Tumor heterogeneity and immune-evasive T follicular cell lymphoma phenotypes at single-cell resolution.

T follicular helper (TFH) cell lymphomas (TFHLs) are characterized by TFH-like properties and accompanied by substantial immune-cell infiltration into tumor tissues. Nevertheless, the comprehensive understanding of tumor-cell heterogeneity and immune profiles of TFHL remains elusive. To address this, we conducted single-cell transcriptomic analysis on 9 lymph node (LN) and 16 peripheral blood (PB) samples from TFHL patients. Tumor cells were divided into 5 distinct subclusters, with significant heterogeneity observed in the expression levels of TFH markers. Copy number variation (CNV) and trajectory analyses indicated that the accumulation of CNVs, together with gene mutations, may drive the clonal evolution of tumor cells towards TFH-like and cell proliferation phenotypes. Additionally, we identified a novel tumor-cell-specific marker, PLS3. Notably, we found a significant increase in exhausted CD8+ T cells with oligoclonal expansion in TFHL LNs and PB, along with distinctive immune evasion characteristics exhibited by infiltrating regulatory T, myeloid, B, and natural killer cells. Finally, in-silico and spatial cell-cell interaction analyses revealed complex networking between tumor and immune cells, driving the formation of an immunosuppressive microenvironment. These findings highlight the remarkable tumor-cell heterogeneity and immunoevasion in TFHL beyond previous expectations, suggesting potential roles in treatment resistance.

Follicular lymphoma microenvironment: insights provided by single-cell analysis.

Follicular lymphoma (FL) is the most frequent indolent lymphoma and is characterized by the abundant infiltration of tumor microenvironment (TME) cells. The activity of TME cells reportedly plays an important role in the biology of FL. TME cells that reside within neoplastic follicles, such as T-follicular helper cells and follicular dendritic cells, have been shown to aid in FL development and progression through interactions with malignant B cells, whereas regulatory T cells have unexpectedly shown an apparently favorable prognostic impact in FL. Unfortunately, the understanding of the FL TME, particularly regarding minor cell subsets, has been hampered by unknown cell heterogeneity. As with other solid and hematologic cancers, novel single-cell analysis technologies have recently been applied to FL research and have uncovered previously unrecognized heterogeneities, not only in malignant B cells but also in TME cells. These reports have greatly increased the resolution of our understanding of the FL TME and, at the same time, raised questions about newly identified TME cells. This review provides an overview of the unique aspects of FL TME cells with a clinical viewpoint and highlights recent discoveries from single-cell analysis, while also suggesting potential future directions.

Clonal germinal center B cells function as a niche for T-cell lymphoma.

Angioimmunoblastic T-cell lymphoma (AITL) is proposed to be initiated by age-related clonal hematopoiesis (ACH) with TET2 mutations, whereas the G17V RHOA mutation in immature cells with TET2 mutations promotes the development of T follicular helper (TFH)-like tumor cells. Here, we investigated the mechanism by which TET2-mutant immune cells enable AITL development using mouse models and human samples. Among the 2 mouse models, mice lacking Tet2 in all the blood cells (Mx-Cre × Tet2flox/flox × G17V RHOA transgenic mice) spontaneously developed AITL for approximately up to a year, while mice lacking Tet2 only in the T cells (Cd4-Cre × Tet2flox/flox × G17V RHOA transgenic mice) did not. Therefore, Tet2-deficient immune cells function as a niche for AITL development. Single-cell RNA-sequencing (scRNA-seq) of >50 000 cells from mouse and human AITL samples revealed significant expansion of aberrant B cells, exhibiting properties of activating light zone (LZ)-like and proliferative dark zone (DZ)-like germinal center B (GCB) cells. The GCB cells in AITL clonally evolved with recurrent mutations in genes related to core histones. In silico network analysis using scRNA-seq data identified Cd40-Cd40lg as a possible mediator of GCB and tumor cell cluster interactions. Treatment of AITL model mice with anti-Cd40lg inhibitory antibody prolonged survival. The genes expressed in aberrantly expanded GCB cells in murine tumors were also broadly expressed in the B-lineage cells of TET2-mutant human AITL. Therefore, ACH-derived GCB cells could undergo independent clonal evolution and support the tumorigenesis in AITL via the CD40-CD40LG axis.

A single-cell atlas of non-haematopoietic cells in human lymph nodes and lymphoma reveals a landscape of stromal remodelling.

The activities of non-haematopoietic cells (NHCs), including mesenchymal stromal cells and endothelial cells, in lymphomas are reported to underlie lymphomagenesis. However, our understanding of lymphoma NHCs has been hampered by unexplained NHC heterogeneity, even in normal human lymph nodes (LNs). Here we constructed a single-cell transcriptome atlas of more than 100,000 NHCs collected from 27 human samples, including LNs and various nodal lymphomas, and it revealed 30 distinct subclusters, including some that were previously unrecognized. Notably, this atlas was useful for comparative analyses with lymphoma NHCs, which revealed an unanticipated landscape of subcluster-specific changes in gene expression and interaction with malignant cells in follicular lymphoma NHCs. This facilitates our understanding of stromal remodelling in lymphoma and highlights potential clinical biomarkers. Our study largely updates NHC taxonomy in human LNs and analysis of disease status, and provides a rich resource and deeper insights into LN and lymphoma biology to advance lymphoma management and therapy.

Tet2 deficiency in immune cells exacerbates tumor progression by increasing angiogenesis in a lung cancer model.

Immune cells harboring somatic mutations reportedly infiltrate cancer tissues in patients with solid cancers and accompanying clonal hematopoiesis. Loss-of-function TET2 mutations are frequently observed in clonal hematopoiesis in solid cancers. Here, using a mouse lung cancer model, we evaluated the activity of Tet2-deficient immune cells in tumor tissues. Myeloid-specific Tet2 deficiency enhanced tumor growth in mice relative to that seen in controls. Single-cell sequencing analysis of immune cells infiltrating tumors showed relatively high expression of S100a8/S100a9 in Tet2-deficient myeloid subclusters. In turn, treatment with S100a8/S100a9 promoted Vegfa production by cancer cells, leading to a marked increase in the tumor vasculature in Tet2-deficient mice relative to controls. Finally, treatment of Tet2-deficient mice with an antibody against Emmprin, a known S100a8/S100a9 receptor, suppressed tumor growth. These data suggest that immune cells derived from TET2-mutated clonal hematopoiesis exacerbate lung cancer progression by promoting tumor angiogenesis and may provide a novel therapeutic target for lung cancer patients with TET2-mutated clonal hematopoiesis.

Alternative Evaluation of an ELISPOT Assay Using Cytokine Activity as a Novel Parameter.

BACKGROUND/AIM: The enzyme-linked immunospot (ELISPOT) assay is a well-established method used to evaluate the strength of T cell-mediated immune activity, and accepted as a standard functional immunological assay. Cytokine activity is a novel parameter reflecting spot size and intensity, which has not been used in ELISPOT assay before. MATERIALS AND METHODS: In the present study, from 113 ELISPOT assay data derived from previous clinical trials with dendritic cell vaccines, both spot number count and cytokine activity data for IFN-γ secretion were obtained using an ELISPOT reader. Comparing the new parameter cytokine activity with the existing parameter spot number, the feasibility of cytokine activity was investigated. RESULTS: There were no significant differences in sensitivity and specificity between spot number and cytokine activity among ELISPOT assay data from CMVpp65 and other antigen peptide-stimulated cytotoxic T lymphocytes. CONCLUSION: Although cytokine activity is a novel parameter unreported so far, it did not show any advantages in the evaluation T cell immune responses compared to the existing spot number parameter.

Alpha-type-1 Polarized Dendritic Cell-based Vaccination in Newly Diagnosed High-grade Glioma: A Phase II Clinical Trial.

BACKGROUND/AIM: Glioblastoma multiforme (GBM) is an intractable tumor that has a very poor prognosis despite intensive treatment with temozolomide plus radiotherapy. PATIENTS AND METHODS: Sixteen newly diagnosed patients with high-grade gliomas were enrolled in a phase II study of the α-type-1 DC vaccine. Briefly, DCs obtained from the culture of enriched monocytes in the presence of a cytokine cocktail, were pulsed with a cocktail of 5 synthetic peptides and cryopreserved until injection into patients. RESULTS: The amount of IL-12 produced by activated DCs was higher than that previously reported. Among 15 evaluable patients, 10 showed positive CTL responses to any peptides in an ELISPOT assay. After 6 years of observation, five patients were still alive, and two of these patients were relapse-free. Moreover, a significant survival-prolonging effect was verified in DC-treated glioma patients. CONCLUSION: Peptide-cocktail-pulsed α-type-1 DC vaccines have a potential therapeutic effect on survival when used in combination with the standard regimen, which is partly based on IL-12-IFN-γ-mediated T-cell activation.

Baseline total lesion glycolysis combined with interim positron emission tomography-computed tomography is a robust predictor of outcome in patients with peripheral T-cell lymphoma.

BACKGROUND: Peripheral T-cell lymphoma (PTCL) represents a heterogeneous and rare subgroup of aggressive lymphomas that generally demonstrate poor clinical outcomes with conventional treatment. Since the prognosis of PTCL is heterogeneous, more accurate risk assessment, and risk-adapted treatment strategies are required. In this study, we examined whether interim positron emission tomography (iPET)-computed tomography (PET/CT) results can be combined with baseline volume-based metabolic assessments including total metabolic tumor volume (TMTV) and total lesion glycolysis (TLG) for risk stratification in PTCL. METHODS: The data of 63 patients with nodal PTCL, who had analyzable baseline PET/CT and iPET, were retrospectively reviewed. We calculated the baseline TMTV and TLG values. All iPET responses were analyzed using the Deauville 5-point scale. RESULTS: On univariate analysis, a prognostic index for PTCL (PIT) higher than 2 (hazard ratio [HR], 2.03; P = .026), high TMTV (>389 cm3 ; HR, 2.24; P = .01), high TLG (>875; HR, 3.77; P = .0005), and positive iPET (HR, 2.18; P = .009) were significantly associated with poorer progression-free survival (PFS). On multivariate analysis, only high TLG and positive iPET independently predicted both poorer overall survival (OS) and PFS. A model combining TLG and iPET showed that patients with low TLG and negative iPET had superior outcomes, with a 5-year PFS and OS of 72% and 90%, respectively. Conversely, both 5-year PFS and OS for those with high TLG and positive iPET were 0%. CONCLUSIONS: In summary, TLG combined with iPET predicted survival in PTCL more accurately. This information may help in the development of risk-adapted treatment strategies for PTCL.

Association between serum erythropoietin levels and renal reversibility in patients with renal impairment from multiple myeloma.

Renal impairment (RI) is a common clinical presentation in patients with multiple myeloma (MM). Despite treatment with novel agents or management strategies that focus on the disease response, some patients develop irreversible RI. This study aimed to determine the influencing, clinical variables of renal reversibility in patients with RI treated with novel drugs. We analyzed 244 patients newly diagnosed with MM retrospectively. Maximum renal response was assessed according to the criteria for the definition of renal response proposed by the International Myeloma Working Group. Major renal response was defined as the occurrence of complete renal response or partial renal response. RI (a glomerular filtration rate < 50 mL/min/1.73 m2 ) was observed in 110 patients (45%). In total, 56 patients (51%) achieved a major renal response. Serum erythropoietin (EPO) levels >25 mIU/mL (P < .001) and a percentage of urinary albumin excretion ≤25% (P < .001) were both significant factors that influenced the occurrence of major renal responses. Both remained significant factors associated with renal reversibility in the multivariate analysis. Patients were assigned a score of 1 each for EPO >25 mIU/mL and urinary albumin ≤25%. The estimated 6-month rates of major renal responses of patients with scores of 2, 1, and 0 were 78.6%, 30.6%, and 0%, respectively (P < .001). In conclusion, a serum EPO level >25 mIU/mL is an independent predictive factor for major renal response and may predict renal reversibility more accurately when urinary albumin level is congruently ≤25%.

Bone marrow infiltration pattern in patients with intravascular large B-cell lymphoma diagnosed by random skin biopsy.

We retrospectively analyzed bone marrow (BM) infiltration pattern in consecutive 30 intravascular large B-cell lymphoma (IVLBCL) patients diagnosed by random skin biopsy (RSB). BM infiltration of lymphoma was observed in 18 patients (60.0%), including five patients with the intrasinusoidal pattern with minimal extravasation, eight patients with the mixed of intrasinusoidal and scattered/interstitial or nodular infiltration, and five patients with the nodular/diffuse pattern. Twelve patients were negative for lymphoma infiltration. BM histology of patients with IVLBCL were diverse and frequently discordant with those of other site of IVLBCL lesions. BM biopsy had a poorer diagnostic performance for detecting intravascular features.

18F-FDG PET/CT-Guided Diagnosis and Evaluation of Treatment Response to Cyclosporine A in Maldistributed Subcutaneous Panniculitis-like T-Cell Lymphoma Without Typical Skin Rash.

A 30-year-old woman presented with persistent fever and elevated lactate dehydrogenase levels without skin rash. F-FDG PET/CT revealed FDG uptake in subcutaneous tissues in the forearm, buttocks, and lower limbs, whereas the trunk was unaffected. She was diagnosed with subcutaneous panniculitis-like T-cell lymphoma based on a PET/CT-guided subcutaneous tissue biopsy from the thigh. Although conventional cytotoxic agent-based chemotherapies failed to achieve disease remission, subsequent cyclosporine A treatment promptly resolved the fever and laboratory abnormalities. Remarkably, abnormal FDG uptake disappeared entirely on follow-up PET/CT, demonstrating its utility in the evaluations of responses to emerging cyclosporine A-based treatments in subcutaneous panniculitis-like T-cell lymphoma.

Diagnosis of intravascular large B cell lymphoma: novel insights into clinicopathological features from 42 patients at a single institution over 20 years.

This study aimed to clarify the comprehensive clinical, laboratory, pathological and imaging features of intravascular large B-cell lymphoma (IVLBCL) using data on 42 IVLBCL patients diagnosed at our hospital over the past 20 years. The majority of patients were diagnosed via random skin biopsy (29/42, 69·0%) followed by bone marrow biopsy alone (8/42, 19·0%). Characteristic features included persistent fever (41/42, 97·6%), decreased performance status (≥2) (100%), hypoxaemia (32/40, 80·0%), impaired consciousness (19/42, 45·2%), hypoalbuminemia (42/42, 100%) and extreme elevation of lactate dehydrogenase and soluble interleukin 2 receptor levels. Brain magnetic resonance imaging showed abnormal findings in 32/37 patients (86·4%). Hyperintense lesion in the pons was a peculiar finding that was unrelated to the neurological deficits. Positron emission tomography-computed tomography revealed a high incidence of bone marrow (26/34, 76·5%), spleen (19/34, 55·9%) and adrenal gland (9/34, 26·5%) involvement. Neurolymphomatosis was noted in 6 patients during the course of the disease. About 60% of IVLBCL patients in whom in vivo diagnosis was possible survived more than 5 years with combination chemotherapy. Our observations provide additional insight into the diagnosis of IVLBCL and indicate that early disease recognition via random skin biopsy combined with imaging, enables in vivo diagnosis of the disease and improved survival for many patients.

Higher frequency of false-positive serum galactomannan tests among older subjects and the association with elevated serum immunoglobulin G levels.

BACKGROUND: The incidence of false-positive serum galactomannan (GM) enzyme-linked immunosorbent assay test results has been scarcely examined among older subjects. Additionally, previous studies have highlighted the influence of serum immunoglobulin G (IgG) levels on GM test results. We hypothesised that age-related IgG level elevation might also be associated with false-positive GM test results in older subjects. OBJECTIVES: This study aimed to examine the association between false-positive GM test results and age or serum IgG levels. PATIENTS/METHODS: We investigated the association between false-positive serum GM test results and age in 1071 healthy adult subjects. Then, we validated this association and further explored the correlation with serum IgG levels by retrospectively identifying 700 patients with newly diagnosed haematologic disorders without probable or proven invasive aspergillosis. RESULTS: Healthy subjects with false-positive GM test results were significantly older than those without false-positive results (P < 0.001). Among patients with haematologic disorders, IgG myeloma patients showed significantly higher false-positive rates (57/125 [45.6%]) than patients with other haematologic disorders (non-Hodgkin lymphoma: 48/315 [15.2%], myelodysplastic syndrome/aplastic anaemia: 19/141 [13.5%]; both P < 0.001) or other types of myeloma (IgA myeloma: 13/60 [21.7%], light chain myeloma: 9/52 [17.3%]; both P < 0.01). Furthermore, among non-multiple myeloma patients, advanced age and higher IgG level were also significantly associated with the high frequency of false-positive GM test results. CONCLUSIONS: False-positive serum GM test results were frequent among older subjects and patients with elevated serum IgG levels. These results suggested that age- and/or disease-related IgG level elevation could induce this phenomenon.

Medullary Abnormalities in Appendicular Skeletons Detected With 18F-FDG PET/CT Predict an Unfavorable Prognosis in Newly Diagnosed Multiple Myeloma Patients With High-Risk Factors.

OBJECTIVE. The prognostic value of medullary abnormalities in the appendicular skeleton (AS) of patients with multiple myeloma (MM) has recently been suggested. However, functional evaluation of these abnormalities using PET/CT has not been investigated to date. This study aimed to explore the prevalence and prognostic relevance of AS medullary abnormalities depicted by PET/CT in patients with MM. MATERIALS AND METHODS. This retrospective study included 228 consecutive patients with newly diagnosed, symptomatic MM who were treated with novel agents. All patients underwent pretreatment 18F-FDG PET/CT. RESULTS. There were 157 (68.9%) patients with zero AS focal lesions, 33 (14.5%) with one to three AS focal lesions, and 38 (16.7%) with more than three AS focal lesions on pre-treatment PET/CT. Patients with more than three AS focal lesions showed significantly shorter progression-free survival (PFS) and overall survival (OS) than did those with fewer lesions (both, p < 0.001). In multivariate analysis, the presence of more than three AS focal lesions remained prognostic for both PFS and OS (both, p < 0.001). Furthermore, the presence of more than three AS focal lesions discriminated patients with both significantly shorter PFS and significantly shorter OS even among patients with established high-risk parameters, including high-risk cytogenetic abnormalities, advanced disease stage, and established high-risk PET/CT findings. CONCLUSION. The presence of more than three focal lesions in the AS on pretreatment PET/CT was an independent predictor of poor survival in patients with newly diagnosed MM. Remarkably, this finding discriminated patients with shorter survival from among those with established high-risk factors. Evaluation of findings in the AS may complement and improve the prognostic performance of known stratification systems as well as PET/CT.

Evaluation of the safety and efficacy of daratumumab outside of clinical trials.

Daratumumab-based therapy has been shown to have significant clinical efficacy in phase 3 trials of patients with relapse or refractory multiple myeloma. Outside of clinical trials, however, clinical data on daratumumab remain limited. We reviewed medical records of patients who received daratumumab combination therapy at our institute (median age 74 years; median lines of prior therapy 4). The overall response rate was 69.4%, and 36.7% of patients achieved complete response (CR) or better. The proportion of patients who attained CR or better was significantly higher among patients with < 4 prior therapies than those with ≥ 4 (56.5% vs 19.2%, P = 0.009). Estimated median progression-free survival (PFS) was 12.4 months (95% confidence interval 8.6-not reached). The median PFS was significantly worse in patients who were refractory to bortezomib and lenalidomide and had received ≥ 4 lines of prior therapy. Twelve of 49 patients attained negative minimal residual disease. Common adverse events included hematological toxicities including neutropenia and lymphopenia; however, the rate of febrile neutropenia was low (3.8%). Infusion-related reactions occurred in 32.1% of patients, but were grade 1 or 2. Daratumumab combination therapies therefore appear to be effective and safe as salvage regimens in clinical practice, especially when used in the early phase.

Diagnostic and Prognostic Value of Using 18F-FDG PET/CT for the Evaluation of Bone Marrow Involvement in Peripheral T-Cell Lymphoma.

PURPOSE: No study has analyzed a sizeable cohort that comprised solely peripheral T-cell lymphoma (PTCL) patients for the diagnostic and prognostic performance of F-FDG PET/CT for bone marrow (BM) involvement. This study aimed to investigate the utility of PET/CT for the identification of BM involvement and to explore its prognostic relevance in patients with PTCL. METHODS: Eighty-three consecutive patients with newly diagnosed PTCL were enrolled in this retrospective study. The diagnosis of BM involvement was confirmed on the basis of positive BM histology or disappearance of marrow abnormalities on follow-up PET/CT concurrently with a successful treatment response. RESULTS: Of 28 patients with confirmed BM involvement, BM biopsy (BMB) and PET/CT detected an involvement in 17 and 25 patients. Among 66 patients with negative BM histology, 11 patients had BM involvement detected by PET/CT and furthermore showed significantly shorter progression-free and overall survival than patients without BM involvement. We recategorized the International Prognostic Index (IPI) risk groups based on the presence of BM involvement using the combined assessment of iliac crest marrow biopsy histology and PET/CT (PET/BMB-based IPI). PET/BMB-based IPI tended to perform better than conventional BMB-based IPI. CONCLUSIONS: This study included the largest population of PTCL patients for PET/CT evaluation of BM involvement so far. PET/CT exhibited a higher sensitivity for BM involvement than BMB. Furthermore, BM assessment using PET/CT identified patients at high risk of disease progression and mortality among those with negative BM histology, suggesting that PET/CT may have a potential to improve existing prognostic strategies in PTCL.