Peripheral T-cell lymphomas expressing CD30 and CD15 expand the spectrum of anaplastic large cell lymphoma, ALK-negative.

Peripheral T-cell lymphomas (PTCL) are morphologically and biologically heterogeneous and a subset expresses CD30, including anaplastic large cell lymphomas (ALCL) and a minority of PTCL, not otherwise specified (PTCL, NOS). ALCL with ALK translocations (ALCL, ALK+) are readily identified by routine diagnostic methods, but differentiating ALCL without ALK translocation (ALCL, ALK-) and PTCL, NOS expressing CD30 (PTCL CD30+) can be challenging. Furthermore, rare PTCL co-express CD30 and CD15 (PTCL CD30+CD15+); some resemble ALCL, ALK- while others resemble classic Hodgkin lymphoma. To explore the relationship between PTCL CD30+CD15+ and ALCL, ALK-, we analysed 19 cases of PTCL with CD30 expression, previously diagnosed as ALCL, ALK- (nine cases) and PTCL CD30+CD15+ (10 cases) for DUSP22/IRF4 rearrangements, coding RNA expression and selected transcriptome analysis using the NanoString nCounter gene expression analysis platform. Unsupervised clustering showed no clear segregation between ALCL, ALK- and PTCL CD30+CD15+. Three cases previously classified as PTCL CD30+CD15+ showed DUSP22/IRF4 rearrangements, favouring a diagnosis of ALCL, ALK-. Our results suggest that cases previously designated PTCL CD30+CD15+, likely fall within the spectrum of ALCL, ALK-; additionally, a subset of ALCL, ALK- with DUSP22/IRF4 rearrangement expresses CD15, consistent with previous reports and expands the immunophenotypic spectrum of this lymphoma subgroup.

CD30 plays a role in T-dependent immune response and T cell proliferation.

CD30 is a member of the tumor necrosis factor receptor (TNFR) superfamily and expressed in both normal and malignant lymphoid cells. However, the role of CD30 in lymphopoiesis is not known. In this study, we showed CD30 was expressed both in T and B cells, but its deficiency in mice had no effect on T- and B-cell development. In fact, CD30 deficiency attenuated B-cell response to T-cell-dependent antigens. The impaired B cell response in CD30-deficient mice is caused by the reduction of activation-induced cytidine deaminase (AID) expression. Moreover, CD30-deficient mice exhibited decreased TCR-mediated T cell proliferation and slightly impaired TCR signaling. High-throughput RNA sequencing analysis revealed that CD30 deficiency led to a decrease of FOXO-autophagy axis in T cells upon TCR stimulation. Thus, CD30 positively regulates T-cell-dependent immune response and T cell proliferation.

Differential molecular programs of cutaneous anaplastic large cell lymphoma and CD30-positive transformed mycosis fungoides.

Background: Discriminating between cutaneous anaplastic large cell lymphoma (cALCL) and CD30-positive transformed mycosis fungoides (CD30+ TMF) is challenging, particularly when they arise in the context of pre-existing mycosis fungoides. The development of molecular diagnostic tools was hampered by the rarity of both diseases and the limited understanding of their pathogenesis. Methods: In this study, we established a cohort comprising 25 cALCL cases and 25 CD30+ TMF cases, with transcriptomic data obtained from 31 samples. We compared the clinicopathological information and investigated the gene expression profiling between these two entities. Furthermore, we developed an immunohistochemistry (IHC) algorithm to differentiate these two entities clinically. Results: Our investigation revealed distinct clinicopathological features and unique gene expression programs associated with cALCL and CD30+ TMF. cALCL and CD30+ TMF displayed marked differences in gene expression patterns. Notably, CD30+ TMF demonstrated enrichment of T cell receptor signaling pathways and an exhausted T cell phenotype, accompanied by infiltration of B cells, dendritic cells, and neurons. In contrast, cALCL cells expressed high levels of HLA class II genes, polarized towards a Th17 phenotype, and exhibited neutrophil infiltration. An IHC algorithm with BATF3 and TCF7 staining emerged as potential diagnostic markers for identifying these two entities. Conclusions: Our findings provide valuable insights into the differential molecular signatures associated with cALCL and CD30+ TMF, which contribute to their distinct clinicopathological behaviors. An appropriate IHC algorithm could be used as a potential diagnostic tool.

CD30 induces Reed-Sternberg cell-like morphology and chromosomal instability in classic Hodgkin lymphoma cell lines.

Classic Hodgkin lymphoma (cHL) is characterized by multinucleated cells called Reed-Sternberg (RS) cells and genetic complexity. Although CD30 also characterizes cHL cells, its biological roles are not fully understood. In this report, we examined the link between CD30 and these characteristics of cHL cells. CD30 stimulation increased multinucleated cells resembling RS cells. We found chromatin bridges, a cause of mitotic errors, among the nuclei of multinucleated cells. CD30 stimulation induced DNA double-strand breaks (DSBs) and chromosomal imbalances. RNA sequencing showed significant changes in the gene expression by CD30 stimulation. We found that CD30 stimulation increased intracellular reactive oxygen species (ROS), which induced DSBs and multinucleated cells with chromatin bridges. The PI3K pathway was responsible for CD30-mediated generation of multinucleated cells by ROS. These results suggest that CD30 involves generation of RS cell-like multinucleated cells and chromosomal instability through induction of DSBs by ROS, which subsequently induces chromatin bridges and mitotic error. The results link CD30 not only to the morphological features of cHL cells, but also to the genetic complexity, both of which are characteristic of cHL cells.

CD30 Expression and Its Functions during the Disease Progression of Adult T-Cell Leukemia/Lymphoma.

CD30, a member of the tumor necrosis factor receptor superfamily, plays roles in pro-survival signal induction and cell proliferation in peripheral T-cell lymphoma (PTCL) and adult T-cell leukemia/lymphoma (ATL). Previous studies have identified the functional roles of CD30 in CD30-expressing malignant lymphomas, not only PTCL and ATL, but also Hodgkin lymphoma (HL), anaplastic large cell lymphoma (ALCL), and a portion of diffuse large B-cell lymphoma (DLBCL). CD30 expression is often observed in virus-infected cells such as human T-cell leukemia virus type 1 (HTLV-1). HTLV-1 is capable of immortalizing lymphocytes and producing malignancy. Some ATL cases caused by HTLV-1 infection overexpress CD30. However, the molecular mechanism-based relationship between CD30 expression and HTLV-1 infection or ATL progression is unclear. Recent findings have revealed super-enhancer-mediated overexpression at the CD30 locus, CD30 signaling via trogocytosis, and CD30 signaling-induced lymphomagenesis in vivo. Successful anti-CD30 antibody-drug conjugate (ADC) therapy for HL, ALCL, and PTCL supports the biological significance of CD30 in these lymphomas. In this review, we discuss the roles of CD30 overexpression and its functions during ATL progression.

CD30 Regulation of IL-13-STAT6 Pathway in Breast Implant-Associated Anaplastic Large Cell Lymphoma.

BACKGROUND: Breast implant-associated anaplastic large cell lymphoma (BIA-ALCL) is a rare, usually indolent CD30+ T-cell lymphoma with tumor cells, often surrounded by eosinophils, expressing IL-13 and pSTAT6. OBJECTIVES: The aim of this study was to understand the unique tumor pathology and growth regulation of BIA-ALCL, leading to potential targeted therapies. METHODS: We silenced CD30 and analyzed its effect on IL-13 signaling and tumor cell viability. IL-13 signaling receptors of BIA-ALCL cell lines were evaluated by flow cytometry and pSTAT6 detected by immunohistochemistry. CD30 was deleted by CRISPR/Cas9 editing. Effects of CD30 deletion on transcription of IL-13 and IL-4, and phosphorylation of STAT6 were determined by real-time polymerase chain reaction and western blotting. The effect of CD30 deletion on p38 mitogen-activated protein kinase (MAPK) phosphorylation was determined. Suppression of IL-13 transcription by a p38 MAPK inhibitor was tested. Tumor cell viability following CD30 deletion and treatment with a pSTAT6 inhibitor were measured in cytotoxicity assays. RESULTS: BIA-ALCL lines TLBR1 and TLBR2 displayed signaling receptors IL-4Rα, IL-13Rα1 and downstream pSTAT6. Deletion of CD30 by CRISPR/Cas9 editing significantly decreased transcription of IL-13, less so Th2 cytokine IL-4, and phosphorylation of STAT6. Mechanistically, we found CD30 expression is required for p38 MAPK phosphorylation and activation, and IL-13-STAT6 signaling was reduced by an inhibitor of p38 MAPK in BIA-ALCL tumor cells. Tumor cell viability was decreased by silencing of CD30, and a specific inhibitor of STAT6, indicating STAT6 inhibition is cytotoxic to BIA-ALCL tumor cells. CONCLUSIONS: These findings suggest reagents targeting the IL-13 pathway, pSTAT6 and p38 MAPK, may become useful for treating BIA-ALCL patients.

Angioimmunoblastic T-Cell Lymphoma with Exuberant CD30-Positive Follicular Dendritic Cell Proliferation in a SARS-CoV-2 Patient: The Role of Mutational Analysis to Exclude an Associated Follicular Dendritic Cell Sarcoma.

Follicular dendritic cell (FDC) proliferation in angioimmunoblastic T-cell lymphoma (AITL) is still not well defined, challenging the accurate differential diagnosis between the AITL with expanded follicular dendritic cell meshwork and the combined AITL and follicular dendritic cell sarcoma (FDCS). Herein, we reported the case of a 58-year-old male with coexisting SARS-CoV-2 infection and AITL with an exuberant CD30-positive FDC proliferation, in which genetic analysis identified mutations of genes commonly involved in AITL but not in FDC sarcoma (i.e., RHOA, TET2, DNMT3A, and IDH2), thus supporting the reactive nature of the CD30-positive FDC expansion.

The Expressions of CD30 and CD123 of Mastocytosis in Taiwan.

Mastocytosis is a rare disease with a low incidence in Asia-Pacific populations. CD30 and CD123 may have potential prognostic and therapeutic value, but the results are inconsistent. Because racial disparities may exist, we aim to evaluate the expressions of CD30 and CD123 in a series of mastocytosis cases in Taiwan. Twelve patients with systemic and 7 with cutaneous forms of mastocytosis were studied. The expressions of CD30 and CD123 were correlated with the clinical features of the patients. Eighty-three percent (10/12) of patients with systemic mastocytosis (SM) had an associated hematological neoplasm. Four of the SM patients had both "B" and "C" findings, and they had a median survival time of 0.9 months. CD30 expression was positive in 50% (6/12) of SM cases and 100% (6/6) of cutaneous mastocytosis cases. CD123 was expressed focally or weakly in only 2 SM-associated hematological neoplasm cases. The distribution of mastocytosis subtypes and the expression of CD30 and CD123 in Taiwan differed from those reported in North America and Europe. However, mastocytosis, especially indolent forms, is easily overlooked as its heterogeneous and nonspecific clinical manifestations. A high index of suspicion and improved diagnostic methods can be helpful.

CD30-targeted therapy induces apoptosis of inflammatory cytokine-stimulated synovial fibroblasts and ameliorates collagen antibody-induced arthritis in mice.

OBJECTIVE: It has been reported that levels of soluble CD30 in serum and joint fluid are significantly elevated in patients with rheumatoid arthritis (RA). This study aimed to investigate whether CD30 could be a therapeutic target for RA. METHODS: The expression and localization of CD30 were examined by immunohistochemical and double immunofluorescence staining on synovial tissue samples obtained from patients with RA or osteoarthritis (OA) during surgery. Changes in CD30 expression of fibroblast-like synoviocytes (FLS) from RA patients with or without TNFα and IL-1ß stimulation were examined by the polymerase chain reaction (PCR) and flow cytometry. Collagen antibody-induced arthritis (CAIA) was created in DBA/1 mice, and the therapeutic effect of brentuximab vedotin (BV) was examined by clinical score, histological findings and measurement of serum levels of SAA, IL-6, and TNFα. RESULTS: CD30 expression was significantly higher in samples from patients with RA than from those with OA. Double immunofluorescence showed a low rate of co-localization of CD30 with CD20 or CD90, but a high rate of co-localization of CD30 and CD138. CD30 mRNA expression was upregulated 11.7-fold in FLS following stimulation by inflammatory cytokines. The clinical scores of CAIA mice were significantly lower following both BV treatments, however, the histological scores of CAIA mice were significantly lower only following treatment with high dose BV (70 mg/kg). CONCLUSIONS: CD30 was expressed on immunocompetent cells in synovial tissue from RA patients and in cytokine-stimulated FLS in vitro. High dose BV (70 mg/kg) showed significant therapeutic effects in ameliorating inflammation and joint destruction in CAIA mice, but low dose BV (30 mg/kg) was insufficient.

A new immunotherapy strategy targeted CD30 in peripheral T-cell lymphomas: CAR-modified T-cell therapy based on CD30 mAb.

Chimeric antigen receptor T-cell immunotherapy (CAR-T) has shown remarkable efficacy in treating tumors of lymphopoietic origin. Herein, we demonstrate an effective CAR-T cell treatment for recurrent and malignant CD30-positive peripheral T-cell lymphomas (PTCL) has been demonstrated. The extracellular fragment gene sequences of CD30 were obtained from tumor tissues of PTCL patients and cloned into a plasmid vector to express the CD30 antigen. The CD30 targeting single-chain antibody fragment (scFv) was obtained from CD30-positive monoclonal hybridoma cells, which were obtained from CD30 antigen immunized mice. After a second-generation of CAR lentiviral construction, CD30 CAR T cells were produced and used to determine the cytotoxicity of this construct toward Karpas 299 cells. The results of CD30 CAR T-mediated cell lysis show that 9C11-2 CAR T cells could significantly promote the lysis of CD30-positive Karpas 299 cells in both LDH and real-time cell electronic sensing (RTCA) assays. In vivo data show that 9C11-2 CAR T cells effectively suppress the tumor growth in a Karpas 299 cell xenograft NCG mouse model. The CD30 CAR T cells exhibited an efficient cytotoxic effect after being co-cultured with the target cells and they also exhibited a significant tumor-inhibiting ability after being intravenously injected into PTCL xenograft tumors; these observations suggest that the new CD30 CAR-T cell may be a promising therapeutic candidate for cancer therapy.

CD153/CD30 signaling promotes age-dependent tertiary lymphoid tissue expansion and kidney injury.

Tertiary lymphoid tissues (TLTs) facilitate local T and B cell interactions in chronically inflamed organs. However, the cells and molecular pathways that govern TLT formation are poorly defined. Here, we identified TNF superfamily CD153/CD30 signaling between 2 unique age-dependent lymphocyte subpopulations, CD153+PD-1+CD4+ senescence-associated T (SAT) cells and CD30+T-bet+ age-associated B cells (ABCs), as a driver for TLT expansion. SAT cells, which produced ABC-inducing factors IL-21 and IFN-γ, and ABCs progressively accumulated within TLTs in aged kidneys after injury. Notably, in kidney injury models, CD153 or CD30 deficiency impaired functional SAT cell induction, which resulted in reduced ABC numbers and attenuated TLT formation with improved inflammation, fibrosis, and renal function. Attenuated TLT formation after transplantation of CD153-deficient bone marrow further supported the importance of CD153 in immune cells. Clonal analysis revealed that SAT cells and ABCs in the kidneys arose from both local differentiation and recruitment from the spleen. In the synovium of aged rheumatoid arthritis patients, T peripheral helper/T follicular helper cells and ABCs also expressed CD153 and CD30, respectively. Together, our data reveal a previously unappreciated function of CD153/CD30 signaling in TLT formation and propose targeting the CD153/CD30 signaling pathway as a therapeutic target for slowing kidney disease progression.

Super-enhancer-based identification of a BATF3/IL-2R-module reveals vulnerabilities in anaplastic large cell lymphoma.

Anaplastic large cell lymphoma (ALCL), an aggressive CD30-positive T-cell lymphoma, comprises systemic anaplastic lymphoma kinase (ALK)-positive, and ALK-negative, primary cutaneous and breast implant-associated ALCL. Prognosis of some ALCL subgroups is still unsatisfactory, and already in second line effective treatment options are lacking. To identify genes defining ALCL cell state and dependencies, we here characterize super-enhancer regions by genome-wide H3K27ac ChIP-seq. In addition to known ALCL key regulators, the AP-1-member BATF3 and IL-2 receptor (IL2R)-components are among the top hits. Specific and high-level IL2R expression in ALCL correlates with BATF3 expression. Confirming a regulatory link, IL-2R-expression decreases following BATF3 knockout, and BATF3 is recruited to IL2R regulatory regions. Functionally, IL-2, IL-15 and Neo-2/15, a hyper-stable IL-2/IL-15 mimic, accelerate ALCL growth and activate STAT1, STAT5 and ERK1/2. In line, strong IL-2Rα-expression in ALCL patients is linked to more aggressive clinical presentation. Finally, an IL-2Rα-targeting antibody-drug conjugate efficiently kills ALCL cells in vitro and in vivo. Our results highlight the importance of the BATF3/IL-2R-module for ALCL biology and identify IL-2Rα-targeting as a promising treatment strategy for ALCL.

A Poorly Differentiated Non-keratinizing Sinonasal Squamous Cell Carcinoma with a Novel ETV6-TNFRSF8 Fusion Gene.

Squamous cell carcinoma of the sinonasal tract is relatively rare and morphologically and genetically heterogeneous. We report the case of an adult male with a left sphenoid sinus mass. A biopsy revealed an undifferentiated carcinoma composed of sheets of epithelioid cells lacking keratinization and glandular formation. The tumor was associated with a prominent lymphoplasmacytic inflammatory infiltrate. Immunohistochemical staining demonstrated diffuse expression of pankeratin and p63; it was negative for p16. In addition, EBER was also negative. Morphologically the findings raised the possibility of non-keratinizing squamous cell carcinoma. RNA sequencing was undertaken to exclude the possibility of NUT carcinoma; interestingly, this revealed a novel ETV6-TNFRSF8 fusion transcript, which was independently confirmed by fluorescence in situ hybridization. The current case is illustrative because it broadens our understanding of the molecular pathogenesis of non-keratinizing squamous cell carcinoma and adds to the diversity of ETV6-rearranged malignancies.

Multicentric MFI30 study: Standardization of flow cytometry analysis of CD30 expression in non-Hodgkin lymphoma.

CD30 transmembrane receptor, a member of the tumor necrosis factor receptor family, is expressed in different lymphomas. Brentuximab vedotin (BV), a CD30 monoclonal antibody (Ab)-drug conjugate, is effective in CD30-positive lymphomas. However, the response to BV is not always correlated to CD30 expression detected by immunohistochemistry (IHC). The objectives of this study were to standardize and evaluate CD30 intensity by flow cytometry (FCM) in non-Hodgkin's lymphomas. Twelve centers analyzed 161 cases on standardized cytometers using normalized median fluorescence intensity (nMFI30) of three different Abs, of which one clone can recognize the same epitope as BV. FCM distinguished four groups of cases: negative group (n = 110) which showed no expression with the three clones; high positive group (n = 13) which gave nMFI30 > 5% with all tested clones; dim positive group (n = 17) which showed nMFI30 > 1% with all tested clones and <5% for at least one; discordant group (n = 21) with positive and negative expression of the different clones. In consistency with the literature, CD30 was positive in all anaplastic large cell lymphomas, in some diffuse large B-cell lymphomas (DLBCL), and in other rare lymphomas. FCM results were concordant with those of IHC in 77% of cases. Discrepancies could be explained by clones-related differences, microenvironment, or intracytoplasmic staining. Interestingly, FCM was more sensitive than IHC in 11% of cases, especially in DLBCL. Multicenter standardized FCM of specific CD30 could improve case detection and extend the treatment of BV to various CD30-positive lymphomas.

A Nested Asymmetric PCR Melting Curve Assay for One-Step Genotyping of Nondeletional α-Thalassemia Mutations.

A rapid DNA-based assay is essential for clinical diagnosis and mass screening in thalassemia-prevention programs. Because of high homology and guanine-cytosine-rich and complex second structure of α-globin genes, it is rather difficult to develop a feasible and simple method for α-thalassemia genotyping. In this study, a strategy of nested asymmetric PCR melting curve analysis was designed to tackle these factors and ensure sensitivity and accuracy. Herein, a novel one-step assay for genotyping of nondeletional α-thalassemia mutations, including hemoglobin (Hb) Westmead (HBA2: c.369C>G), Hb Quong Sze (HBA2: c.377T>C), Hb Constant Spring (HBA2: c.427T>C), CD30 (HBA2: c.91-93delGAG), and CD31 (HBA2: c.95G>A) in a single closed tube, was established and evaluated. All five mutations were accurately determined with the concordance rate of 100% in a blind analysis of 255 genotype-known samples and 1250 clinical samples. In conclusion, this assay is useful for rapid and reliable genotyping of nondeletional α-thalassemia mutations in clinical practice. Especially, the strategy may have the potential to be a versatile scheme for rapid genotyping of other gene mutations because of its high throughput, sufficient stability, low cost, and simple operation.

CD30L/CD30 signaling regulates the formation of the tumor immune microenvironment and inhibits intestinal tumor development of colitis-associated colon cancer in mice.

Inflammatory bowel disease is one of the major causes of colitis-associated colon cancer (CAC). Therefore, it is necessary to explore new therapies to prevent colon cancer (CRC) in view of the relationship between chronic inflammation and tumor development. Previous studies on the correlation between CD30L/CD30 and cancer were mostly limited to lymphoid or homogenous tumors, while there have been only a few reports on the role of CD30L/CD30 signal transduction in the pathogenesis of CAC. In this study, we established an AOM/DSS-induced CAC model with CD30LKO mice to explore the effect of CD30L/CD30 signal transduction on the formation of the intestinal tumor immune microenvironment (TIME) during the development of intestinal tumors. Our results revealed that CD30L deficiency promoted the accumulation of myeloid derived suppressor cells (MDSCs), increased the expression of PD-L1 on MDSCs and tumor associated macrophages (TAMs), and enhanced the secretion of various inflammatory and immunosuppressive factors in the intestinal mucosa of CAC mice. Furthermore, CD30L gene deletion could selectively promote the upregulation of PD-1 expression on CD4+ and CD8+ T cells and inhibit their activation, differentiation and secretion of effector cytokines, which led to an attenuation of antitumor immune responses mediated by TEM (CD44+CD62L-) cells. Thus, our data suggest that CD30L/CD30 signaling might be a potential candidate target for immunological therapy in CAC.

Eighty-eight variants highlight the role of T cell regulation and airway remodeling in asthma pathogenesis.

Asthma is one of the most common chronic diseases affecting both children and adults. We report a genome-wide association meta-analysis of 69,189 cases and 702,199 controls from Iceland and UK biobank. We find 88 asthma risk variants at 56 loci, 19 previously unreported, and evaluate their effect on other asthma and allergic phenotypes. Of special interest are two low frequency variants associated with protection against asthma; a missense variant in TNFRSF8 and 3' UTR variant in TGFBR1. Functional studies show that the TNFRSF8 variant reduces TNFRSF8 expression both on cell surface and in soluble form, acting as loss of function. eQTL analysis suggests that the TGFBR1 variant acts through gain of function and together with an intronic variant in a downstream gene, SMAD3, points to defective TGFßR1 signaling as one of the biological perturbations increasing asthma risk. Our results increase the number of asthma variants and implicate genes with known role in T cell regulation, inflammation and airway remodeling in asthma pathogenesis.