechanisms to prolong cell viability (NFKB1, NFKB2 and RELA, IL1B, CAST, CDK2,GADD45B, BCL3, BIRC3, CDK2, IL1A, PBEF1, IL6, CXCL1, CCL4 and VEGF). The microarray data were confirmed by qPCR and pathway analysis. Moreover, we demonstrate that the X-linked inhibitor of apoptosis protein remained abundant in polymorphonuclear leukocytes over 48 h of LVS infection, whereas BAX mRNA and protein were progressively downregulated. These data strongly suggest that antiapoptotic and prosurvival mechanisms collaborate to sustain the viability of F. tularensis--infected neutrophils.

Natural IgM mediates complement-dependent uptake of Francisella tularensis by human neutrophils via complement receptors 1 and 3 in nonimmune serum.

A fundamental step in the life cycle of Francisella tularensis is bacterial entry into host cells. F. tularensis activates complement, and recent data suggest that the classical pathway is required for complement factor C3 deposition on the bacterial surface. Nevertheless, C3 deposition is inefficient and neither the specific serum components necessary for classical pathway activation by F. tularensis in nonimmune human serum nor the receptors that mediate infection of neutrophils have been defined. In this study, human neutrophil uptake of GFP-expressing F. tularensis strains live vaccine strain and Schu S4 was quantified with high efficiency by flow cytometry. Using depleted sera and purified complement components, we demonstrated first that C1q and C3 were essential for F. tularensis phagocytosis, whereas C5 was not. Second, we used purification and immunodepletion approaches to identify a critical role for natural IgM in this process, and then used a wbtA2 mutant to identify LPS O-Ag and capsule as prominent targets of these Abs on the bacterial surface. Finally, we demonstrate using receptor-blocking Abs that CR1 (CD35) and CR3 (CD11b/CD18) acted in concert for phagocytosis of opsonized F. tularensis by human neutrophils, whereas CR3 and CR4 (CD11c/CD18) mediated infection of human monocyte-derived macrophages. Altogether, our data provide fundamental insight into mechanisms of F. tularensis phagocytosis and support a model whereby natural IgM binds to surface capsular and O-Ag polysaccharides of F. tularensis and initiates the classical complement cascade via C1q to promote C3 opsonization of the bacterium and phagocytosis via CR3 and either CR1 or CR4 in a phagocyte-specific manner.

EccA1, a component of the Mycobacterium marinum ESX-1 protein virulence factor secretion pathway, regulates mycolic acid lipid synthesis.

Pathogenic mycobacteria, which cause multiple diseases including tuberculosis, secrete factors essential for disease via the ESX-1 protein export system and are partially protected from host defenses by their lipid-rich cell envelopes. These pathogenic features of mycobacterial biology are believed to act independently of each other. Key ESX-1 components include three ATPases, and EccA1 (Mycobacterium marinum MMAR_5443; M. tuberculosis Rv3868) is the least characterized. Here we show that M. marinum EccA1's ATPase activity is required for ESX-1-mediated protein secretion, and surprisingly for the optimal synthesis of mycolic acids, integral cell-envelope lipids. Increased mycolic acid synthesis defects, observed when an EccA1-ATPase mutant is expressed in an eccA1-null strain, correlate with decreased in vivo virulence and intracellular growth. These data suggest that two mycobacterial virulence hallmarks, ESX-1-dependent protein secretion and mycolic acid synthesis, are critically linked via EccA1.

Francisella tularensis inhibits the intrinsic and extrinsic pathways to delay constitutive apoptosis and prolong human neutrophil lifespan.

Francisella tularensis is a facultative intracellular bacterium that infects many cell types, including neutrophils. We demonstrated previously that F. tularensis inhibits NADPH oxidase assembly and activity and then escapes the phagosome to the cytosol, but effects on other aspects of neutrophil function are unknown. Neutrophils are short-lived cells that undergo constitutive apoptosis, and phagocytosis typically accelerates this process. We now demonstrate that F. tularensis significantly inhibited neutrophil apoptosis as indicated by morphologic analysis as well as annexin V and TUNEL staining. Thus, ∼80% of infected neutrophils remained viable at 48 h compared with ∼50% of control cells, and ∼40% of neutrophils that ingested opsonized zymosan. In keeping with this finding, processing and activation of procaspases-8, -9, and -3 were markedly diminished and delayed. F. tularensis also significantly impaired apoptosis triggered by Fas crosslinking. Of note, these effects were dose dependent and could be conferred by either intracellular or extracellular live bacteria, but not by formalin-killed organisms or isolated LPS and capsule, and were not affected by disruption of wbtA2 or FTT1236/FTL0708-genes required for LPS O-antigen and capsule biosynthesis. In summary, we demonstrate that F. tularensis profoundly impairs constitutive neutrophil apoptosis via effects on the intrinsic and extrinsic pathways, and thereby define a new aspect of innate immune evasion by this organism. As defects in neutrophil turnover prevent resolution of inflammation, our findings also suggest a mechanism that may in part account for the neutrophil accumulation, granuloma formation, and severe tissue damage that characterizes lethal pneumonic tularemia.