Hodgkin Lymphoma Cell Research Articles

Identification of two unannotated miRNAs in classic Hodgkin lymphoma cell lines.

MicroRNAs (miRNAs) are small non coding RNAs responsible for posttranscriptional regulation of gene expression. Even though almost 2000 precursors have been described so far, additional miRNAs are still being discovered in normal as well as malignant cells. Alike protein coding genes, miRNAs may acquire oncogenic properties in consequence of altered expression or presence of gain or loss of function mutations. In this study we mined datasets from miRNA expression profiling (miRNA-seq) of 7 classic Hodgkin Lymphoma (cHL) cell lines, 10 non-Hodgkin lymphoma (NHL) cell lines and 56 samples of germinal center derived B-cell lymphomas. Our aim was to discover potential novel cHL oncomiRs not reported in miRBase (release 22.1) and expressed in cHL cell lines but no other B-cell lymphomas. We identified six such miRNA candidates in cHL cell lines and verified the expression of two of them encoded at chr2:212678788-212678849 and chr5:168090507-168090561 (GRCh38). Interestingly, we showed that one of the validated miRNAs (located in an intron of the TENM2 gene) is expressed together with its host gene. TENM2 is characterized by hypomethylation and open chromatin around its TSS in cHL cell lines in contrast to NHL cell lines and germinal centre B-cells respectively. It indicates an epigenetic mechanism responsible for aberrant expression of both, the TENM2 gene and the novel miRNA in cHL cell lines. Despite the GO analysis performed with the input of the in silico predicted novel miRNA target genes did not reveal ontologies typically associated with cHL pathogenesis, it pointed to several interesting candidates involved in i.e. lymphopoiesis. These include the lymphoma related BCL11A gene, the IKZF2 gene involved in lymphocyte development or the transcription initiator GTF2H1.

Differentiating fulminant EBV infection complicated by HLH from Lymphoma: report of a case and a brief literature review

Epstein-Barr virus (EBV) infection may present with fulminant constitutional symptoms, cytopenia(s), and systemic lymphadenopathy, raising clinical suspicion for lymphoma and prompting lymph node and bone marrow biopsies. At the microscopic level, the histopathologic findings in cases of acute EBV lymphadenitis may mimic certain lymphoid neoplasms, creating a range of differential diagnoses and diagnostic pitfalls.We present a case of fulminant EBV infection in an adolescent whose clinical and radiographic findings led to lymph node and bone marrow biopsies to rule out lymphoma. One week after being diagnosed with acute EBV infection (infectious mononucleosis), a 17-year-old Caucasian male presented with worsening symptoms including persistent fever, progressive, painful lymphadenopathy, and splenomegaly. A peripheral blood smear showed lymphocytosis with many reactive lymphocytes, anemia, and thrombocytopenia. Laboratory studies showed elevated ferritin, triglycerides, and soluble IL-2/CD25. A cervical lymph node biopsy demonstrated an EBV-positive, reactive B-immunoblast proliferation with large atypical lymphoid cells mimicking Reed-Sternberg cells of Hodgkin lymphoma, in addition to patchy vasculitis, coagulative necrosis, and prominent hemophagocytic activity. Bilateral bone marrow biopsies showed a hypercellular marrow with patchy infiltrates of similar EBV-positive, large atypical lymphoid cells, as well as prominent hemophagocytic activity. The diagnosis of acute EBV associated lymphoproliferation with concurrent hemophagocytic lymphohistiocytosis (HLH) was rendered.Recognition of common and uncommon clinical presentations of acute EBV infection is essential, particularly when histopathologic findings raise suspicion for a possible hematolymphoid neoplasm. Both the lymph node architectural and viral cytopathic changes observed in EBV lymphadenitis exhibit significant morphologic overlap with classic Hodgkin lymphoma (cHL) and several other lymphomas, including anaplastic large cell lymphoma, diffuse large B cell lymphoma, and angioimmunoblastic T cell lymphoma. Recognition of immunohistochemical staining patterns in EBV lymphadenitis is critical to avoid misdiagnosis. Conversely, bona fide lymphoma, particularly cHL, can masquerade as EBV infection. We provide a concise discussion and tables of the histopathologic differential diagnosis of EBV lymphadenitis, including cHL and other lymphomas. Pathologists should include acute EBV infection within the differential diagnosis when confronted with clinical and pathologic findings concerning for lymphoma, particularly in adolescents and young adults.

Hodgkin Lymphoma Cell Lines and Tissues Express mGluR5: A Potential Link to Ophelia Syndrome and Paraneoplastic Neurological Disease.

Ophelia syndrome is characterized by the coincidence of severe neuropsychiatric symptoms, classical Hodgkin lymphoma, and the presence of antibodies to the metabotropic glutamate 5 receptor (mGluR5). Little is known about the pathogenetic link between these symptoms and the role that anti-mGluR5-antibodies play. We investigated lymphoma tissue from patients with Ophelia syndrome and with isolated classical Hodgkin lymphoma by quantitative immunocytochemistry for mGluR5-expression. Further, we studied the L-1236, L-428, L-540, SUP-HD1, KM-H2, and HDLM-2 classical Hodgkin lymphoma cell lines by FACS and Western blot for mGluR5-expression, and by transcriptome analysis. mGluR5 surface expression differed significantly in terms of receptor density, distribution pattern, and percentage of positive cells. The highest expression levels were found in the L-1236 line. RNA-sequencing revealed more than 800 genes that were higher expressed in the L-1236 line in comparison to the other classical Hodgkin lymphoma cell lines. High mGluR5-expression was associated with upregulation of PI3K/AKT and MAPK pathways and of downstream targets (e.g., EGR1) known to be involved in classical Hodgkin lymphoma progression. Finally, mGluR5 expression was increased in the classical Hodgkin lymphoma-tissue of our Ophelia syndrome patient in contrast to five classical Hodgkin lymphoma-patients without autoimmune encephalitis. Given the association of encephalitis and classical Hodgkin lymphoma in Ophelia syndrome, it is possible that mGluR5-expression in classical Hodgkin lymphoma cells not only drives tumor progression but also triggers anti-mGluR5 encephalitis even before classical Hodgkin lymphoma becomes manifest.

"Coexistence of Classical Hodgkin Lymphoma and Langerhans Cell Proliferation Presenting as a Tumor in Tumor Pattern"

"Coexistence of Classical Hodgkin Lymphoma and Langerhans Cell Proliferation Presenting as a Tumor in Tumor Pattern"

The Medium Obtained from the Culture of Hodgkin Lymphoma Cells Affects the Biophysical Characteristics of a Fibroblast Cell Model

The neoplastic Hodgkin-Reed-Sternberg (HRS) cells in Hodgkin lymphoma (HL) represent only 1-10% of cells and are surrounded by an inflammatory microenvironment. The HL cytokine network is a key point for the proliferation of HRS cells and for the maintenance of an advantageous microenvironment for HRS survival. In the tumor microenvironment (TME), the fibroblasts are involved in crosstalk with HRS cells. The aim of this work was to study the effect of lymphoma cell conditioned medium on a fibroblast cell population and evaluate modifications of cell morphology and proliferation. Hodgkin lymphoma-derived medium was used to obtain a population of "conditioned" fibroblasts (WS-1 COND). Differences in biophysical parameters were detected by the innovative device Celector®. Fibroblast-HL cells interactions were reproduced in 3D co-culture spheroids. WS-1 COND showed a different cellular morphology with an enlarged cytoplasm and enhanced metabolism. Area and diameter cell values obtained by Celector® measurement were increased. Co-culture spheroids created with WS-1 COND showed a tighter aggregation than those with non-conditioned WS-1. The presence of soluble factors derived from HRS cells in the conditioned medium was adequate for the proliferation of fibroblasts and conditioned fibroblasts in a 3D HL model allowed to develop a representative model of the in vivo TME.

IL-6 as an Inflammatory Marker in Hodgkin Lymphoma

Hodgkin lymphoma (HL) is a lymphoid tumor that develops from embryological or post-germinal center B-cells. The disease was originally recognized in 1832 by Thomas Hodgkin. Globally, 0.4% of clinically diagnosed cancers in 2018 were caused by HL. Regardless of age, ethnicity, or location, the prevalence of HL varies. It's a particular kind of malignant tumor in which the cancerous Hodgkin and Reed-Sternberg (HRS) cells are surrounded by a significant cellular infiltration of immune cells (lymphocytes, macrophages, eosinophils, mast cells, plasma cells, and collagen cells), which also contribute to the microenvironment of the tumor. Numerous chemotherapy drugs and low-dose field radiation have been found to significantly reduce illness mortality [1].
 High levels of cytokines that stimulate the immune system, such as interleukin (IL)-6, tumor necrosis factor (TNF), and eotaxin are produced by developing tumor cells and/or by reactive immune cells associated with tumors. These markers are anticipated to promote B-cell activation and related high-risk DNA-modifying activities, which will lead to the growth of B-cell lymphoma. IL-6 is a critical cytokine that stimulates the growth of hematological and solid tumors and is synthesized by a wide range of cell types, including fibroblasts, ECs, epithelial cells, and monocytes as hematological tumor lines. Through the activation of JAK, it stimulates the transcription factors STAT. In NHL, lower progression-free survival and overall survival rates have been linked to higher blood IL-6 levels, and IL-6 production in HL cell lines has been demonstrated. Numerous investigations revealed that IL-6 expression correlated with early onset vascular development events, indicating that it may be involved in angiogenesis [2].
 Interleukin-6 (IL-6) is thought to play a crucial pathobiological function in classical HL among the biologic variables like cytokines. According to reports, adult patients with HL who have elevated serum interleukin-6 (IL-6) have better response rates, longer survival times, and B symptoms. TH-17 response may be elicited by HRS cells that express IL-6, but TH-2 response may be elicited by HRS cells that do not express IL-6. T-lymphocytes are known to be attracted by IL-6, and depending on how it interacts with other cytokines like TGF-beta, the lymphocytes may induce a TH-2 or TH-17 response. A significant cancer hallmark known as immune evasion can be brought on in cancer patients by the TH-2 response [3].

A Non-Coding Variant in LOC105371512 Confers Susceptibility to Bleomycin-Induced Lung Injury

Background: Bleomycin has been used as a cornerstone in the treatment of a variety of cancer types including Hodgkin Lymphoma (HL) and Germ Cell Tumor. Unfortunately, bleomycin has its complications, such as Bleomycin-induced pneumonitis (BIP) occurring in ~20% of patients and is fatal in 4-5%. In addition, there are no trials guiding effective treatment for BIP. While brentuximab as an alternative for bleomycin has shown good results in HL, it is still not considered as a standard first line treatment for most of the world. We aimed to investigate the significance of known genetic associations [(HFE rs1799945 (H63D) and BLMH rs1050565 (I443V)] in Asian patients, and identify previously uncharacterized genetic biomarkers associated with the predisposition to BIP. Through functional studies, we also sought to investigate the mechanisms in which these genetic biomarkers led to BIP. Methods: We analyzed 133 HL patients treated with bleomycin in Singapore. Clinical data was extracted from electronic medical records, BIP was characterized and graded by CTCAEv4. Genomic DNA was genotyped using Infinium Global Screening Array-24 v1.0 for 700,078 markers. Alt-R® CRISPR-Cas9 was used for the gene-editing experiment. Results: A total of 21.43% of patients developed clinically significant BIP, defined as CTCAEv4 ≥ Grade 2. Cumulative bleomycin dose was significantly lower in the BIP cases, as BIP patients had bleomycin discontinued from future therapy. All other clinical features were not significantly different between the groups. We replicated an association of HFE rs1799945 (H63D) with BIP and identified 4 new potential genetic predictors. LOC105371512 rs189773 was ranked the top loci. We genotyped 4 lung cancer cell lines. Comparing rs189773 CC and TT genotypes, CC had lower expression of pro-fibrotic markers, while higher expression of anti-fibrotic ones. We next used CRISPR/Cas9 to generate cells with either CC or TT genotype at rs189773 locus using the same cell line (A459). Interestingly, the BLMH protein expression level showed a reduction in A549(TT) compared to A549(CC), with a more remarkable effect in the presence of bleomycin. Screening the fibrotic biomarkers further revealed that growth factors and pro-fibrotic interleukins were up-regulated in A549(TT) compared to A549(CC) cells especially when the cells were treated with bleomycin. Next, we found that this SNP is located in a transcriptionally active region, with a higher activity level for the presence of T at the position compared to C. The rs189773-flanking region was subsequently shown to enhance the transcription level of distal genes with consistent effects on the profibrotic growth factors and cytokines. The qPCR study demonstrated that BLMH was not regulated at the mRNA level. We thus treated the cells with bleomycin and a 26S proteasome inhibitor, bortezomib. Interestingly, the bleomycin-induced down-regulation of BLMH protein was rescued upon co-treating the cells with both bleomycin and bortezomib. This proteasome inhibitor drug consistently diminished the heightened levels of profibrotic markers induced by bleomycin. Conclusion: In conclusion, in the first pharmacogenomics-GWAS study on BIP in Asians, rs189773 was ranked the top likely loci, with functional studies showing that this variant is in an enhancer region, modulating the cellular response to bleomycin via regulation of a distal locus, BLMH, and profibrotic growth factors. At the post-translational level, inhibition of the 26S proteasome by bortezomib restores BLMH expression and suppresses profibrotic growth factors. This study provides insights into future screening targets and potential new treatment options for BIP. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Hyper N-Glycosylated SEL1L3 As B-Cell Receptor Autoantigen of Primary Vitreoretinal Lymphoma

Chronic antigenic stimulation of B lymphocytes through their B-cell receptor (BCR) is thought to play an important role in the genesis of B-cell lymphomas. SAMD14/neurabin-I has been described as auto-antigenic target of primary central nervous system lymphoma (PCNSL) BCRs. Primary vitreoretinal lymphoma (PVRL) is a rare subtype of DLBCL and can progress to PCNSL. Besides manifestation in an immunologically privileged site, PVRL and PCNSL share frequent recurrent genetic alterations like MYD88 and CD79B mutations. Both PVRL and PCNSL are considered being related to the MCD subtype of DLBCLs and may depend on chronic BCR signaling through antigenic stimulation. To investigate the role of chronic antigenic stimulation in PVRL, we cloned and expressed BCRs from PVRL patients as antibody fragments (Fabs) and tested for specific binding against common antigens. All cases with corresponding BCR sequences were previously reported by Belhouachi et al. (Blood Adv. 2020;4(7):1357-1366). For 20 of 55 PVRL BCR sequences both immunoglobulin heavy and light chain variable region (V) genes were available. DNA clones comprising an ApaLI restriction site, the complete light chain V gene, a κ- or λ-constant region gene, a ribosome binding site, a signal sequence, the heavy chain V gene and a BstEII restriction site were ordered from GeneCust® (GeneCust, 5690 Ellange, Luxembourg) in a pUC57 vector. DNA fragments were cloned in front of a γ-1 constant region gene and a Histidine-tag into a modified pCES-1 vector for expression as Fabs. PVRL-BCRs were screened on protein macroarrays representing clones of UniPEx human cDNA expression libraries expressed in E. coli (Bioscience, Dublin, Ireland). SEL1L3 was identified as the auto-antigenic target of the B-cell receptors in 3 out of 20 PVRL cases. None of the PVRL Fabs bound to LRPAP1 (common BCR antigen of mantle cell lymphomas), RpoC (Moraxella catarrhalis bacterial antigen and target of nodular lymphocyte-predominant Hodgkin lymphoma cells) or galectin-3 (member of the lectin family). SEL1L3 is a 1132 amino acid long protein with multiple glycosylation and phosphorylation sites (uniprot.org). This is especially important since previously identified BCR antigens from different lymphoma entities showed post-translational modifications such as hyper-N-glycosylation or hypo-phosphorylation. Furthermore, SEL1L3 is highly expressed in the pigmented layer of the retina. Western Blot analysis of the blood of a PVRL patient with serum antibodies against SEL1L3 showed an increased molecular weight of SEL1L3 as compared to SEL1L3 of controls. After treatment with N-acetyl-β-d-glucosaminidase or PNGase F, this difference in molecular weight disappeared, indicating a hyper N-glycosylation of SEL1L3 in PVRL patients with SEL1L3-reactive BCRs as trigger for its' immunogenicity. SEL1L3 induced proliferation of aggressive lymphoma cell lines transfected with SEL1L3-reactive BCRs. Moreover, the BCR binding epitope of SEL1L3 (aa 560 - 585) conjugated to a truncated form of Pseudomonasaeruginosa exotoxin A, killed exclusively lymphoma cells expressing BCRs with the respective reactivity. Additionally, BCRs of a locally treated PVRL patient with serum antibodies against SEL1L3 were cloned from biopsies of the vitreous body at initial diagnosis and of a lymphoma manifestation in the breast at relapse. Both BCRs showed binding to SEL1L3 suggesting a continued dependence of the BCR on antigen stimulation. These results suggest an important role of chronic antigenic stimulation by the post-translationally modified auto-antigen SEL1L3 in the genesis of a subgroup of patients with PVRL, might give an explanation for the selective tropism of PVRLs and provide the basis for a new treatment approach with high specificity.

Replacing Procarbazine with Dacarbazine in Escalated Beacopp Dramatically Reduces the Post Treatment Haematopoietic Stem and Progenitor Cell Mutational Burden in Hodgkin Lymphoma Patients with No Apparent Loss of Clinical Efficacy

Introduction When treating Hodgkin lymphoma (HL), it is common UK practice to modify escalated BEACOPP (eBPP) by removing oral procarbazine and replacing it with intravenous dacarbazine (250mg/m2 D2-3) to reduce haematopoietic stem cell and gonadal toxicity. A similar replacement of procarbazine in COPP (to COPDac) has reduced gonadal toxicity and conferred a comparable long-term event-free survival to children (EuroNet-PHL-C1 trial; Mauz-Körholz et al Lancet Oncol 2021). Our own retrospective study has shown that adults treated with first-line escalated BEACOPDac (eBPDac) have a reduced blood transfusion requirement and earlier return of menstrual periods compared with real-world eBPP patients (pts) with no loss of clinical efficacy compared to HD18 eBPP pts (Santarsieri et al Blood 2021). To better understand the toxicity differences, we performed this study to compare the haematopoietic stem and progenitor cell (HSPC) mutational burden in pts who have received procarbazine and dacarbazine containing regimens. Chemotherapeutic agents are known to damage DNA and this mutational process likely causes many of the long-term chemotherapy toxicities. Little is known about the mutational signatures and mutation burdens caused by HL therapies, although a novel mutational signature (SBS25) has recently been described in two HL cell lines (Petljak et al Cell 2019). Methods We whole genome sequenced 6-8 single-cell derived HSPC colonies from each of 12 advanced stage HL pts (n = 91; mean sequencing depth 26X). The pts were all in remission > 6 months and had been previously treated with either eBPDac (6 cycles (n=2); 4 cycles (n=2)), eBPP (6 cycles (n=3); 5 cycles (n=1); 4 cycles (n=1)) or ABVD (n=3; all 6 cycles). Single HSPC somatic mutation burdens and mutation spectra from these chemotherapy exposed individuals were compared to those from our previously published normal cohort (n = 110; mean sequencing depth 24X) (Mitchell et al Nature 2022). In addition we have updated the outcomes of our retrospective multi-centre study of pts treated with eBPDac including additional pts and longer follow-up of the entire cohort. Mutational analysis The somatic single nucleotide variant mutation burden in normal adult HSPCs ranges from approximately 400 aged 20yr to 1500 aged 80yr; an average of 18 mutations accumulate with every year (Figure 1A). The HSPCs from ABVD and eBPDac-treated pts had similar minor excess somatic mutation burdens compared to aged-matched normal HSPCs of 183 (CI95%= 110-256) and 291 (CI95%= 242-340) mutations respectively. In contrast, the HSPCs from eBPP-treated pts had a striking and significantly increased excess mutation burden of 1153 (CI95%= 937-1369). Of the eBPP pts, the single patient who received 4 cycles had the lowest number of excess mutations, but noting the inter-patient variation, we cannot yet conclude that eBPP cycle number correlates with the number of mutations. Descriptive analysis of the mutational profiles (Figure 1B) revealed that every patient who received procarbazine had a clear SBS25-like mutational signature, characterised by T>A base substitutions, demonstrating for the first time that this DNA mutational signature is directly attributable to procarbazine. Clinical data analysis The clinical data presented last year have been updated with more pts and longer follow-up (n=288; median 2.3 yr). With this high-risk group of advanced stage pts, our data suggest that the eBPDac pts have reduced red cell transfusion requirements, reduced hospitalisation, improved menstrual period recovery and improved sperm count preservation compared with eBPP pts. The eBPDac 24-month PFS is similar to HD18 3-yr PFS (94.9% vs 92.3%) and appears superior to RATHL 5-yr PFS (81.4%). The eBPDac 24-month OS estimate is 99%. Conclusions We have shown that dacarbazine-containing regimens (ABVD and eBPDac) confer a lower mutation burden to HSPCs compared with procarbazine-containing eBPP and that procarbazine is the agent responsible for the SBS25 mutational signature. This work provides a model example of how genomic analysis could be used to inform therapeutic decision making in cancer. Our retrospective analysis provides strong supportive evidence that eBPDac is a highly efficacious HL treatment and noting the clear benefits in terms of HSPC genomic health, we would encourage clinicians offering eBPP to HL pts to consider replacing procarbazine with dacarbazine. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Phase I Trial of Brentuximab Vedotin Plus Cyclosporine in Relapsed/Refractory Hodgkin Lymphoma

Introduction Treatment of relapsed/refractory (R/R) Hodgkin lymphoma (HL) following failure of brentuximab vedotin (BV) and PD-1 blockade is a major unmet need. Resistance to BV is associated with multidrug resistance gene 1 (MDR1) overexpression. There are pre-clinical data showing that MDR1 inhibitors such as cyclosporine (CsA) or verapamil (VRP) are synergistic with BV in BV-resistant HL cell lines. Preliminary results of a phase I study evaluating the safety and efficacy of combination of BV with CsA and VRP in R/R HL were previously reported. Here we report the final results of the completed study. Methods This is a single center, open-label phase I investigator-initiated study. Patients (pts) with HL whose disease had relapsed or was refractory to ≥1 prior lines of therapy were enrolled. The dose finding portion followed a 3+3 design with 4 planned dose levels (DL). BV was given at 1.2 mg/kg (DL1) or 1.8 mg/kg (DL2-4) intravenously every 3 weeks. Planned dose of CsA was 5 mg/kg (DL1-2) or 7.5 mg/kg (DL3-4) orally twice daily on days 1-5. Planned dose of VRP was 120 mg orally four times daily on days 1-5 (DL4). Each cycle was 3 weeks. Once the maximum tolerated dose (MTD) was determined, BV-refractory pts were enrolled in an expansion cohort at the MTD. Primary objective was to determine the MTD and safety of the combination. Secondary objectives were overall response rate (ORR), complete response (CR) rate, response duration (DOR), overall survival (OS), and progression-free survival (PFS). Results Twenty-nine pts were enrolled onto the study, 14 in the dose-finding portion and 15 in the dose expansion BV-refractory cohort. With respect to baseline characteristics, 38% of patients were female, 69% had advanced stage disease, 45% had extranodal disease, and 24% had B symptoms. Median age was 36 (20-69) and pts had a median of 5 (3-12) prior lines of therapy. All had prior BV (only 2 BV-sensitive) and 27 had prior PD-1 blockade. Four pts were treated on DL1, 22 on DL2, and 3 on DL3. MTD was DL2. DLTs observed were: DL1 10-day CsA (n=1) grade (Gr) 3 hyperbilirubinemia, abdominal pain, and hypertension, DL2 (n=1) Gr3 abdominal pain and Gr3-4 neutropenia, DL3 (n=2) Gr3 bone pain/constipation/Gr4 lymphopenia (n=1) and Gr3 hyperglycemia (n=1). Median duration of treatment was 3 (1-16) cycles. Most frequent adverse events (AEs) were nausea (90%), hypertension (90%), anemia (86%), fatigue (76%), neutropenia (76%), and leukopenia (76%). All pts had Gr3+ AEs with most frequent being neutropenia (62%). Reasons for treatment discontinuation included death (n=4), disease progression (n=10), toxicity (n=2), patient refusal (n=8), loss to follow-up (n=2), MD decision (n=1), proceeding to HCT (n=1), and study completion (n=1). Treatment-related death occurred in 3 pts (pneumonitis at DL1, respiratory failure and hypotension at DL2). The ORR/CR was 62%/24%. The median DOR and PFS was 5 months and median OS was not reached. Discussion The combination of BV and CsA was effective in BV-refractory R/R HL but was also associated with toxicity, including treatment-related deaths that led to early termination of the clinical trial. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A Unusual Case of Classic Hodgkin Lymphoma (cHL) with Aberrant T-Antigens Expression

Abstract Introduction/Objective T-cell-associated antigens (TCA) are rarely expressed by Hodgkin/Reed-Sternberg (HRS) cells of classical Hodgkin lymphoma (cHL) and is associated with uncertain clinical significance. Methods/Case Report We present a very recent case of a 68 year-old female with no significant past medical history presented with a large right groin mass for 1 month. She denied any fever, night sweats, fatigue or weight loss. CT abdomen/pelvis showed inguinal/pelvic lymphadenopathy. Resection showed effaced nodal architecture with a vague nodular appearance and with large, atypical nucleolated cells in a background of small, mature lymphocytes and histiocytes. Eosinophils are conspicuously absent in the background. The large cells were polylobated lymphocyte and histiocyte rich (L & H) like, classic HRS cells as well as multinucleated in appearance. The morphologic features overlapped between Classic Hodgkin Lymphoma, and Nodular Lymphocyte Predominant Hodgkin Lymphoma. Given the immunohistochemical (IHC) features of the neoplastic cells [CD45 negative, PAX5+ (weak), CD30+ (strong), CD15+ (majority) and MUM1 +] the diagnosis was most compatible with CHL. Interestingly, a subset of the Hodgkin cells (10-20%) aberrantly expressed a number of T-cell markers (CD2, CD3, CD4, and CD8). However, the background smaller T-cells did not show cytologic atypia or aberrancies of T-cell antigen markers. The large cells were negative for CD43 and ALK1. TCR gamma gene rearrangement was also negative for clonal T-lymphocytes, thus excluding the possibility of peripheral T-cell lymphoma with Hodgkin-like cells. Based on the morphology and IHC profile, this case proves to be CHL with multiple TCA expression including CD2, CD3, CD4 and CD8. She was planned for chemotherapy. Four months after the initial diagnosis and after completion of 2 cycles of chemotherapy, her PET CT scan showed improvement with right inguinal adenopathy being smaller in size and decreased avidity, indicating a response to treatment. She is due for 2 more cycles. Till todate she is doing well with no further symptoms. Results (if a Case Study enter NA) NA. Conclusion So far reported cases of classic Hodgkin lymphoma (CHL) with aberrant T-cell antigen (TCA) expression were associated with decreased event-free survival and overall survival when compared with TCA-negative CHLs. Our finding contributes to the literature and improves our knowledge of the disease.

Navitoclax Most Promising BH3 Mimetic for Combination Therapy in Hodgkin Lymphoma.

The treatment of young patients with Hodgkin lymphoma (HL) is often successful but a significant proportion of patients suffers from late toxicity. In the current era there are new opportunities for less toxic and more targeted treatment options. In this respect, the anti-apoptotic pathway is an attractive target since Hodgkin tumor cells abundantly express components of this pathway. We measured the effect of BH3 mimetics that interfere with anti-apoptotic proteins in cell lines, also in combination with the standard of care chemotherapeutic doxorubicin and the recently discovered preclinically active tamoxifen. Several anti-apoptotic BCL-2 family proteins were expressed in each case (n = 84) and in HL cell lines (n = 5). Cell lines were checked for sensitivity to BH3 mimetics by BH3 profiling and metabolic assays and monotherapy was only partially successful. Doxorubicin was synergistic with a BCL-XL inhibitor and BCL2/XL/W inhibitor navitoclax. Tamoxifen that targets the estrogen receptor β present in the mitochondria of the cell lines, could induce cell death, and was synergistic with several BH3 mimetics including/as well as navitoclax. In conclusion, targeting the anti-apoptotic pathway by the triple inhibitor navitoclax in combination with doxorubicin or tamoxifen is a promising treatment strategy in HL.

The noncatalytic regions of the tyrosine kinase Tnk1 are important for activity and substrate specificity

Human Tnk1 (thirty-eight negative kinase 1) is a member of the Ack family of nonreceptor tyrosine kinases. Tnk1 contains a sterile alpha motif, a tyrosine kinase catalytic domain, an SH3 (Src homology 3) domain, and a large C-terminal region that contains a ubiquitin association domain. However, specific physiological roles for Tnk1 have not been characterized in depth. Here, we expressed and purified Tnk1 from Sf9 insect cells and established an invitro assay system using a peptide substrate derived from the Wiskott-Aldrich Syndrome Protein (WASP). By Tnk1 expression in mammalian cells, we found that the N-terminal SAM domain is important for self-association and kinase activity. We also studied a fusion protein, originally discovered in a Hodgkin's Lymphoma cell line, that contains an unrelated sequence from the C17ORF61 gene fused to the C-terminus of Tnk1. Cells expressing the fusion protein showed increased tyrosine phosphorylation of cellular substrates relative to cells expressing WT Tnk1. A truncated Tnk1 construct (residues 1-465) also showed enhanced phosphorylation, indicating that the C17ORF61 sequence was dispensable for the effect. Additionally, invitro kinase assays with the WASP peptide substrate showed no increase in intrinsic Tnk1 activity in C-terminally truncated constructs, suggesting that the truncations did not simply remove an autoinhibitory element. Fluorescence microscopy experiments demonstrated that the C-terminus of Tnk1 plays an important role in the subcellular localization of the kinase. Taken together, our data suggest that the noncatalytic regions of Tnk1 play important roles in governing activity and substrate phosphorylation.

P029: Are Reed-Sternberg cells stuck in mitosis by short nucleoplasmic bridges as a consequence of centromeric instability?

Background: Multinucleated giant tumor cells are frequently observed in lymph nodes of untreated lymphoma patients. Malignant Hodgkin lymphoma (HL) cells i. e. small Hodgkin and large bi- or multinucleated Reed-Sternberg (HRS) cells are characterized by genomic instability and downregulation of numerous key regulators of e.g. cell cycle, spindle apparatus, cytokinesis and cell lineage differentiation. So far, the formation of HRS cells remains obscure. We have demonstrated previously that telomere instability is involved in the proliferation of small cells into large binucleated cells. Here, we have used a collection of HL cell lines to analyze the successive steps involved in the transformation of the mononucleated into the multinucleated RS-like cells. Materials and Methods: Seven HL cell lines and 30 HL lymph nodes were used. Cytokinesis-block micronucleus assay and telomere and centromere staining followed by the M-FISH technique were employed to analyze chromosomal instability. DNA repair pathways were investigated. Results: We demonstrate that telomere dysfunction in HL cell lines is associated with the formation of dicentric chromosomes with both centromeres in close proximity, but also with a high rate of formation of micronuclei. Two morphologically different types of nucleoplasmic bridges (NPBs) were observed: (1) Long NPBs related to telomere dysfunction and chromosome fusions, and (2) short NPBs related to centromere breakpoints with configurations of cells looking like “stuck” together as binucleated cells. Short NPBs and binucleated cells were seen in the cultures of all HL cell lines. However, each HL cell line was characterized by an individual signature of the proportion of long and short NPBs, which correlated with centrosome amplification and formation of stable binucleated cells. Similar mechanisms were identified in HL patient lymph nodes prior to treatment, thus underscoring the biological significance of our findings in cell cultures. Transcriptome analysis confirmed the variations in the DNA repair pathways regarding the rate of large cells among the different HL cell lines. Conclusion: The formation of dicentric chromosomes related to centromere instability, and short NPBs were associated with the emergence of binucleated cells through different mechanisms. Our findings open a novel route to understanding the transition from mononucleated cells to multinucleated cells in HL and in other B-cell lymphomas.

P035: CRISPR/Cas9-mediated knockout reveals an important role of CD30 in Hodgkin and Reed-Sternberg cells of classical Hodgkin lymphoma

P035: CRISPR/Cas9-mediated knockout reveals an important role of CD30 in Hodgkin and Reed-Sternberg cells of classical Hodgkin lymphoma

P046: Preclinical evaluation of novel repurposed drug combinations in Hodgkin lymphoma

Although the outcome of patients with Hodgkin lymphoma (HL) is comparably high, it worsens rapidly in relapsed or refractory disease. In the pursuit of improving cancer treatment and finding new therapy options, repurposing drugs has evolved as a promising strategy. This project takes a systematic look at the direct antitumor effect of 12 non-oncological, repurposed drugs in HL that survived initial screening in a broad panel of cell lines. Additionally, we consider their immunological effects and aim to decipher strategies on how to potentially use them in conjunction with immunotherapy. Luciferase toxicity assays in different concentrations were performed with four HL cell lines, narrowing down the list of 12 to the five most toxic agents which were then tested again in three different combinations. The most effective combination was then evaluated in mouse models. To evaluate the influence of the screening survivor drugs on immune response, assays for T-cell activation, macrophage polarization and phagocytosis were conducted. Five compounds, Albendazole (Alb), Auranofin (Aur), Disulfiram (Dis), Fluvastatin (Flu) and Propranolol (Pro) showed markedly elevated in-vitro toxicity. The three combinations Alb+Aur+Dis+Pro, Aur+Dis+Flu+Pro and a combination of all 5 drugs showed synergistic cytotoxicity in HL cell lines. Latter combination clearly reduced tumor growth in mouse models. Except for Doxycycline, which markedly repressed T-cell activation, none of the other tested 11 compounds showed negative effects on either T-cell activation, or macrophage polarization and phagocytosis. On the contrary, Eprosartan (Epro), Metformin (Met), Clofibrate (Clof) and Ornidazole (Orni) increased antibody dependent cellular phagocytosis (ADCP) in in-vitro assays. This project shows that combinations of repurposed, non-oncological drugs at safe doses have a direct cytotoxic effect against HL and a potentially beneficial impact on key immune response mechanisms. Taken together, our results justify further evaluation of combinations of these clinically known drugs with chemotherapy or checkpoint blockade in cases of HL without other treatment options.

P054: The Kinase CK2 is deregulated and targetable in classical Hodgkin Lymphoma

Introduction: CK2 is a pleiotropic kinase consisting of 2 catalytic (a) and 2 regulatory (b) subunits, usually overexpressed, that sustains cancer signaling cascades through the activation of NF-kB, PI3K and STAT pathways. Considering that NF-kB, STAT and PI3K pathways are key players in Hodgkin lymphoma (HL), it is likely that CK2 might play a role in the pathogenesis of this disease. Methods: Experiments were performed and replicated at least for five times employing 4 HL cell lines (L-428, L-540, KM-H2 and HDLM-1) cultured in RPMI. CK2a, CK2b, RelA-Serine (S)529, RelA, PARP, AKT-S473, AKT, STAT3-S727, STAT3, tubulin, ubiquitin expression levels were evaluated by western blot analysis (WB). Silmitasertib and bortezomib were used as CK2a and proteasome inhibitors. Apoptosis was assessed by Annexin V/Propidium iodide assay and PARP cleavage by WB. Immunohistochemistry (IHC) for CK2a and CK2b was performed on formalin fixed paraffin embedded sections of lymph-nodes from patients with HL, indolent and aggressive NHL. Chou-Talalay methods was used to calculate the combination index (C.I.) of two drugs. Results: By WB and IF we found that all the 4 HL cell lines expressed higher levels of CK2a, but not CK2b, as compared to normal B lymphocytes (p<0.001). By IHC on 35 patients’ lymph-nodes, we confirmed that CK2a but not CK2b was highly expressed in Reed-Sternberg cells. In addition, patients with lower levels of CK2b display a better progression-free survival (p<0.05). The unbalance between a and b subunits was not observed in low or high-grade NHL (p<0.001). Furthermore, we observed that mRNA levels of both CSNK2A and CSNK2B were similar to normal B lymphocytes, and that proteasome inhibition with bortezomib caused the upregulation of CK2b. AKT, RelA and STAT3 were constitutively phosphorylated in HL at their activatory S-residue (S473, S529, and S727 respectively). Treatment of HL cell lines with silmitasertib caused down-regulation of these phospho-serine proteins, time and dose-dependent apoptosis (p<0.0001). In addition, we found that silmitasertib did not modulate surface CD30 and had a synergistic anti-apoptotic effect in combination with monomethyl auristatin E (C.I.<1) Conclusions: We demonstrated that CK2a is overexpressed, active, induced key pro-survival signals in HL, and its inhibition trigger apoptosis. CK2b is likely downregulated due to proteasome degradation. These preliminary data suggest that CK2 might be a new therapeutic target in HL.

Phycochemistry and bioactivity of cyanobacterial secondary metabolites.

Microbes are a huge contributor to people’s health around the world since they produce a lot of beneficial secondary metabolites. Cyanobacteria are photosynthetic prokaryotic bacteria cosmopolitan in nature. Adaptability of cyanobacteria to wide spectrum of environment can be contributed to the production of various secondary metabolites which are also therapeutic in nature. As a result, they are a good option for the development of medicinal molecules. These metabolites could be interesting COVID-19 therapeutic options because the majority of these compounds have demonstrated substantial pharmacological actions, such as neurotoxicity, cytotoxicity, and antiviral activity against HCMV, HSV-1, HHV-6, and HIV-1. They have been reported to produce a single metabolite active against wide spectrum of microbes like Fischerella ambigua produces ambigols active against bacteria, fungi and protozoa. Similarly, Moorea producens produces malygomides O and P, majusculamide C and somocystinamide which are active against bacteria, fungi and tumour cells, respectively. In addition to the above, Moorea sp. produce apratoxin A and dolastatin 15 possessing anti cancerous activity but unfortunately till date only brentuximab vedotin (trade name Adcetris), a medication derived from marine peptides, for the treatment of Hodgkin lymphoma and anaplastic large cell lymphoma has been approved by FDA. However, several publications have effectively described and categorised cyanobacterial medicines based on their biological action. In present review, an effort is made to categorize cyanobacterial metabolites on the basis of their phycochemistry. The goal of this review is to categorise cyanobacterial metabolites based on their chemical functional group, which has yet to be described.

Consecutive Inhibition of Telomerase and Alternative Lengthening Pathway Promotes Hodgkin's Lymphoma Cell Death.

Telomere maintenance is key during cancer development. Malignant cells can either use telomerase or an alternative lengthening of telomere (ALT) pathway to maintain their telomere length. In Hodgkin’s Lymphoma (HL), the presence of telomerase activation is established. The activation of ALT has been reported recently. Our data confirm this notion describing co-localization of the phosphorylated form of telomeric repeat-binding factor 1 (pT371-TRF1) with ALT-associated promyelocytic leukemia bodies. Surprisingly, to our knowledge, there are no published studies targeting both telomere maintenance pathways in HL. Consequently, we investigated, for the first time, the effects of both telomerase and ALT inhibition on HL cell viability: We inhibited telomerase and/or ALT, given either individually, simultaneously, or consecutively. We report that the inhibition of telomerase using BIBR1532 followed by ALT inhibition, using trabectedin, caused a decrease of greater than 90% in cell viability in three patient-derived HL cell lines. Our results suggest that HL cells are most vulnerable to the consecutive inhibition of telomerase followed by ALT inhibition.

NFkB Pathway and Hodgkin Lymphoma.

The tumor cells that drive classical Hodgkin lymphoma (cHL), namely, Hodgkin and Reed-Sternberg (HRS) cells, display hallmark features that include their rareness in contrast with an extensive and rich reactive microenvironment, their loss of B-cell phenotype markers, their immune escape capacity, and the activation of several key biological pathways, including the constitutive activation of the NFkB pathway. Both canonical and alternative pathways are deregulated by genetic alterations of their components or regulators, EBV infection and interaction with the microenvironment through multiple receptors, including CD30, CD40, BAFF, RANK and BCMA. Therefore, NFkB target genes are involved in apoptosis, cell proliferation, JAK/STAT pathway activation, B-cell marker expression loss, cellular interaction and a positive NFkB feedback loop. Targeting this complex pathway directly (NIK inhibitors) or indirectly (PIM, BTK or NOTCH) remains a challenge with potential therapeutic relevance. Nodular predominant HL (NLPHL), a distinct and rare HL subtype, shows a strong NFkB activity signature because of mechanisms that differ from those observed in cHL, which is discussed in this review.