Generation and External Validation of a Histologic Transformation Risk Model for Patients with Follicular Lymphoma.

Follicular lymphoma (FL) is the most frequent indolent lymphoma. Some patients (10%-15%) experience histologic transformation (HT) to a more aggressive lymphoma, usually diffuse large B-cell lymphoma (DLBCL). This study aimed to validate and improve a genetic risk model to predict HT at diagnosis.We collected mutational data from diagnosis biopsies of 64 FL patients. We combined them with the data from a previously published cohort (total n = 104; 62 from nontransformed and 42 from patients who did transform to DLBCL). This combined cohort was used to develop a nomogram to estimate the risk of HT. Prognostic mutated genes and clinical variables were assessed using Cox regression analysis to generate a risk model. The model was internally validated by bootstrapping and externally validated in an independent cohort. Its performance was evaluated using a concordance index and a calibration curve. The clinicogenetic nomogram included the mutational status of 3 genes (HIST1HE1, KMT2D, and TNFSR14) and high-risk Follicular Lymphoma International Prognostic Index and predicted HT with a concordance index of 0.746. Patients were classified as being at low or high risk of transformation. The probability HT function at 24 months was 0.90 in the low-risk group vs 0.51 in the high-risk group and, at 60 months, 0.71 vs 0.15, respectively. In the external validation cohort, the probability HT function in the low-risk group was 0.86 vs 0.54 in the high-risk group at 24 months, and 0.71 vs 0.32 at 60 months. The concordance index in the external cohort was 0.552. In conclusion, we propose a clinicogenetic risk model to predict FL HT to DLBLC, combining genetic alterations in HIST1H1E, KMT2D, and TNFRSF14 genes and clinical features (Follicular Lymphoma International Prognostic Index) at diagnosis. This model could improve the management of FL patients and allow treatment strategies that would prevent or delay transformation.

Cutaneous Involvement of Angioimmunoblastic T-Cell Lymphoma Masquerading as B-Cell Reactive Lymphoid Hyperplasia.

ABSTRACT: A 59-year-old woman presented with a persistent eruption manifested as multiple agminated miliary facial papules. Histopathological examination showed prominent nodular dermal lymphoid infiltrates with hyperplastic follicles that were initially interpreted as B-cell reactive lymphoid hyperplasia. Several years later, an additional biopsy showed a dense perifollicular infiltrate with reactive primary and secondary follicles. Accompanying T cells corresponded to CD3/CD4/PD1/CXCL13-positive cells and scattered Epstein-Barr virus-positive B cells were identified by in situ hybridization. A monoclonal T-cell population was demonstrated by TCRγ and TCRß Polymerase Chain Reaction amplification, as well as a minor abnormal circulating T-cell population by flow cytometry (0.62% of the white blood cells, CD4+CD3s-CD7-). A biopsy specimen from an enlarged right supraclavicular lymph node disclosed nodal involvement by angioimmunoblastic T-cell lymphoma. The observation of B-cell dermal nodular infiltrates with well-demarcated lymphoid aggregates forming primary lymphoid follicles may lead to overlook the T-cell component in some cases of angioimmunoblastic T-cell lymphoma. In such cases, a careful assessment of the apparently minor T-cell component is important to establish a correct diagnosis.

Diagnostic Value of Genotypic Analysis in Primary Cutaneous Lymphomas using Standardized BIOMED-2 Polymerase Chain Reaction Protocols: Experience in Daily Clinical Practice.

BIOMED-2 Concerted Action BMH4-CT98-3936 (BIOMED-2) PCR protocols are an important diagnostic tool in the evaluation of cutaneous lymphomas. The aim of this study was to assess the diagnostic value of the genotyping results obtained by these techniques in daily clinical practice. A total of 360 paraffin-embedded skin samples were retrospectively reviewed from 114 cutaneous T-cell lymphomas and 35 cutaneous B-cell lym-phomas. A total of 249 biopsies from 180 patients with benign lymphoid infiltrates served as controls. T-cell receptor and immunoglobulin gene rearrangements were assessed using the BIOMED-2 method. A combined T-cell receptor gamma and beta assay approach reliably distinguished cutaneous T-cell lymphomas from benign skin T-cell infiltrates (sensitivity 89.4%; specificity 81.5%). Analysis of complete immunoglobulin heavy chain rearrangements also differentiated cutaneous B-cell lymphomas from benign B-cell infiltrates (sensitivity 85.7%; specificity 82.4%). In conclusion, the full BIOMED-2 protocol is a useful aid combined with clinical, histological and immunophenotypical findings for assessment of lymphoid clonality in skin lymphoid proliferations.

Monomorphic Epitheliotropic Intestinal T-Cell Lymphoma With Secondary Cutaneous Involvement: A Diagnostic Challenge.

ABSTRACT: A 45-year-old woman presented with a solitary breast nodule that histologically corresponded to a dense dermal/subcutaneous infiltration of atypical cytotoxic T-lymphocytes (CD3+, CD8+, CD56+, TIA-1+, CD5-, CD4-, CD30-, EBV-), resembling subcutaneous panniculitic T-cell lymphoma. The presence of TCRδ gene rearrangement and the absence of ßF1 expression let to suspect the diagnosis of primary cutaneous γδT-cell lymphoma. As a consequence of jejunum perforation following chemotherapy treatment, a mucosal atypical lymphoid infiltration with marked epitheliotropism was observed in the resected intestinal sample, and the diagnosis of monomorphic epitheliotropic intestinal T-cell lymphoma (MEITL) was finally established. Disease progression appeared with multiple erythematous plaques showing a dense lichenoid atypical cytotoxic T-cell infiltrate with intense epidermotropism, mimicking primary cutaneous epidermotropic aggressive CD8+ T-cell lymphoma. MEITL is an uncommon and aggressive peripheral T-cell lymphoma that often presents in adults with gastrointestinal symptoms. Secondary cutaneous involvement is a rare phenomenon that may show clinicopathologic and immunohistochemical features that overlap with different subtypes of primary cutaneous cytotoxic T-cell lymphomas. In the absence of gastrointestinal symptoms, the diagnosis may be challenging, and only the evidence of underlying MEITL may allow to establish the definite diagnosis.

Pharmacological modulation of CXCR4 cooperates with BET bromodomain inhibition in diffuse large B-cell lymphoma.

Constitutive activation of the chemokine receptor CXCR4 has been associated with tumor progression, invasion, and chemotherapy resistance in different cancer subtypes. Although the CXCR4 pathway has recently been suggested as an adverse prognostic marker in diffuse large B-cell lymphoma, its biological relevance in this disease remains underexplored. In a homogeneous set of 52 biopsies from patients, an antibody-based cytokine array showed that tissue levels of CXCL12 correlated with high microvessel density and bone marrow involvement at diagnosis, supporting a role for the CXCL12-CXCR4 axis in disease progression. We then identified the tetra-amine IQS-01.01RS as a potent inverse agonist of the receptor, preventing CXCL12-mediated chemotaxis and triggering apoptosis in a panel of 18 cell lines and primary cultures, with superior mobilizing properties in vivo than those of the standard agent. IQS-01.01RS activity was associated with downregulation of p-AKT, p-ERK1/2 and destabilization of MYC, allowing a synergistic interaction with the bromodomain and extra-terminal domain inhibitor, CPI203. In a xenotransplant model of diffuse large B-cell lymphoma, the combination of IQS-01.01RS and CPI203 decreased tumor burden through MYC and p-AKT downregulation, and enhanced the induction of apoptosis. Thus, our results point out an emerging role of CXCL12-CXCR4 in the pathogenesis of diffuse large B-cell lymphoma and support the simultaneous targeting of CXCR4 and bromodomain proteins as a promising, rationale-based strategy for the treatment of this disease.

HHV8-related lymphoid proliferations: a broad spectrum of lesions from reactive lymphoid hyperplasia to overt lymphoma.

Human herpesvirus 8 (HHV8)-associated lymphoid proliferations are uncommon and poorly characterized disorders mainly affecting immunosuppressed patients, especially with HIV infection. They encompass different diseases with overlapping features that complicate their classification. In addition, the role of HHV8 in reactive lymphoid hyperplasia is not well known. To analyze the clinicopathological spectrum of these lesions, we have reviewed 66 biopsies of 61 patients with HHV8 infection. All cases were also investigated for Epstein-Barr virus (EBV) and HIV infection. We identified 13 (20%) cases of HHV8-related reactive lymphoid hyperplasia, 2 (3%) HHV8 plasmablastic proliferations of the splenic red pulp, 28 (42%) multicentric Castleman disease, 6 (9%) germinotropic lymphoproliferative disorders, and 17 (26%) HHV8-related lymphomas. As expected, the pathologic subtype was predictive of overall survival (P<0.05). Forty-seven of our cases were HIV positive (77%). In addition to the classical presentation of the different entities, we identified novel and overlapping features. Reactive HHV8 proliferations were frequently associated with systemic symptoms but never progressed to overt HHV8-positive lymphoma. Two cases had a plasmablastic proliferation limited to spleen. Eight cases of multicentric Castleman disease had a previously unrecognized presentation shortly after the diagnosis of HIV infection, six cases had cavity effusions, and three showed plasmablast enriched proliferations. One germinotropic lymphoproliferative disorder was EBV negative and three occurred in HIV-positive patients, who had distinctive clinical and morphological features. Two of the HHV8-related lymphomas did not fulfill the criteria for previously recognized entities. All these findings expand the clinical and pathological spectrum of HHV8-related lymphoid proliferations, which is broader than current recognized.

In vivo intratumoral Epstein-Barr virus replication is associated with XBP1 activation and early-onset post-transplant lymphoproliferative disorders with prognostic implications.

Post-transplant lymphoproliferative disorders are life-threatening complications following hematopoietic or solid organ transplantation. They represent a spectrum of mostly EBV-driven lymphoplasmacytic proliferations. While the oncogenic effect of EBV is related to latent infection, lytic infection also has a role in lymphomagenesis. In vitro, EBV replication is linked to plasma cell differentiation and XBP1 activation, although this phenomenon has never been addressed in vivo. We analyzed for the first time latent and lytic intratumoral EBV infection in a series of 35 adult patients with a diagnosis of post-transplant lymphoproliferative disorder (26M/9F, median age 54 years). A complete EBV study was performed including the analysis of the latent EBER, latent membrane protein-11, and EBV nuclear antigens as well as the immediate-early BZLF1/ZEBRA and early BMRF1/EADE31 lytic genes. XBP1 activation was assessed by nuclear protein expression. EBV infection was observed in 28 (80%) cases being latency II and III the most frequently observed 22 (79%). Intratumoral EBV replication was detected in 17 (60%) cases. Among these, XBP1 activation was observed in 11/12 evaluable cases associated with strong cytoplasmic immunoglobulin expression consistent with plasma cell differentiation. Intriguingly, the combination of latency III infection and EBV replication identified a high-risk subgroup of patients with significantly shorter survival (overall survival at 1 year 18% vs 48%) and early-onset (median of 7 vs 26 months) post-transplant lymphoproliferative disorder. Moreover, these patients appear to be more heavily immunosuppressed, so they exhibit lower rates of rejection and graft vs host disease but higher rates of cytomegalovirus reactivation. In conclusion, EBV replication is associated with plasma cell differentiation and XBP1 activation with prognostic implications. Both latency III and lytic EBV infection are related to aggressive and early-onset post-transplant lymphoproliferative disorder. These results suggest that immunohistochemical study of latent and lytic EBV genes in the clinical practice may help to select higher-risk patients to new therapies including antiviral treatments.

A new biologic prognostic model based on immunohistochemistry predicts survival in patients with diffuse large B-cell lymphoma.

Biologic factors that predict the survival of patients with a diffuse large B-cell lymphoma, such as cell of origin and stromal signatures, have been discovered by gene expression profiling. We attempted to simulate these gene expression profiling findings and create a new biologic prognostic model based on immunohistochemistry. We studied 199 patients (125 in the training set, 74 in the validation set) with de novo diffuse large B-cell lymphoma treated with rituximab and CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) or CHOP-like therapies, and immunohistochemical stains were performed on paraffin-embedded tissue microarrays. In the model, 1 point was awarded for each adverse prognostic factor: nongerminal center B cell-like subtype, SPARC (secreted protein, acidic, and rich in cysteine) < 5%, and microvascular density quartile 4. The model using these 3 biologic markers was highly predictive of overall survival and event-free survival in multivariate analysis after adjusting for the International Prognostic Index in both the training and validation sets. This new model delineates 2 groups of patients, 1 with a low biologic score (0-1) and good survival and the other with a high score (2-3) and poor survival. This new biologic prognostic model could be used with the International Prognostic Index to stratify patients for novel or risk-adapted therapies.

Chromogenic LMO2 mRNA ISH Expression Correlates with LMO2 Protein and Gene Expression and Captures Their Survival Impact in Diffuse Large B-Cell Lymphoma, NOS.

BACKGROUND: LMO2 is a relevant gene involved in B-cell ontogeny and a survival predictor of aggressive large B-cell lymphomas (aLBCL). Most studies assessing LMO2 mRNA expression have relied on microarray platforms or qRT-PCR methods, overlooking tissue morphology. In this study, we evaluate LMO2 RNA expression by chromogenic in situ hybridization (CISH) in normal tissue and in a series of 82 aLBCL. METHODS: LMO2 CISH was performed in formalin-fixed paraffin-embedded tissues, scored by three different methods, and correlated with a transcriptome panel. RESULTS: We obtained statistically significant results correlating the methods of evaluation with LMO2 protein expression and gene expression results. Normal tonsil tissue showed high levels of LMO2, particularly within the light zone of the germinal center. Conversely, in aLBCL, a notable reduction in LMO2 expression was noted, remarkably in cases carrying MYC rearrangements. Furthermore, significant results were obtained through overall survival and Cox regression survival analysis, incorporating International Prognostic Index data alongside LMO2 expression levels. CONCLUSIONS: We show a reliable method to identify LMO2 mRNA expression by CISH, effectively capturing many of the reported biologic features of LMO2.

Cell-Free DNA as a Biomarker at Diagnosis and Follow-Up in 256 B and T-Cell Lymphomas.

BACKGROUND: Cell-free DNA (cfDNA) analysis has become a promising tool for the diagnosis, prognosis, and monitoring of lymphoma cases. Until now, research in this area has mainly focused on aggressive lymphomas, with scanty information from other lymphoma subtypes. METHODS: We selected 256 patients diagnosed with lymphomas, including a large variety of B-cell and T-cell non-Hodgkin and Hodgkin lymphomas, and quantified cfDNA from plasma at the time of diagnosis. We further selected 49 large B-cell lymphomas (LBCL) and analyzed cfDNA levels at diagnosis (pre-therapy) and after therapy. In addition, we performed NGS on cfDNA and tissue in this cohort of LBCL. RESULTS: Lymphoma patients showed a statistically significant higher cfDNA concentration than healthy controls (mean 53.0 ng/mL vs. 5.6 ng/mL, p < 0.001). The cfDNA concentration was correlated with lymphoma subtype, lactate dehydrogenase, the International Prognostic Index (IPI) score, Ann Arbor (AA), and B-symptoms. In 49 LBCL cases, the cfDNA concentration decreased after therapy in cases who achieved complete response (CR) and increased in non-responders. The median cfDNA at diagnosis of patients who achieved CR and later relapsed was higher (81.5 ng/mL) compared with levels of those who did not (38.6 ng/mL). A concordance of 84% was observed between NGS results in tumor and cfDNA samples. Higher VAF in cfDNA is correlated with advanced stage and bulky disease. CONCLUSIONS: cfDNA analysis can be easily performed in almost all lymphoma cases. The cfDNA concentration correlated with the characteristics of the aggressiveness of the lymphomas and, in LBCL, with the response achieved after therapy. These results support the utility of cfDNA analysis as a complementary tool in the management of lymphoma patients.

Gene-expression profiling and not immunophenotypic algorithms predicts prognosis in patients with diffuse large B-cell lymphoma treated with immunochemotherapy.

Diffuse large B-cell lymphomas (DLBCLs) can be divided into germinal-center B cell-like (GCB) and activated-B cell-like (ABC) subtypes by gene-expression profiling (GEP), with the latter showing a poorer outcome. Although this classification can be mimicked by different immunostaining algorithms, their reliability is the object of controversy. We constructed tissue microarrays with samples of 157 DLBCL patients homogeneously treated with immunochemotherapy to apply the following algorithms: Colomo (MUM1/IRF4, CD10, and BCL6 antigens), Hans (CD10, BCL6, and MUM1/IRF4), Muris (CD10 and MUM1/IRF4 plus BCL2), Choi (GCET1, MUM1/IRF4, CD10, FOXP1, and BCL6), and Tally (CD10, GCET1, MUM1/IRF4, FOXP1, and LMO2). GEP information was available in 62 cases. The proportion of misclassified cases by immunohistochemistry compared with GEP was higher when defining the GCB subset: 41%, 48%, 30%, 60%, and 40% for Colomo, Hans, Muris, Choi, and Tally, respectively. Whereas the GEP groups showed significantly different 5-year progression-free survival (76% vs 31% for GCB and activated DLBCL) and overall survival (80% vs 45%), none of the immunostaining algorithms was able to retain the prognostic impact of the groups (GCB vs non-GCB). In conclusion, stratification based on immunostaining algorithms should be used with caution in guiding therapy, even in clinical trials.

MYC protein expression and genetic alterations have prognostic impact in patients with diffuse large B-cell lymphoma treated with immunochemotherapy.

MYC alterations influence the survival of patients with diffuse large B-cell lymphoma. Most studies have focused on MYC translocations but there is little information regarding the impact of numerical alterations and protein expression. We analyzed the genetic alterations and protein expression of MYC, BCL2, BCL6, and MALT1 in 219 cases of diffuse large B-cell lymphoma. MYC rearrangement occurred as the sole abnormality (MYC single-hit) in 3% of cases, MYC and concurrent BCL2 and/or BCL6 rearrangements (MYC double/triple-hit) in 4%, MYC amplifications in 2% and MYC gains in 19%. MYC single-hit, MYC double/triple-hit and MYC amplifications, but not MYC gains or other gene rearrangements, were associated with unfavorable progression-free survival and overall survival. MYC protein expression, evaluated using computerized image analysis, captured the unfavorable prognosis of MYC translocations/amplifications and identified an additional subset of patients without gene alterations but with similar poor prognosis. Patients with tumors expressing both MYC/BCL2 had the worst prognosis, whereas those with double-negative tumors had the best outcome. High MYC expression was associated with shorter overall survival irrespectively of the International Prognostic Index and BCL2 expression. In conclusion, MYC protein expression identifies a subset of diffuse large B-cell lymphoma with very poor prognosis independently of gene alterations and other prognostic parameters.

Round-robin testing for LMO2 and MYC as immunohistochemical markers to screen MYC rearrangements in aggressive large B-cell lymphoma.

Aggressive large B-cell lymphomas (aLBCL) include a heterogeneous group of lymphomas with diverse biological features. One of the approaches to the diagnosis of aLBCL is based on the identification of MYC rearrangements (MYC-R), in addition to BCL2 and BCL6 rearrangements by genetic techniques, mainly fluorescent in situ hybridization (FISH). Because of the low incidence of MYC-R, the identification of useful immunohistochemistry markers to select cases for MYC FISH testing may be useful in daily practice. In a previous work, we identified a strong association between the profile CD10 positive/LMO2 negative expression and the presence of MYC-R in aLBCL and obtained good intralaboratory reproducibility. In this study, we wanted to evaluate external reproducibility. To evaluate whether LMO2 can be a reproducible marker between observers 50 aLBCL cases were circulated among 7 hematopathologists of 5 hospitals. Fleiss' kappa index for LMO2 and MYC were 0.87 and 0.70, respectively, indicating high agreement between observers. In addition, during 2021-2022, the enrolled centers included LMO2 in their diagnostic panels to evaluate prospectively the utility of the marker, and 213 cases were analyzed. Comparing LMO2 with MYC, the group of CD10 positive cases showed higher specificity (86% vs 79%), positive predictive value (66% vs 58%), likelihood positive value (5.47 vs 3.78), and accuracy (83% vs 79%), whereas the negative predictive values remained similar (90% vs 91%). These findings place LMO2 as a useful and reproducible marker to screen MYC-R in aLBCL.

SOX11 is useful in differentiating cyclin D1-positive diffuse large B-cell lymphoma from mantle cell lymphoma.

AIMS: To characterize the frequency and clinicopathological features of cyclin D1-positive diffuse large B-cell lymphoma (DLBCL) and the usefulness of SOX11 in the differential diagnosis from mantle cell lymphoma (MCL). METHODS AND RESULTS: We retrospectively stained 206 consecutive DLBCLs for cyclin D1, and identified three (1.5%) positive cases, comprising two in the elderly with necrosis, and a third with a starry-sky pattern. All three cases shared the same non-germinal centre B-cell (non-GCB) phenotype [CD5-/CD10-/bcl-6+/MUM1+/SOX11-], Epstein-Barr virus (EBV) negativity, and absence of CCND1 aberrations by fluorescence in-situ hybridization. The third case showed both BCL6 and MYC rearrangements: a double-hit lymphoma. In the same period there were 22 MCLs, all expressing cyclin D1, with 89% cases expressing SOX11, a frequency that is statistically different from cyclin D1-positive DLBCL. Notably, we identified a pleomorphic MCL initially misdiagnosed as DLBCL. A separate cohort of 98 DLBCL cases was negative for SOX11, with only one case expressing cyclin D1 with a GCB phenotype (CD10+/bcl-6+/MUM1-). The two patients with tumour necrosis rapidly died of disease. The other two were in complete remission after immunochemotherapy. CONCLUSION: Cyclin D1-positive DLBCLs are rare, and they are negative for SOX11 or CCND1 aberration. SOX11 is useful in differentiating cyclin D1-positive DLBCL from MCL.

T cell lymphoblastic lymphoma with uncommon CD20 expression.

We report an interesting case of a T lymphoblastic lymphoma (T-LBL) with uncommon CD20 expression. Immunophenotype analysis showed TdT expression with positivity of CD3 and CD7 among other T-lineage markers. Other non-T markers such as CD79a or myeloid-associated antigens were negative. Molecular studies were performed evidencing clonal rearrangements for gamma and beta chain T cell receptor were clonal in the lymph node biopsy. Cytogenetic analysis showed deletions in CDKN2A and NOTCH2 genes. CD20 when present in T-LBL can lead to a false diagnosis of B-LBL. In this scenario, analysis of rearrangements of both immunoglobulin heavy chain B cell receptor and rearrangement of the T cell receptor can be very helpful.

PCM1::JAK2 fusion associates with an atypical form of mycosis fungoides.

Deregulation of JAK-STAT pathway seems to be relevant in mycosis fungoides (MFs). We report the case of a 23-year-old woman diagnosed of atypical MF carrying isolated PCM1::JAK2 fusion and eosinophilia. The disease was refractory to common treatments and progressed increasing the number of large CD30 positive T-cells. After progression, treatment with brentuximab vedotin was decided and decreased the proportion of large cells, but the low-grade component persisted, and the skin lesions worsened. Immunohistochemical expression of p-STAT3 detected in most tumor cells demonstrated the abnormal activation of JAK-STAT pathway. Very few cases of mature T-cell lymphomas carrying PCM1::JAK2 gene fusion have been reported to date, and we review previous cases described with this alteration. Described cases shared similar clinicopathological features and low genetic complexity, and the presence of PCM1::JAK2 fusion associates with a distinctive form of the disease.

Outcomes and molecular profile of oligomonocytic CMML support its consideration as the first stage in the CMML continuum.

Patients with oligomonocytic chronic myelomonocytic leukemia (OM-CMML) are currently classified according to the 2017 World Health Organization myelodysplastic syndromes classification. However, recent data support considering OM-CMML as a specific subtype of chronic myelomonocytic leukemia (CMML), given their similar clinical, genomic, and immunophenotypic profiles. The main purpose of our study was to provide survival outcome data of a well-annotated series of 42 patients with OM-CMML and to compare them to 162 patients with CMML, 120 with dysplastic type (D-CMML), and 42 with proliferative type (P-CMML). OM-CMML had significantly longer overall survival (OS) and acute myeloid leukemia-free survival than did patients with CMML, considered as a whole group, and when compared with D-CMML and P-CMML. Moreover, gene mutations associated with increased proliferation (ie, ASXL1 and RAS-pathway mutations) were identified as independent adverse prognostic factors for OS in our series. We found that at a median follow-up of 53.47 months, 29.3% of our patients with OM-CMML progressed to D-CMML, and at a median follow-up of 46.03 months, 28.6% of our D-CMML group progressed to P-CMML. These data support the existence of an evolutionary continuum of OM-CMML, D-CMML, and P-CMML. In this context, we observed that harboring more than 3 mutated genes, carrying ASXL1 mutations, and a peripheral blood monocyte percentage >20% significantly predicted a shorter time of progression of OM-CMML into overt CMML. These variables were also detected as independent adverse prognostic factors for OS in OM-CMML. These data support the consideration of OM-CMML as the first evolutionary stage within the proliferative continuum of CMML.

Cell-Free DNA for Genomic Analysis in Primary Mediastinal Large B-Cell Lymphoma.

High-throughput sequencing of cell-free DNA (cfDNA) has emerged as a promising noninvasive approach in lymphomas, being particularly useful when a biopsy specimen is not available for molecular analysis, as it frequently occurs in primary mediastinal large B-cell lymphoma (PMBL). We used cfDNA for genomic characterization in 20 PMBL patients by means of a custom NGS panel for gene mutations and low-pass whole-genome sequencing (WGS) for copy number analysis (CNA) in a real-life setting. Appropriate cfDNA to perform the analyses was obtained in 18/20 cases. The sensitivity of cfDNA to detect the mutations present in paired FFPE samples was 69% (95% CI: 60-78%). The mutational landscape found in cfDNA samples was highly consistent with that of the tissue, with the most frequently mutated genes being B2M (61%), SOCS1 (61%), GNA13 (44%), STAT6 (44%), NFKBIA (39%), ITPKB (33%), and NFKBIE (33%). Overall, we observed a 75% concordance to detect CNA gains/losses between DNA microarray and low-pass WGS. The sensitivity of low-pass WGS was remarkably higher for clonal CNA (18/20, 90%) compared to subclonal alterations identified by DNA microarray. No significant associations between cfDNA amount and tumor burden or outcome were found. cfDNA is an excellent alternative source for the accurate genetic characterization of PMBL cases.

Sézary syndrome patient-derived models allow drug selection for personalized therapy.

Current therapeutic approaches for Sézary syndrome (SS) do not achieve a significant improvement in long-term survival of patients, and they are mainly focused on reducing blood tumor burden to improve quality of life. Eradication of SS is hindered by its genetic and molecular heterogeneity. Determining effective and personalized treatments for SS is urgently needed. The present work compiles the current methods for SS patient-derived xenograft (PDX) generation and management to provide new perspectives on treatment for patients with SS. Mononuclear cells were recovered by Ficoll gradient separation from fresh peripheral blood of patients with SS (N = 11). A selected panel of 26 compounds that are inhibitors of the main signaling pathways driving SS pathogenesis, including NF-kB, MAPK, histone deacetylase, mammalian target of rapamycin, or JAK/STAT, was used for in vitro drug sensitivity testing. SS cell viability was evaluated by using the CellTiter-Glo_3D Cell Viability Assay and flow cytometry analysis. We validated one positive hit using SS patient-derived Sézary cells xenotransplanted (PDX) into NOD-SCID-γ mice. In vitro data indicated that primary malignant SS cells all display different sensitivities against specific pathway inhibitors. In vivo validation using SS PDX mostly reproduced the responses to the histone deacetylase inhibitor panobinostat that were observed in vitro. Our investigations revealed the possibility of using high-throughput in vitro testing followed by PDX in vivo validation for selective targeting of SS tumor cells in a patient-specific manner.

Worse outcome and distinct mutational pattern in follicular lymphoma with anti-HBc positivity.

Epidemiological studies have demonstrated the association between hepatitis B virus (HBV) infection and B-cell non-Hodgkin lymphoma (NHL), mainly for diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL). We studied a cohort of 121 patients with FL for HBV infection status, clinical features, and gene mutational profile. Anti-HBc was detectable in 16 patients (13.2%), although all had undetectable HBV DNA. Anti-HBcore+ (anti-HBc+) cases presented with older age at diagnosis than anti-HBc- cases (68.1 vs 57.2 years; P = .007) and higher ß2-microglobulin (56.3% vs 28.9%; P = .04). All patients included in the study fulfilled criteria for treatment and received therapy with rituximab or rituximab-containing chemotherapy. There were no episodes of HBV reactivation or HBV hepatitis during treatment and/or maintenance. Remarkably, anti-HBc+ patients had significantly lower 10-year progression-free survival (PFS; 12.9% vs 58.3%; P < .0001) and overall survival (OS; 22.0% vs 86.2%; P < .0001), that remained at multivariate analysis. Gene mutational profiling of all cases showed that anti-HBc+ cases had higher incidence of ARID1A mutations and absence of EP300 mutations, 2 key epigenetic regulators in FL. Overall, our study shows that FL patients with resolved HBV infection have a worse outcome independently of other well-known clinical risk factors and a distinct gene mutational profile.