The role of next-generation sequencing (NGS) for minimal residual disease (MRD) assessment in pediatric acute lymphoblastic leukemia (ALL) is still under consideration. Fifty pediatric patients were prospectively evaluated for specific clonal rearrangements of immunoglobulin and T-cell receptor genes using NGS analysis at diagnosis and on days 33 and 78 from therapy onset. The prognostic value or the NGS-MRD status was analyzed after a median follow-up of 4 years. All but one patient with negative NGS-MRD status on day 33 are in clinical remission. A total of 29 (58%) patients were NGS-MRD positive on day 33, of which 9 (18%) patients remained positive on day 78. However, only a small percentage of the patients with positive NGS-MRD status on day 33 and day 78 relapsed: 21% (6/29) and 33% (3/9), respectively. Positive NGS-MRD status is not a reliable prognostic biomarker in children with ALL and warrants careful consideration in disease stratification.

Fibrin-associated large B-cell lymphoma (FA-LBCL) has been recognized as a distinct entity in the 5th WHO classification of hematolymphoid tumors. It is a rare Epstein-Barr virus-positive B-cell neoplasia that arises in various sites of chronic fibrin deposition associated with (pseudo-)cystic cavities, chronic hematomas, cardiac myxomas, or prostheses. Cystic lymphangiomatous lesions of the adrenal gland are rare, benign vascular lesions that are most commonly asymptomatic. However, they are occasionally discovered incidentally and then often removed to rule out other, more serious diagnoses. The present case report documents a highly unusual instance of a 45-year-old female patient who was diagnosed with a FA-LBCL arising from a long-standing cystic lymphangiomatous lesion of the adrenal gland, representing a co-occurence of two rare diagnoses. We provide a comprehensive report of the morphological, immunohistochemical, and molecular features of this case of FA-LBCL and give a brief overview of this emerging lymphoma entity.

Pure red cell aplasia (PRCA) is a rare hematologic syndrome characterized by anemia with marked reticulocytopenia and, in Asia, is often accompanied by T-cell large granular lymphocyte leukemia (T-LGL). Minimal research has been done on the epidemiology and sequential events of PRCA combined with T-LGL. This study identified 2801 PRCA and 840 T-LGL patients by using big data of the National Health Insurance Service between 2003 and 2022. The average annual crude incidence of PRCA was 2.77 per million and remained stable over 20 years, while T-LGL incidence was 0.82 per million with an increasing trend, possibly reflecting improved diagnostic accessibility. The average age for PRCA and T-LGL onset increased over the study period, consistent with aged society. Associated PRCA conditions are rheumatic diseases (10.5%), thymoma (4.7%), parvovirus infection (1.0%), inflammatory bowel diseases (0.8%), T-LGL (0.6%) and no specific cause (82.4%). Among 18 patients with both PRCA and T-LGL, PRCA preceded T-LGL (50%) or diagnosed concurrently (44%), suggesting that autoreactive T cells in PRCA which suppress erythropoiesis and sequentially evolve into clonal T cell proliferation and, eventually, T-LGL occurrence. This observation supports the hypothesis that both conditions might share a common pathogenic pathway. Further study should identify the causal relationship of PRCA diagnosis followed by T-LGL diagnosis.

Bcl2-positive germinal centres (GCs) have never been documented in reactive lymphoid hyperplasia (RLH). Here we describe such phenomenon in paediatric chronic tonsillitis (PCT), addressing the differential diagnosis with paediatric lymphomas.

Six PCT cases with Bcl2-positive GCs were retrieved from a retrospective series of 166 tonsillectomies from children and adolescents. Clinical-pathological data were collected, also considering the status of IG rearrangements and BCL2 translocations. PCT with Bcl2-positive GCs mostly occurred in males (5/6 cases; median age: 5 years). Histologically, tonsil architecture was preserved and Bcl2 positivity was documented in a minority of GCs. Bcl2-positive GCs expressed CD10 and Bcl6 and had a high proliferation index. All cases had polyclonal IG rearrangements without evidence of monotypic kappa and lambda chains by RNAscope. BCL2 translocations were lacking in all the cases.

Bcl2-positive GCs in PCT are a rare and benign phenomenon, expanding the spectrum of lymphoma-mimicking paediatric RLH.

T-cell chronic lymphoproliferative disorders (T-CLPD) are a heterogeneous group of mature T-cell malignancies, the classification of which remains challenging. In this study, we classified tumor cells from 86 patients diagnosed with either T-CLPD (n = 81) or T-cell acute lymphoblastic leukemia (n = 5) into precise functional and maturation-associated compartments, based on their phenotypic similarities with their normal maturation-related and functional associated T-cell counterparts. A database was generated using blood samples from 6 sex- and age-matched healthy donors as a template for normal T-cell subset flow cytometric immunophenotypes, to which tumor cells of individual patients were compared. Except for nodal T follicular-helper cell lymphoma and adult T-cell leukemia/lymphoma, which showed phenotypes overlapping with that of T follicular-helper and T regulatory cells, respectively, all other T-CLPD displayed immunophenotypic profiles consistent with conventional T helper (Th) cells, with different maturation-associated profiles per diagnostic category. These included predominant naive/naive-central memory phenotypes in T-cell prolymphocytic leukemia to terminal effector cytotoxic cellular profiles in T-cell large granular lymphocytic leukemia; other T-CLPD diagnostic categories (mostly Sézary syndrome/mycosis fungoides) resembled the diverse memory T-cell subsets. Interestingly, immunophenotypically less-mature tumor cells (T-cell prolymphocytic leukemia) displayed more heterogeneous Th profiles, whereas those with memory T-cell profiles showed more consistent Th-associated patterns (eg, Th2 or Th17 in Sézary syndrome/mycosis fungoides), and the most mature neoplasms (eg, T-cell large granular lymphocytic leukemia) systematically displayed a Th1-like pattern, reflecting progressively lower plasticity for the more advanced tumor-associated maturation stages. These findings confirm the presence of distinct phenotypic patterns resembling specific maturation-associated and Th-related profiles of normal T cells among distinct diagnostic categories of T-CLPD, which might contribute to a more precise classification of T-CLPD.

Sjögren's Disease (SjD) is a systemic autoimmune disease with an increased risk of developing hematolymphoid neoplasms, with mucosa-associated marginal zone lymphoma being the most common. This could be related to the lack of a diagnostic test to achieve early diagnosis. Clonal rearrangements are molecular tests used in the diagnosis of lymphoid neoplasms that have proven to be supportive in the diagnosis and may be useful in the early diagnosis process when applied to minor salivary gland biopsies (MSGB).

Cross-sectional study including MSGB with a diagnosis of SjD between 2019 and 2022 at a university hospital in Bogota, Colombia. Sociodemographic data and histopathological characteristics were collected. Immunohistochemical studies and rearrangement tests were then performed according to the BIOMED-2 protocol.

Rearrangements were performed for T cell receptors and B cell immunoglobulins. A polyclonal result was found in most cases. Four cases were oligoclonal in IgH and two isolated clonal results: one in the TCR β segment and the other in the kappa light chain segment of immunoglobulin.

Clonal rearrangements may be a useful tool for the early diagnosis of hematolymphoid neoplasms. Further studies with longer follow-up and application to MSGB are needed to better define a clonal pattern.

The clonality of 14 feline intestinal small and large T-cell lymphomas were examined, which consisted of concurrent mucosal and transmural lymphomas, respectively. Histologically, the small cell lymphomas were localized to the mucosal region and were observed adjacent to large T-cell lymphoma lesions. The large T-cell lymphomas were spread throughout the submucosal region, forming transmural lesions. Both cell types were immunohistochemically stained using anti-cluster of differentiation 3 antibody. For clonality analysis, genomic DNA was extracted from formalin-fixed, paraffin-embedded sections of the mucosal and transmural lesions, separately. Clonality analysis was performed using primer sets targeting T cell receptor beta, T cell receptor delta, and T cell receptor gamma loci. In 12/14 cats, the results of the clonality analysis for T-cells differed between the mucosal and transmural lesions. Despite the fact that the cellular morphologies differed between lesion types, the T-cell clonality was consistent in 1/14 cats, suggesting a common clonal origin. In the remaining case, the clonal relationship between the 2 lesions could not be determined. These results indicate that concurrent lymphomas with small and large T-cells in their mucosal and transmural lesions, respectively, develop via separate pathogenic mechanisms.

Bone marrow involvement (BMI) is a poor prognostic factor in diffuse large B cell lymphoma (DLBCL), and accurate evaluation of BMI is crucial for determining stages and prognosis. This study aimed to identify the most effective examinations for evaluating BMI in DLBCL, including positron emission tomography-computed tomography (PET/CT), immunoglobulin gene rearrangement (IGR), flow cytometry (FCM), bone marrow cytology (BMC) and bone marrow biopsy pathology (BMB), and to further explore its prognostic significance in DLBCL patients.

This retrospective study included 364 newly diagnosed DLBCL patients, all of whom underwent PET/CT, IGR, FCM, BMC, and BMB at diagnosis. Survival outcomes were analyzed via Kaplan-Meier and Cox regression models. Novel prognostic models incorporating combined IGR and BMB results were developed in a training cohort.

Compared to other detection methods, Clonal IGR BMI-positive were found the highest rate of 114 patients (31.3%), and IGR BM involvement-positive patients of DLBCL had the worst survival outcomes, especially among patients in stages I to III (P<0.001). Notably, PET/CT existed some limitations in BMI diagnosis, particularly in stage IV patients (P>0.05). Additionally, the combination of IGR and BMB demonstrated superior prognostic predictive capability for the patients in stage IV (PP<0.001). Multivariate analysis further confirmed that double-positive BMI of IGR and BMB was an independent prognostic factors of PFS (P=0.026) and OS (P=0.042). In addition, the novel IPI and NCCN-IPI stratification models were established by incorporating the combination of IGR and BMB in training group. The C-index of novel models were increased when IGR and BMB were supplemented in our cohort.

Our results suggest that IGR is the most valuable methods for evaluating BMI compared to traditional detection methods. Adding the combination of IGR and BMB to the IPI and NCCN-IPI score may improve their predictive ability. In summary, IGR is essental for evaluation of BMI and provide an ideal method for disease staging and risk stratification in DLBCL patients in the rituximab era.

We examined the epidemiology of parotid gland lymphomas (PGL), the incidence, survival rates, clinical features, and association with primary Sjögren's syndrome (pSS).

This retrospective nationwide cohort study analyzed data from Danish patients diagnosed with PGL between 2000 and 2020. Data were collected from medical records, the National Pathology Register, and the Danish lymphoma database. Statistical analyses included Kaplan-Meier curves, log-rank tests, and Cox proportional hazards models.

A total of 433 patients were included. The incidence rate was 0.39 per 100,000 person-years, with PGL constituting 1.9% of all non-Hodgkins lymphoma in Denmark. The average annual incidence was 2.7% (incidence rate ratio = 1.027, p < 0.01). Follicular lymphoma (FL) was the most common subtype with 154 cases (35.6%), followed by large B-cell lymphoma (LBCL) with 119 cases (27.5%), and extranodal marginal zone lymphoma (EMZL) with 84 cases (19.4%). The median overall survival (OS) for FL was 9.5 years (95% CI 6.9-10.2), with 5-year and 10-year OS rates of 70% and 44%, respectively. For LBCL, the median OS was 7.8 years (95% CI 5.0-8.8), with 5-year and 10-year OS rates of 59% and 33%. EMZL had a median OS of 12.8 years (95% CI 9.0-16.3), with 5-year and 10-year OS rate of 83% and 55%. EMZL was significantly associated with pSS, relative risk 21.97 (95% CI 2.81-171.53). Advanced age, B symptoms, and elevated LDH levels were significantly linked to poorer overall survival.

This study offers new epidemiological, clinical, and prognostic insights, with a focus on their association with pSS.

3 Laryngoscope, 135:1391-1400, 2025.

Lymphocytic variant hypereosinophilic syndrome (L-HES) is a rare disorder characterized by persistent eosinophilia driven by aberrant T-cell populations. Diagnosis remains challenging due to the lack of standardized diagnostic criteria.

We retrospectively analyzed 18 patients diagnosed with L-HES between 2016 and 2023. Comprehensive flow cytometry was performed on peripheral blood samples.

Nine patients demonstrated the classic sCD3-CD4+CD5+CD2+CD45RO+CD45RA- immunophenotype, ranging from 0.6% to 70% of total lymphocytes. Two patients showed variant sCD3-CD4+ phenotypes, five had expanded (> 10%) sCD3+CD4+CD7- T-cells, and two displayed aberrant CD8+ T-LGL populations. Clonality was established in all patients with nonclassic phenotypes by molecular TCR testing or based on uniform TRBC1. We assessed a serial gating strategy to quantify the classic L-HES phenotype and found this to be highly sensitive and specific with an estimated limit of detection of 0.06% of lymphocytes. Using this strategy, we identified decreased but detectable abnormal T-cells in all classic phenotype patients reassessed posttreatment, down to as low as 0.3% of lymphocytes. The identification of T-LGL phenotypes with eosinophilia is a novel finding.

Our study highlights the diverse immunophenotypic spectrum of L-HES, emphasizing the importance of comprehensive flow cytometry analysis for accurate diagnosis.

This study explores the epidemiology, incidence, and survival outcomes associated with lymphomas of the submandibular gland (SMG) and examines the influence of autoimmune diseases on these parameters.

This retrospective nationwide cohort study analysed data from patients diagnosed with SMG lymphomas in Denmark between 2000 and 2020. Information was extracted from medical records, the National Pathology Register, and the Danish Lymphoma Database. Survival analyses were conducted using Kaplan-Meier curves, log-rank tests, and Cox proportional hazards models, focusing on lymphoma subtypes and autoimmune diseases.

The cohort consisted of 101 patients with a lymphoma diagnosis and involvement of the SMG. Large B-cell lymphoma (LBCL) was diagnosed in 33 cases (32.7%), follicular lymphoma (FL) in 29 cases (28.7%), extranodal marginal zone lymphoma (EMZL) in 27 cases (26.7%), and 12 cases (11.9%) with other subtypes. EMZL had a significantly longer overall survival (OS) compared to other subtypes, with a median OS of 12.4 years (95% CI 11.2-12.4) vs. 8.4 years (95% CI 6.0-12.2). EMZL and FL showed favourable 5-year OS rates of 95% and 89%, respectively. LBCL had a 5-year OS rate of 65%. Age over 60 significantly negatively impacted OS. Traditional poor prognostic indicators did not significantly affect OS. A notable association between EMZL and autoimmune diseases was observed, particularly with Sjögren's syndrome, indicated by an increased relative risk of 2.67 (CI 95% 0.45-16.01).

Lymphomas of the SMG are rare and have ambiguous clinical presentations. This study provides novel epidemiological, clinical, and prognostic information.

Primary cutaneous marginal zone lymphoma (PCMZL) is considered a lymphoproliferative disorder (International Consensus Classification, ICC) or an overt lymphoma (WHO-HAEM5). Seeking evidence for a reactive process or true lymphoma, we retrieved recurrent PCMZLs from the French Study Group of Cutaneous Lymphoma (GFELC) database.

Histology, phenotype (light-chain restriction, immunoglobulin, and immune-receptor translocation-associated protein-1 [IRTA1] expression) and B-cell clonality at diagnosis and recurrence were compared according to recurrence site (local, locoregional, or distant) and outcomes.

Initial lesions of the 61 patients (mean age 52) were mostly isolated on the trunk (48%) and classified T1 (70%). Times to first recurrence for local, locoregional, and distant recurrences, were 20, 29, and 37 months, respectively. Light-chain restriction type did not differ significantly between local/locoregional recurrences and distal recurrences (P = 0.06; n = 60). The same B-cell clones were identified for 23/42 local/locoregional recurrences, while 5/19 distant recurrences showed different clonal profiles (P = 0.0003). No tumour expressed IRTA1. Fifty-eight tumours were heavy-chain (IgG/IgG4) class-switched PCMZLs and 3 IgM+/IgD- PCMZLs. All IgM+ tumours underwent either transformation (skin or brain) into diffuse large B-cell lymphomas (DLBCLs) and extracutaneous spreading.

As suggested by WHO-HAEM5, immunoglobulin phenotype assessment (IgM alongside IgD) appears to be a possible valuable tool in the initial diagnosis of PCMZL to differentiate between the indolent class-switched PCMZL (IgM-negative) and IgM+ (IgD-) PCMZL, which has an uncertain prognosis. The variation in B-cell rearrangements and light chain restriction observed in distant recurrences of PCMZL may suggest different antigen-driven stimulation processes.

The interactions between T-cell chronic lymphoproliferative disorder (T-CLPD) tumor cells and the bystander immune cells may play a critical role in the failure of immune surveillance and disease progression, but the altered blood immune profiles of T-CLPD remain unknown.

Here we analyzed the distribution of residual non-tumoral immune cells in blood of 47 T-CLPD patients -14 T-prolymphocytic leukemia (T-PLL), 7 Sézary syndrome/mycosis fungoides (SS/MF) and 26 T-large granular lymphocytic leukemia (T-LGLL)-, as tumor models of neoplastic T-cells that resemble naive/central memory (N/CM), memory and terminal effector T-cells, respectively, compared to 110 age- and sex-matched healthy donors, using spectral flow cytometry.

Overall, our results showed deeply altered immune cell profiles in T-PLL, characterized by significantly increased counts of monocytes, dendritic cells, B-cells, NK-cells and innate lymphoid cells (ILC) -particularly ILC3-, together with reduced normal T-cells. In contrast, SS/MF showed neutrophilia, associated with decreased numbers of dendritic cells and NK-cells, potentially reflecting their increased migration from blood to the skin. In turn, T-LGLL displayed the mildest immune impairment, dependent on the TCD4+ vs. TCD8+ nature of the clonal T-cells and presence of STAT3 mutations among TαβCD8+ T-LGLL cases. Further dissection of the normal T-cell compartment showed a significant reduction of the earliest T-cell maturation compartments (N/CM) in T-PLL and SS/MF, whereas T-cells remained within normal ranges in T-LGLL, with only a minor reduction of N/CM T-cells.

These findings point out the existence of differentially altered innate and adaptive immune cell profiles in the distinct diagnostic subtypes of T-CLPD, with progressively less pronounced alterations from T-PLL and SS/MF to T-LGLL.

Peripheral T-cell lymphomas with gamma-delta phenotype (GDTCL) are rare lymphoid malignancies. Beyond the well-recognized entities of extranodal lymphomas with gamma-delta phenotype as defined by the fifth edition of the World Health Organization Classification of Hematolymphoid Tumors and 2022 International Consensus Classification, there is a group of poorly defined gamma-delta T-cell lymphomas with predominantly nodal presentation, termed as nodal GDTCL (nGDTCL). In this study, we present a series of 12 cases of Epstein-Barr virus-negative nGDTCL, highlighting the clinical, histopathological, and molecular features of this rare entity. Seven cases reported in the literature were included in the analysis. Of the 12 cases, nGDTCL shows an increased incidence in elderly men, with a median age of 65.5 years. All cases presented primarily with enlarged lymph nodes, and 4 cases (4/12, 33.3%) showed involvement of extranodal sites, including skin, liver, spleen, and bone marrow. Histologically, 9 cases showed a diffuse and monomorphic proliferation of mostly medium-to-large lymphoid cells, whereas 3 cases demonstrated lymphoepithelioid morphology. All cases (12/12, 100%) were positive for CD3 and TCRγδ. CD4, CD8, and CD56 were positive in 66.7% (8/12), 25% (3/12), and 8.3% (1/11) of cases, respectively. Most cases (8/12, 66.7%) showed a noncytotoxic phenotype. Using immunohistochemistry, the majority of cases (6/8, 75.0%) belonged to the peripheral T-cell lymphoma-GATA3 subtype with GATA3 and/or CCR4 expression and a noncytotoxic CD4-positive phenotype. Two cases (2/8, 25%) belonged to the peripheral T-cell lymphoma-TBX21 subtype, of which 1 displayed a cytotoxic CD8-positive phenotype. Next-generation sequencing was performed in 9 cases, and TP53 mutation was detected in 66.7% (6/9) of the cases. Mutations of ATM and KSR2 were identified in 2 cases each. It remains uncertain whether nGDTCL represents a distinct entity, and further studies are needed for better characterization. Nonetheless, nodal-based GDTCL should be distinguished from secondary nodal involvement by other extranodal GDTCL and Epstein-Barr virus-positive T/NK-cell lymphoproliferative diseases.

Mycosis fungoides (MF) is a rare malignancy that is characterized by the presence of circulating tumour cells (CTCs) in a subgroup of patients. Reliably distinguishing MF from inflammatory skin conditions is challenging.

To evaluate the potential benefits of next-generation sequencing (NGS)-based T-cell receptor rearrangement repertoire analysis in detecting clonal rearrangements in MF and inflammatory skin conditions.

Skin biopsies and blood samples from 33 patients with MF and 10 patients with inflammatory skin conditions were analysed using TRB and TRG NGS. Twenty-seven patients had early-stage IA (n = 19) and IB (n = 8) MF, and six had advanced-stage disease (IIB, n = 5; IIIA, n = 1).

Analysis applying standard abundance thresholds identified at least one clonal rearrangement in the skin DNA of 97% (n = 32/33) of patients with MF and in 90% (n = 9/10) of those with inflammatory skin conditions. To enhance specificity, an abundance and distribution-based approach was applied, which considered only rearrangements that significantly stood out from the physiological background as clonal (MF, n = 29/33; inflammatory skin conditions, n = 1/10), allowing for highly sensitive (88%) and specific (90%) discrimination between MF and other inflammatory skin conditions. CTCs were detected in 46% (n = 11/24) of patients with early-stage MF and in 60% (n = 3/5) of those with late-stage MF.

NGS-based T-cell receptor repertoire analysis is a highly sensitive and specific method for the differential diagnosis of early-stage MF vs. inflammatory skin conditions, and for the sensitive molecular detection of CTCs.

Algae, a crucial constituent of marine systems, serve an indispensable function as primary producers, supporting the marine food web, contributing to carbon sequestration, and providing habitats that sustain biodiversity. This review focuses on the bioactive constituents of algae, including polysaccharides, polyphenols, polypeptides, and terpenoid compounds, and discusses their potential applications in treating immune-related diseases, as well as the mechanisms through which they modulate immune responses. The bioactive substances derived from algae, including polyphenols, bioactive peptides, terpenes, polysaccharides and other compounds, may play a preventive role by modulating allergic responses and reducing the incidence of inflammation and cancer.

The group of patients with DNA-repair-defects increases susceptibility to infections due to impaired repertoire diversity. In this context, we aimed to investigate the TCRvβ-repertoire by flow cytometric analysis and its correlation with clinical entities in a group of IEI patients with DNA repair defects. Peripheral lymphocyte subset and TCRvβ-repertoire analyses were performed by flow cytometric analysis. The aim was to explore the changing TCR-Vβ-repertoire that can predict some clinical entities by investigating the repertoire using flow-cytometric-analysis-based TCR-Vβ and its interaction with clinical entities in a group of IEI patients with DNA repair defects. TCR-repertoire of the patients with DNA-repair-defects and healthy controls was analysed with flow-cytometer. The potential of flow-cytometric analysis of the TCR repertoire as a practical and easily accessible clinical prediction method was investigated. Thirty-nine-IEI patients with DNA-repair-defects and 15 age-matched healthy-controls were included in this study. Peripheral lymphocyte subset and TCR-Vβ repertoire analyses were performed by flow cytometry. Compared to the control group, 9 out of 24 clones (37.5%) exhibited a statistically significant reduction, while only 3 clones showed a statistically significant increase (p < 0.05). Preferential use of vβ-genes was associated with some clinical entities. Lower TCR-vβ-9 and TCR-vβ23, higher TCR-vβ7.2 were found in the patients with pneumonia (n = 13) (p = 0.018, p = 0.044 p = 0.032). AT patients with pneumonia had lower TCR-vβ-9 clone than patients without pneumonia (p = 0.008). Skewed proliferation of most TCR-vβ clones was seen DNA-repair-defects, especially AT. In addition, this study showed that preferential use of TCR-vβ genes could be predictive for some clinical entities.

Mycosis fungoides (MF) and Sezary syndrome (SS) are common entities among primary cutaneous lymphomas. Large cell transformation is challenging for diagnosis and therapy. Molecular mechanisms by which these lymphomas undergo this transformation are poorly defined. We studied the immunohistochemical status of p53 in these entities and assessed whether p53 expression could be a useful tool for diagnosis and assessment of transformation. We extracted patients with transformed and untransformed SS or MF from the French Study Group on Cutaneous Lymphoma database between 2014 and 2021, followed in the Rennes University Hospital. An immunohistochemical study of p53 expression was performed on the biopsies sampled as part of routine care. We compared p53 overexpression in the different groups. We included 25 patients with MF, 7 patients with transformed MF (T-MF), 11 patients with SS, and 5 patients with transformed SS (T-SS). Using a cut-off set at 30% expression of neoplastic cells, we noted an overexpression of p53 in T-MF and T-SS compared with nontransformed forms (47% vs. 12%, respectively, P < 0.01) and in MF compared with SS (23% vs. 7%, respectively, P < 0.01). Overexpression of p53 with a cut-off at 30% therefore seems to be a discriminating tool in the differential diagnosis of MF/SS versus their transformed forms as well as the differential diagnosis between MF and SS.

Epstein-Barr virus (EBV)-associated post-transplant lymphoproliferative disease (PTLD) is predominantly of B cell origin. The concept of clonal evolution from poly- to monoclonal lymphoproliferation has been put forward, but T-cell PTLDs are rare with an unknown etiology. In a unique autopsy case of a 53-year-old man with EBV-associated T-cell PTLD, we observed polymorphic T-cell proliferation across several organs and monomorphic T-cell proliferation in the perforated ileum. Interestingly, both manifestations exhibited identical monoclonal peaks in the T-cell receptor rearrangement polymerase chain reaction (PCR) analyses. These findings suggest the existence of clonal evolution in EBV-associated T-cell PTLD, leading to the proposal of the novel concept of polymorphic T-cell PTLD.

Lymphatic filariasis is caused by infections of thread-like filarial worms, namely Wuchereria bancrofti, Brugia Malayi and Brugia timori. However, in-depth analysis of the antibody repertoire against Lymphatic filariasis is lacking. Using high-throughput sequencing of antibody repertoires, immunome analysis of IgG (LG) and IgM (LM) repertoires were studied. Despite significant differences between LG and LM in V(D)J gene usage, IGHV4-34, IGHV6-1, IGHD3-10 and IGHJ4 were preferred in both repertoires. The CDR3 in the LG repertoire showed a longer length than LM. Higher SHM level were observed in LG sequences and presence of oligoclonal sequences indicates the extent of clonal expansion. The prevalence of rare clonotypes in LM repertoire depicts the high clonal diversity when compared to LG repertoire. Monoclonal antibodies against closely related parasitic infections were present within the LG repertoire suggesting that immune repertoires may not be as exclusive and biased against the target infection as initially thought. The characterization of the immune repertoire can provide critical insight into the antibody response patterns in disease state, antibody generation process during infections and future antibody designs.

Plasma cell neoplasia and POEMS syndrome patients may present Castleman disease (CD)-type features in lymph nodes. Our aim was to better characterize the histopathological patterns found in plasma cell neoplasia associated CD and to improve the detection of clonal plasma cell populations in the lymph node biopsies of these patients. Lymph node and bone marrow samples from six cases with plasma cell neoplasia associated CD, including POEMS syndrome and multiple myeloma were analyzed. A complete analysis including morphology, IHC and PCR-based clonality detection in the lymph node biopsies and morphology; IHC and FCM in the bone marrow biopsies was done. Correlation with clinical and laboratory features was performed. Both plasma cell rich and hyper vascular Castleman disease like histopathological features were found. In two out of six cases, sheets of plasma cells with light chain and isotype restriction were identified in the lymph nodes. B cell clonality analysis provided evidence of clonal populations in 5 out of 6 cases, increasing the sensitivity for the detection in cases without morphologically identifiable monotypic plasma cells. None of the twelve control cases with iMCD and HHV-8 positive CD showed clonal populations. In conclusion, the combination of morphology, complete immunophenotyping and B cell clonality analysis in the lymph node biopsy may provide evidence of clonal populations in up to 83 % of cases with plasma cell neoplasia/POEMS syndrome associated Castleman disease. Clinical work-up for the confirmation of plasma cell dyscrasia is advisable in such cases.

T-cell clonality assessment constitutes an essential part of the diagnostic evaluation of suspected T-cell neoplasms. Recent advances in flow cytometry-based analysis of TCR β-chain constant region 1 (TRBC1) have introduced an accurate method of assessment of T-cell clonality. Its broader applicability is constrained due to the requirement of viable cells. Furthermore, the utility of the TRBC1 antibody in tissue immunohistochemistry (IHC) has not been comprehensively addressed. Herein, we validated an IHC-based approach to assess T-cell clonality using formalin-fixed, paraffin-embedded tissue. Utilizing DeepLIIF image analysis, we quantified TRBC1 positivity among CD3-positive cells in a training cohort comprising 34 cases of α/β T-cell neoplasms and 29 cases of reactive lymphoid tissue as controls. In an independent validation cohort comprising 29 T-cell neoplasms and 20 controls, similar image quantification was conducted by a pathologist uninvolved in the analysis of the training cohort and blinded to the diagnoses. Receiver operating characteristic analysis of the training cohort established the optimal cutoff points for monotypic TRBC1 expression-79.0% or higher indicating monotypic positivity and 36.3% or lower denoting negativity. These thresholds demonstrated robust metrics in both the training (sensitivity 88.2%, specificity 93.1%, positive predictive value 93.8%, negative predictive value 87.1%) and the validation cohorts (sensitivity 93.1%, specificity 95.0%, positive predictive value 96.4%, negative predictive value 90.5%). TRBC1 IHC was correlated with flow cytometry in 52 cases, which demonstrated a strong quantitative correlation of TRBC1 positivity (r = 0.78; P <.001) and a high categoric agreement (85.9%) in classifying monotypic versus polytypic staining. Discrepancies in categorization were associated with low tumor percentages. Furthermore, multiplex immunofluorescence was performed in 15 cases for targeted quantification of TRBC1 expression in CD3-positive, PAX5-negative cells, achieving a concordance of 86.7% with IHC. In summary, TRBC1 IHC offers a reliable and practical complementary method for assessing T-cell clonality.

Assessment of the presence of clonal lymphoproliferations via polymerase chain reaction (PCR)-based analysis of rearranged immunoglobulin (IG) or T-cell receptor (TR) genes is a valuable method in the diagnosis of suspect lymphoproliferative disorders. Additionally, this methodology can be used for evaluating dissemination of lymphoma cells and for studying the clonal relationship between multiple (different locations) and consecutive (over time) lymphomas. Here we describe an integrated approach to assess clonality via analysis of Ig heavy chain (IGH), Ig kappa (IGK), TCR beta (TRB), and TCR gamma (TRG) gene rearrangements, based on the standardized multiplex PCRs as originally developed by the European BIOMED-2 consortium (currently named EuroClonality). The described protocol covers the pre-analytical phase of DNA isolation (from formalin-fixed paraffin-embedded and fresh tissues, body fluids, peripheral blood, and bone marrow), the analytical phase of PCR GeneScan analysis, and the post-analytical interpretation of the obtained profiles, following established guidelines.

Flow cytometric (FC) immunophenotyping and T-cell receptor (TCR) gene rearrangement studies are essential ancillary methods for the characterisation of T-cell lymphomas. Traditional manual gating and polymerase chain reaction (PCR)-based analyses can be labour-intensive, operator-dependent, and have limitations in terms of sensitivity and specificity. The objective of our study was to investigate the efficacy of the Phenograph and t-SNE algorithms together with an antibody specific for the TCR β-chain constant region 1 (TRBC1) to identify monoclonal T-cell populations. FC- and PCR-based clonality analyses were performed on 275 samples of T-cell lymphomas, B-cell lymphomas, and reactive lymphocytic proliferations. Monotypic T-cell populations were identified in 65.1% of samples by manual gating and 72.4% by algorithm-driven analysis, while PCR-based analysis detected clonal T cells in 68.0%. Of the 262 monotypic populations identified, 46.6% were classified as T-cell lymphomas and 53.4% as T-cell populations of uncertain significance (T-CUS). Algorithm-driven gating identified monotypic populations that were overlooked by manual gating or PCR-based methods. The study highlights the difficulty in distinguishing monotypic populations as T-cell lymphoma or T-CUS. Further research is needed to establish criteria for distinguishing between these populations and to improve FC diagnostic accuracy.

The clinical presentation of T-cell large granular lymphocytic leukemia (T-LGLL) is extremely variable: 30% of patients have neutropenia with no associated symptoms, others present with bacterial infections and sepsis may occur. Tools to predict patient outcome are lacking. Stemming from preliminary results obtained by single cell-RNAseq we investigated by qPCR HSP and IFIT gene families in 27 LGLL patients (23T-LGLL and 4 NK-LGLL), including 11 with neutropenia and/or thrombocytopenia and 16 asymptomatic for the disease. HSP90AA1 and HSPA1B, among HSP family and CD279 exhibited a significantly higher expression in CD3 + CD57 + sorted cells of symptomatic LGLL patients compared to asymptomatic patients and healthy controls. Also, monocytes derived from symptomatic LGLL patients expressed high levels of CCL3, CCL4 and CCL5 mRNA and of IL-1β, IL-6, TNF, and PD-L1 mRNA, thus confirming a pro-inflammatory cytokine profile reminiscent of a non-classical phenotype. Overall, these data provide a rationale for considering HSP and CD279 genes as potential biomarkers for distinguishing symptomatic LGLL patients from asymptomatic ones, emphasizing the importance of further research to explore their implications for targeted therapy development.

Mature T-cell neoplasms arise from the neoplastic transformation of a single T lymphocyte, and all cells in a neoplastic clone share the same V segment in the beta chain of the T-cell receptor (TCR). These segments may represent an innovative target for the development of targeted therapies.

A specific V segment of the TCR beta chain (TRBV5-1) was analyzed using bioinformatic tools, identifying three potential antigenic peptides. One of these peptides, selected for synthesis, was used to screen a library of human single-chain variable fragments (scFv) through phage display. One fragment demonstrated high affinity and specificity for the antigen and was used to produce a human monoclonal antibody of the IgG1 class.

Surface plasmon resonance (SPR) studies confirmed the high affinity of the monoclonal antibody for the antigen in the nanomolar range. Flow cytometry analysis on patients' samples demonstrated that the antibody, conjugated with a fluorochrome, selectively binds to tumor T lymphocytes expressing TRBV5-1, without binding to other lymphocytes or blood cell components.

The development of fully human IgG1 monoclonal antibodies targeting specific V segments of the TCR beta chain represents a potential therapeutic option for patients with mature T-cell neoplasms.

Hematopoietic neoplasms are common in dogs; however, their association with pregnancy has not been previously reported in veterinary medicine. This rare occurrence presents a variety of diagnostic, therapeutic, prognostic, and ethical challenges. We report a case of a 3-year-old pregnant Bernese Mountain Dog diagnosed with multicentric aggressive unclassified hematopoietic cancer associated with paraneoplastic hypercalcemia during pregnancy. The dog died 7 days after diagnosis, and at Day 36 of pregnancy before any treatment decision could be made. Post-mortem evaluation, including histology, immunohistochemistry, and clonality analysis, led to the diagnosis of an unclassified hematopoietic cancer affecting the uterus and placenta, with no evidence of fetal involvement. The placenta likely acted as a barrier, preventing neoplastic involvement of the fetuses. Alternatively, the pregnancy might have been too early for the hematopoietic neoplasm to affect the labyrinth zone of the placenta and the fetuses. The dramatic disease progression could be explained by compromised cell-mediated immunity during pregnancy. This immunodeficient state is induced by embryonic, maternal, and hormonal factors, which suppress the response to mitogens to prevent rejection of the placenta and the conceptuses. Thus, pregnant dogs might exhibit increased vulnerability to cancer and infectious diseases that rely on cell-mediated immunity for host defense.

Primary cutaneous marginal zone lymphoma (PCMZL) and primary cutaneous CD4+ small/medium T-cell lymphoproliferative disease (CD4+ TLPD) are two distinct entities with excellent prognosis; however, they show profound clinical and histopathological similarities, leading to differential diagnostic uncertainty.

Our aim was to review and reanalyze cases of primary cutaneous lymphoproliferations diagnosed at Semmelweis University, featuring characteristics of PCMZL and CD4+ TLPD.

Cutaneous lymphoma biopsy specimens between 2018 and 2022 were collected and re-evaluated. Medical history, clinical picture, imaging, and laboratory findings were collected. Immunohistochemical staining for CD20, CD3, BCL6, CD10, PD1, CD3, CD4, CD8, and PCR tests for IGH, IGK, TCRB, and TCRG were repeated in selected cases.

Among 55 cases diagnosed as PCMZL (16) or CD4+ TLPD (39), 3 patients had been diagnosed with both LPDs at different time points of their disease course. Four additional patients were identified with single lesions featuring overlapping histopathological characteristics of both LPDs and both monoclonal IGH and TCR rearrangements. All patients are currently in complete remission with local treatment.

We propose that besides the overlapping histopathological, molecular, and clinical features, the subsequent appearance of PCMZL and CD4+ TLPD in a short timeframe in the same patients may suggest a common pathogenic background.

T- and natural killer (NK)-cell lymphomas are neoplasms derived from immature T cells (lymphoblastic lymphomas), or more commonly, from mature T and NK cells (peripheral T-cell lymphomas, PTCLs). PTCLs are rare but show marked biological and clinical diversity. They are usually aggressive and may present in lymph nodes, blood, bone marrow, or other organs. More than 30 T/NK-cell-derived neoplastic entities are recognized in the International Consensus Classification and the classification of the World Health Organization (fifth edition), both published in 2022, which integrate the most recent knowledge in hematology, immunology, pathology, and genetics. In both proposals, disease definition aims to integrate clinical features, etiology, implied cell of origin, morphology, phenotype, and genetic features into biologically and clinically relevant clinicopathologic entities. Cell derivation from innate immune cells or specific functional subsets of CD4+ T cells such as follicular helper T cells is a major determinant delineating entities. Accurate diagnosis of T/NK-cell lymphoma is essential for clinical management and mostly relies on tissue biopsies. Because the histological presentation may be heterogeneous and overlaps with that of many benign lymphoid proliferations and B-cell lymphomas, the diagnosis is often challenging. Disease location, morphology, and immunophenotyping remain the main features guiding the diagnosis, often complemented by genetic analysis including clonality and high-throughput sequencing mutational studies. This review provides a comprehensive overview of the classification and diagnosis of T-cell lymphoma in the context of current concepts and scientific knowledge.

PCR-based fragment analysis of the T-cell receptor (TCR) gene is used extensively in diagnostic labs to assess clonality in T-cell populations in multiple tissue sites. Of the numerous TCR assays that have been reported, studies assessing use on biopsies suspicious for mycosis fungoides specifically are lacking. We compared clonality findings from a previously run 2-tube/2-fluorochrome dye assay to a redesigned 1-tube/1-fluorochrome dye assay on formalin-fixed skin biopsies. Overall, the accuracy of the 2-tube assay was marginally better (75.7% vs. 71.4%), when using clinical history combined with histologic diagnosis as the gold standard. The 2-tube assay had better sensitivity (73.7% vs. 65.8%), while the 1-tube assay had superior specificity (93.8% vs. 87.5%). Clonality results were easier to interpret with the 1-tube assay. In nearly 19% of cases, a change of assays on the same biopsy resulted in a change of clonality interpretation. For laboratories that change TCR-γ clonality assays, follow-up biopsies for mycosis fungoides assessment may result in a change of diagnosis.

Leading from a two-case series, including two patients receiving a diagnosis of epidermotropic T-cell lymphoma, featuring a mycosis fungoides (MF)-like clinical pattern and ALK expression and molecular alteration, we performed a critical appraisal of ALK+ primary cutaneous T-cell lymphomas (pcTCL). Considering our patients and the literature, 32 cases were retrieved, 7 of which featured an MF-like clinical picture over a 4-to-20-year period. MF-like cases show distinctive histology, comprising a predominantly epidermotropic infiltration of small-to-large, atypical-to-pleomorphic, with few anaplastic cells, negligible-to-intense CD30-expression, and a CD4+/cytotoxic granule+ phenotype. These features should prompt a search for ALK expression captured by the ALK D5F3 clone. Bona fide ALK+ pcTCL is very rare, and existent data suggest the presence of a broader pattern of disease, including instances mimicking MF and/or primary cutaneous CD8+ aggressive epidermotropic cytotoxic T-cell lymphoma. The major challenges in dealing with this subset include prodromal phases, misinterpreted as inflammatory dermatosis or parapsoriasis/early phase MF both clinically and histologically, while recognition of its ALK-driven biology is hampered both by the unusual clinic-pathologic pattern of the disease, which stands apart from the classical (i.e., nodal) picture of ALK+ anaplastic large cell lymphoma and by the low sensitivity of ALK1 clone. Data on its optimal management are far from being conclusive: An MF-like approach is currently chosen, but depending on CD30 and, most notably, ALK expression, a targeted therapy could be envisaged in advanced stages, as clinical response to ALK inhibition was documented in one patient.

Cats have the highest incidence of lymphoma among all animal species. Lymphoma accounts for 41% of all malignant tumors in cats and is responsible for 90% of hematopoietic tumors in felines. Biopsies are considered the gold standard for diagnosis. Polymerase chain reaction (PCR)-based clonality assessment of antigen receptor gene rearrangements can be a valuable complementary tool for identifying infiltrating B-and T-lymphocyte clones. Many studies have focused on intestinal cases but few have addressed mediastinal lymphoma. This study aims to: (1) investigate the clonality patterns of lymphoma samples from various anatomical sites, with a particular focus on mediastinal lymphoma, and (2) evaluate the sensitivity and specificity of the clonality analysis of pleural effusion samples in comparison with cytology, histology, immunohistochemistry, and immunocytochemistry for diagnosing mediastinal lymphoma. There were 82 cases, divided into 49 formalin-fixed and paraffin-embedded biopsy specimens (FFPE), 22 cell pellets, and 11 fresh tissue. This study examined the sensitivity and specificity of PCR for antigen receptor rearrangement (PARR) compared to immunohistochemistry (IHC) and immunocytochemistry. For T-cell receptor gamma chain genes, PARR demonstrated a sensitivity of 58.33% for both fresh tissue and FFPE samples, with a specificity of 100%. Cell pellet analysis exhibited a sensitivity of 64.71% and maintained 100% specificity. A combined analysis of fresh tissue and FFPE with cell pellets showed a sensitivity of 62.07%. For IGH, the sensitivity for fresh tissue and FFPE samples was 56.25%, while cell pellet analysis showed a sensitivity of 62.50%. When considering fresh tissue and FFPE samples, the sensitivity was 57.14%. In conclusion, molecular techniques have emerged as valuable tools for detecting lymphoma, especially in cases where traditional diagnostic methods yield inconclusive results, such as mediastinal lymphoma. While biopsy may not always be feasible, cytology and cell pellets obtained from pleural effusion offer alternative immunocytochemistry and molecular analysis samples, provided they are of sufficient quality and quantity. All sample types considered in this study were suitable for PARR to aid in cases with inconclusive results. Therefore, the sample selection should be tailored to the clinical situation.

It is known that a subset of cases of classic Hodgkin lymphoma (CHL) with B-cell-rich nodules (lymphocyte-rich CHL) exhibits morphologic and immunophenotypic features that overlap with nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL), raising diagnostic difficulties that can be resolved in most cases by performing an adequate battery of immunohistochemical studies.

To fully characterize cases of T-cell-rich Hodgkin lymphoma where a specific diagnosis of NLPHL (ie, pattern D) or CHL could not be made even after complete immunophenotypic investigation.

The clinical, immunomorphologic, and molecular (when applicable) presentation of 3 cases of T-cell-rich Hodgkin lymphoma was thoroughly investigated.

These 3 cases harbored lymphocyte-predominant-like and Hodgkin and Reed-Sternberg-like cells that partially expressed B-cell and CHL markers and were negative for Tiftein-Barr virus-encoded small RNA, in a T-cell-rich background with residual follicular dendritic cell meshworks; 1 case had frequent and the other 2 cases scant/absent eosinophils and plasma cells. Two patients with advanced-stage (III or IV) disease presented with axillary and supraclavicular lymphadenopathy, respectively, and without B symptoms. These patients underwent NLPHL-like therapeutic management with 6 cycles of R-CHOP (rituximab, cyclophosphamide, doxorubicin hydrochloride [hydroxydaunorubicin], vincristine sulfate [Oncovin], and prednisone) chemotherapy; both are in complete remission 7 years posttherapy. One patient presented with stage I disease involving an internal mammary lymph node without B-symptoms and was treated with surgical excision alone; this patient is also in complete remission 1 year later.

These cases illustrate overlapping features of T-cell-rich NLPHL and CHL with neoplastic cells expressing both B-cell program and CHL markers. This underrecognized overlap has not been fully illustrated in the literature, although it portrays a therapeutic challenge. These neoplasms may deserve in-depth investigation in the future that may bring up diagnostic or theragnostic implications.