Hodgkin Lymphoma Tumor Research Articles

Circulating Tumor DNA Sequencing for Biologic Classification and Individualized Risk Stratification in Patients With Hodgkin Lymphoma.

Current clinical challenges in Hodgkin lymphoma (HL) include difficult-to-treat relapsed/refractory disease and considerable long-term toxicities of treatment. Since clinical risk factors lack discriminatory power, intensity of therapy is mainly based on tumor burden. Exploring HL genetics and tumor microenvironment (TME) might provide valuable insights for improved risk stratification. In this study, we applied circulating tumor DNA sequencing to 243 patients obtained from pivotal German Hodgkin Study Group trials to identify subtypes of HL. Independent validation of the subtypes was performed in 96 patients treated in the EuroNet-PHL-C2 study. Outcome differences of subtypes were assessed in an event-enriched clinical validation cohort comprising 72 patients from the HD21 trial, using a refined, validated, and clinically feasible assay. We propose a biologic classification of HL consisting of three distinct subtypes: inflammatory immune escape HL is characterized by frequent copy-number variations including immune escape variants such as high-level amplifications of the PD-L1 locus and an inflammatory TME. Virally-driven HL is associated with Epstein-Barr virus and/or human herpesvirus 6 and an inflammatory TME with neutrophils and macrophages, while the tumor mutational burden (TMB) is low. Oncogene-driven HL is defined by a high TMB, recurrent mutations in oncogenic drivers such as TNFAIP3, ITPKB, and SOCS1, and a cold TME. A refined and validated assay version aiming at clinically feasible risk stratification showed significant progression-free survival differences between subtypes. In addition, assessment of minimal residual disease (MRD) allowed for the detection of patients at very high risk of relapse within the subtypes. We propose a clinically feasible, noninvasive method for individualized risk stratification and MRD monitoring in patients with HL on the basis of circulating tumor DNA sequencing.

EXPRESSION OF VITAMIN D3 RECEPTOR IN TUMOR TISSUE AND RELATIONSHIP BETWEEN CLINICAL FEATURES, PROGNOSTIC INDICATORS AND EXPRESSION OF VITAMIN D3 RECEPTOR IN HODGKIN LYMPHOMA

<p><strong>Background and objectives:</strong> In our study, we aimed to investigate the expression of vitamin D receptor and the relationship between clinical features, prognostic indicators, and expression of vitamin D receptor in Hodgkin lymphoma. <strong>Materials and Methods:</strong> Pathology records and medical files of patients with Hodgkin lymphoma were evaluated. Forty-two tissue samples were analyzed retrospectively. Vitamin D receptor staining was performed with immunohistochemistry. The patients were divided into low, medium, and intense staining subgroups. The demographic data, clinical and biochemical values of patients were reviewed retrospectively, and the relationship between clinical features, prognostic indicators, and vitamin D receptor expressions were examined. <strong>Results:</strong> VDR staining was positive in Reed-Sternberg cells of all Hodgkin lymphoma patients. The cases were divided into three groups according to VDR staining intensity as low, medium and intense staining and compared with known clinical prognostic parameters. Only the presence of symptom B was statistically significantly different. Gender, bone marrow involvement, extralymphatic involvement, mediastinal mass, bulky mass, mean leukocyte count, and treatment response were associated with vitamin D receptor staining intensity, but they were statistically insignificant. <strong>Conclusion: </strong>Our study is the second study showing VDR in tumor Hodgkin lymphoma tumor tissues and is meant to be the first to evaluate its association with clinical and biochemical prognostic laboratory tests. Vitamin D receptor staining in all Hodgkin lymphoma patients led to the conclusion that VDR can be used in the histopathological differential diagnosis of HL. More studies are needed to assess and demonstrate the relationship between prognostic and diagnostic indicators and vitamin D receptor intensity of immunohistochemically detected VDR in tumor tissues of Hodgkin lymphoma. Also, further studies with serum vitamin D levels and other histologic subtypes can provide more reliable results.</p><p> </p><p><strong> Article visualizations:</strong></p><p><img src="/-counters-/soc/0715/a.php" alt="Hit counter" /></p>

Pulmonary Infundibular Stenosis Secondary to a Mediastinal Hodgkin Lymphoma Tumor Invasion in a Young Adult Male: What a Rare Occurrence!

Introduction: Cardiac involvement is a rare unusual localization of Hodgkin lymphoma, as it’s known that primary lymphomas of the heart are rare. They develop primarily in the right atrium, and extend to the pericardium in about one third of the cases. Pulmonary infundibular stenosis secondary to mediastinal mass Hodgkin lymphoma invasion is rare but not impossible especially in relapse cases. Case Report: A rare case of a young adult patient diagnosed with Hodgkin Lymphoma under chemotherapy presents a relapse of his pathology associated with heart failure; predominantly, right heart failure due to extension of the mediastinal mass tumor to the right cavities of the heart causing pulmonary infundibular stenosis which is a rare occurrence. He was put under an intense chemotherapy and symptomatic heart failure treatment with close follow-up after multi-disciplinary discussions including cardiologist, onco-hematologists and cardiothoracic surgeons. Three months later, a significant reduction of the mass was observered. Conclusion : Pulmonary infundibular stenosis secondary to mediastinal Hodgkin lymphoma invasion is a rare entity of secondary tumors of the heart. Multimodal imagery is the key tool for diagnosis. There prognosis depend on multiple factors but generally good in younger adult patients compared to aged individuals.

Thyroid Functionsand Thyroid Lesions in Children with Hodgkin Lymphoma and Central Nervous System Tumors Who Received Radiotherapy to the Head and Neck Region

Objective: The aim of this study was to assess thyroid function and lesions after radiotherapy to the head and neck region in children with Hodgkin lymphoma (HL) or central nervous system (CNS) tumors. Material and Methods: The study included children diagnosed with HL or CNS tumors who were in complete remission at least one year after completion of chemotherapy (CT) and who had all received radiotherapy (RT) to the head and/or neck. All patients were requested to undergo thyroid function tests and thyroid ultrasonography (USG). Results: A total of 37 patients were included. The mean age was 13.7±3.8 years. The mean follow-up time was 5.09±2.5 years. All patients had CT and RT. Seven (18.9%) patients had subclinical hypothyroidism, and 7 (18.9%) had thyroid USG abnormalities. None of the patients had thyroid malignancy. Age under 10 years at diagnosis, follow-up time of 3 years or more, and an RT dose of 25 Gy or more were found as effective factors for subclinical hypothyroidism development. Only an RT dose of 25 Gy or more was found to be related to thyroid USG abnormalities. Conclusion: Thyroid function tests and thyroid imaging should be routinely examined in any patient who has been treated for cancer to evaluate thyroid dysfunction regardless of clinical findings and the follow-up of these patients should be lifelong.

Deficit Accumulation Index and Biological Markers of Aging in Survivors of Childhood Cancer

Survivors of childhood cancer experience premature aging compared with community controls. The deficit accumulation index (DAI) uses readily available clinical data to measure physiological age in survivors; however, little data exist on how well deficit accumulation represents underlying biological aging among survivors of cancer. To examine the associations between the DAI and epigenetic age acceleration (EAA) and mean leukocyte telomere length (LTL). This cross-sectional study analyzed data from the St Jude Lifetime Cohort, an assessment of survivors of childhood cancer who were treated at St Jude Children's Research Hospital in Memphis, Tennessee. Data were collected between 2007 and 2016, assayed between 2014 and 2019, and analyzed between 2022 and 2023. Participants were adult survivors who were diagnosed between 1962 and 2012 and who survived 5 years or more from time of diagnosis. The analyses were restricted to survivors with European ancestry, as there were too few survivors with non-European ancestry. The DAI included 44 aging-related items, such as chronic health conditions and functional, psychosocial, and mental well-being. Item responses were summed and divided by the total number of items, resulting in a ratio ranging from 0 to 1. These DAI results were categorized based on reported associations with hospitalization and mortality: low, defined as a DAI less than 0.2; medium, defined as a DAI of 0.2 to less than 0.35; and high, defined as a DAI of 0.35 or higher. Genome-wide DNA methylation was generated from peripheral blood mononuclear cell-derived DNA. The EAA was calculated as the residuals from regressing the Levine epigenetic age on chronological age. The mean LTL was estimated using whole-genome sequencing data. This study included 2101 survivors of childhood cancer (1122 males [53.4%]; mean [SD] age, 33.9 [9.1] years; median [IQR] time since diagnosis, 25.1 [18.7-31.9] years) with European ancestry. Compared with survivors in the low DAI group, those in the high DAI group experienced 3.7 more years of EAA (β = 3.66; 95% CI, 2.47-4.85; P < .001), whereas those in the medium DAI group experienced 1.8 more years of EAA (β = 1.77; 95% CI, 0.84-2.69; P < .001), independent of treatment exposures. The EAA and DAI association was consistent across 3 common diagnoses (acute lymphoblastic leukemia, Hodgkin lymphoma, and central nervous system tumors) and across chronological age groups. For example, among acute lymphoblastic leukemia survivors, those in the medium DAI group (β = 2.27; 95% CI, 0.78-3.76; P = .001) experienced greater EAA vs those in the low DAI group. Similarly, among survivors younger than 30 years, the high DAI group experienced 4.9 more years of EAA vs the low DAI group (β = 4.95; 95% CI, 2.14-7.75; P < .001). There were no associations between mean LTL residual and the DAI. This cross-sectional study of survivors of childhood cancer showed that the DAI was associated with EAA, suggesting an underlying biological process to the accumulation of deficits. Both the DAI and EAA were effective at identifying aging phenotypes, and either may be used to measure aging and response to interventions targeting aging pathways.

Whole slide imaging of tumour microenvironment in classical Hodgkin’s lymphoma: development of a clinical prediction model based on programmed death-ligand 1 and tumorous Reed-Sternberg cells

AimsThe prognostic impact of programmed death-ligand 1 (PD-L1) cells in classic Hodgkin lymphoma (cHL) tumour microenvironment remains undefined.MethodsModel development via Transparent Reporting of a multivariable prediction model for Individual Prognosis...

Survival of Hematopoietic Stem Cell Transplantation in the National Institute of Neoplastic Diseases, Lima-Perú between 2012 to 2022: 10 Years of Experience

Introduction: Hematopoietic stem cell transplantation (HSCT) is part of standard treatment in a great number of hematologic malignancies and some solid tumors. The National Institute of Neoplastic Diseases (INEN) started the HSCT program in 2012, performing over 600 HSCT so far. In this study, we report the overall survival (OS) at 5 years of patients who undergone this procedure in our institution through 10 years of experience. Methods: We analyzed retrospectively the survival of all patients admitted to the HCST program at INEN between 2012 and 2022. Clinical and pathological data were collected from the medical records and the unit's transplant patient registry. The OS was determined through the Kaplan-Meier method and the comparisons of survival curves were performed using the log-rank or Breslow test. Results: During the study period, a total of 647 transplants were performed. The median age was 39 years (range: 1 - 70), 73.4% of patients were between 18 and 60 years, and 57.1% were men. Most transplants (n = 591, 91.3%) were performed in the adult population and 8.6% (n = 56) in pediatric population. According to the type of transplant, 68% (n=440) were autologous and 32% (n=207) allogeneic from related donor. The most frequent diagnosis were acute leukemia (n = 221, 34.2%), multiple myeloma (n = 190, 29.3%), non-Hodgkin lymphoma (n = 105, 16,2%; of these, 22 were T- cell lymphomas), Hodgkin lymphoma (n = 69, 10.7%) and germinal cell tumor (n = 33, 5.1%); in the pediatric population, leukemia was the most common diagnosis (66.1%). The median follow-up of the patients was 3.7 years (95% CI: 3.4, 3.9), 25.2% (n = 163) of the patients died due to disease progression. The median overall survival (OS) for the entire cohort was not reached and the OS rate at 5 years was 68.3%. The 5-year OS rate by transplant subtype was 53.9% and 75.3% (p &amp;lt; 0.001) for allogeneic and autologous transplants, respectively; and according to the pathology, the patients who had a better 5-year OS were HL (87.5%) and NHL (77.5%), while in leukemias it was 56.1%. Survival by age group (&amp;lt;40 vs &amp;gt;40) and sex (female vs. male) did not present a significant difference (p &amp;gt; 0.05). Conclusions: The survival of the transplanted patients was similar to that reported in other series. Leukemia was the most frequent hematological malignancy admitted to the HSCT program. HL and NHL were the malignant tumors with the highest survival rates at 5 years. Nearly a third of transplants were allogeneic, all from matched related donors. HSCT in our series has shown an improvement in OS at 5 years in both autologous and allogeneic transplant patients compared to non-transplanted patients, according to international reports.

P-401 Is cancer type associated with semen quality and infertility among men who desire fertility preservation?

Abstract Study question To investigate the association between cancer-types and semen-parameters before gonadotoxic-treatments, and to evaluate which patients have the highest risk of diminished spermatogenesis prior to therapy. Summary answer sperm quality among cancer patients before gonadotoxic treatment can differ according to the type of cancer. What is known already Many studies have reported impaired sperm quality at cancer diagnosis before any given gonadotoxic regiments. Except for testicular cancer and Hodgkin’s Lymphoma, baseline data on semen quality in case of different types of malignancies among male cancer patients are limited or based on low sample size Study design, size, duration A retrospective population-based cohort analysis was performed, 237 oncological male patients who were referred for fertility preservation between 1995-2020 within 90 days of cancer diagnosis and prior to cancer treatment Participants/materials, setting, methods The study population included 237oncological male patients who were referred to the Sperm Bank, Fertility, and IVF unit for the purpose of fertility preservation. Sperm analysis was performed within 90 days of cancer diagnosis and prior to therapy. The pre-treatment sperm analyses were based on the World Health Organization (WHO) guidelines Main results and the role of chance Histological examination of malignant diseases demonstrated 20 subtypes of cancers, the major groups included 49 patients with testicular cancer (TC), 72 patients with Hodgkin lymphoma (HD), 35 patients with other lymphoma, 29 patients with other hematological cancer, 16 with sarcoma and 36 with other solid malignant tumors. Our results show significant difference in sperm concentration between the different cancer groups (P value 0.007), the lowest median concentration of spermatozoa was found in the testicular cancer and sarcoma cancer patients 10 million per ml compared to the other cancer groups (other hematologic, Hodgkin lymphoma, other lymphoma and other solid tumors). Oligospermia was observed in 59% of testicular cancer patients and 50% of the sarcoma patients. Fertility outcomes were analyzed as a secondary objective. We found that only 37 patients' partners conceived and delivered offspring. Only 16.2% of those patients used their banked sperm and conceived via IVF-ICSI. We found that 20% of testicular cancer patients and other lymphoma patients including NHL conceived spontaneously after cancer diagnosis and treatment. Limitations, reasons for caution This is a retrospective cohort study and the oncology patients sample size is relatively small. Wider implications of the findings Our study emphasizes that sperm quality among cancer-patients before gonadotoxic-treatment can differ according to the cancer type. Significant reduction in sperm quality in testicular cancer patients was demonstrated, with a significant association was found between sperm concentration and various cancer-type. Further studies are warranted to confirm our results. Trial registration number not applicable

17th International Conference on Malignant Lymphoma, Palazzo dei Congressi, Lugano, Switzerland, 13 - 17 June, 2023.

Introduction: While nearly all younger patients (pts) with Hodgkin's lymphoma (HL) are cured with conventional chemotherapy, outcomes are significantly worse in those over age 60. Intriguingly, among older HL pts, those with EBV+ disease have even poorer outcomes (Keegan et al., JCO 2005), yet the biological basis for this phenomenon is not understood. The paucity of malignant cells in HL tumors has hampered genomic studies. Circulating tumor DNA (ctDNA) profiling can overcome these limitations, allowing noninvasive genotyping and risk stratification in HL (Spina et al., Blood 2018; Alig et al., ASH 2022). Here, we use a fully non-invasive approach for genomic profiling of elderly HL pts, focusing on the impact of ctDNA and EBV status on clinical outcomes. Methods: We profiled 57 pts (64%) from NCT02414568, a prospective, multicenter phase II LYSA-PVAB study of newly diagnosed classical HL >60 years (Ghesquières et al., ASH2019). All pts received 6 cycles of PVAB (prednisone, vinblastine, doxorubicin, and bendamustine). Plasma cfDNA was genotyped using Cancer Personalized Profiling by Deep Sequencing (CAPP-Seq); MRD was monitored using Phased variant Enrichment and Detection Sequencing (PhasED-Seq). Pts were classified into high and low pretreatment ctDNA levels using a threshold of 2.5 log hGE/mL (Alig et al., ASH2022). We defined EBV status either by LMP1 staining of FFPE tumor tissue or by VirCAPP-Seq targeting 180 viral species, where a predefined threshold of 32 genomes/ml was previously validated as 94% accurate (Garofalo et al., ASH 2022). Results: Pretreatment ctDNA was detected in 94% of pts with a median concentration of 2.2% (MAF), which was significantly correlated with disease burden measured by TMTV (p = 0.034). Pts with high pre-treatment ctDNA levels (39%) had significantly worse PFS (HR = 2.3, p = 0.039). EBV+ pts also had inferior PFS (HR = 2.2, p = 0.04) and OS (HR = 3.1, p = 0.028) (Figures A-B). In multivariate analysis, both indices remained independently associated with worse outcomes (HR = 2.5, p = 0.01 for pre-treatment ctDNA and HR = 2.7, p = 0.01 for EBV status). Interestingly, pre-treatment ctDNA levels did not differ between EBV+ and EBV- tumors (p = 0.92), while specific genetic aberrations were significantly associated with respective EBV status (Figure C). Among evaluable pts for MRD monitoring, detection of ctDNA after four cycles of chemotherapy was highly associated with risk of death (Figure D). The research was funded by: NIH/NCI R01 R01CA257655 Keywords: Diagnostic and Prognostic Biomarkers, Genomics, Epigenomics, and Other -Omics, Hodgkin lymphoma Conflicts of interests pertinent to the abstract. C. Rossi Consultant or advisory role: Genmab, Janssen, Incyte Educational grants: Kyte, Abbvie S. K. Alig Consultant or advisory role: Takeda Pharmaceuticals D. M. Kurtz Consultant or advisory role: Adaptive Biotechnologies, Roche, Foresight O. Casasnovas Consultant or advisory role: Roche, Takeda Educational grants: Roche, Takeda, BMS, Janssen A. A. Alizadeh Consultant or advisory role: Adaptive Biotechnologies, Foresight Diagnostics, Roche, BMS, Gilead, Genentech, Karyopharm

Betulin and Crinum asiaticum L. bulbs extract attenuate pulmonary fibrosis by down regulating pro-fibrotic and pro-inflammatory cytokines in bleomycin-induced fibrosis mice model

BackgroundPulmonary fibrosis (PF) is a lung disease characterized by scaring of lung tissue that impairs lung functions. The estimated survival time of patients with pulmonary fibrosis is 3–5 years. Bleomycin (BLM) is used clinically in the treatment of Hodgkin lymphoma and testicular germ-cell tumors. Bleomycin’s mechanism of action is the inhibition of DNA and protein synthesis. This happens when leukocytes induce the release of cytokines and chemokines which increase the pro-fibrotic and pro-inflammatory cytokines such as IL-6, TNF-alpha, IL-13, IL-1β and transforming growth factor-beta 1 (TGF-β). Crinum asiaticum L. bulbs (CAE) are widely found in parts of Africa, Asia and Indian Ocean Island. It is also prevalent in southern part of Ghana and traditionally used by the indigenes to treat upper respiratory tract infections, and for wound healing. Betulin (BET) is found in the bulbs of Crinum asiaticum L. but widely isolated from the external bark of birches and sycamore trees. Betulin as a lupine type triterpenes has been researched for their pharmacological and biological activities including anticancer, anti-inflammatory, antimicrobial activities and anti-liver fibrosis effects.Aim of the study: The aim was to study the anti-pulmonary fibrosis effect of Crinum asiaticum L. bulbs extract and betulin in bleomycin-induced pulmonary fibrosis in mice. Materials and methodThere was a single oropharyngeal administration of bleomycin (80 mg/kg) in mice followed by the treatment of CAE and BET after 48 h of exposure to BLM. ResultsThere was increased survival rate in CAE and BET treatment groups compared to the BLM induced group. There was a marked decreased in the levels of hydroxyproline, collagen I and III in the CAE and BET treatment groups compared to BLM-treated group. The treatment groups of CAE and BET significantly down regulated the levels of pro-fibrotic and pro-inflammatory cytokines concentrations such as TGF-β1, MMP9, IL-6, IL-1β and TNF-alpha compared to an increased in the BLM treated groups. The histological findings of the lungs suggested the curative effects of CAE and BET following BLM induced pulmonary fibrosis in mice, the study showed improved lung functions with wide focal area of viable alveolar spaces and few collagen fibers deposition on lungs of treatment groups. ConclusionCAE and BET attenuated pulmonary fibrosis by down regulating pro-fibrotic and pro-inflammatory cytokines as well as improving lung function. This could be a lead in drug discovery where compounds with anti-fibrotic effects could be developed for the treatment of lung injury.

Measles Virus is Associated with Hodgkin Lymphoma and Additional Tumors -A Never Ending Story

Measles Virus is Associated with Hodgkin Lymphoma and Additional Tumors -A Never Ending Story

Aberrant synaptophysin expression in classic Hodgkin lymphoma

BackgroundSynaptophysin is an immunohistochemical marker for neuroendocrine differentiation and is widely used in pathologic diagnosis. Its expression in malignant lymphoma has not yet been described. However, we experienced an index case of classic Hodgkin lymphoma with synaptophysin expression. This experience prompted us to investigate synaptophysin expression in classic Hodgkin lymphoma.MethodImmunohistochemical staining of synaptophysin was performed in 59 diagnosed cases of classic Hodgkin lymphoma, 10 anaplastic large cell lymphomas, 16 diffuse large B-cell lymphomas, and 5 extranodal marginal zone lymphoma of the mucosa-associated tissue. Synaptophysin-positive cases were stained for both chromogranin and CD56a.ResultOf 59 classic Hodgkin lymphoma cases, 11 (19%) were positive for synaptophysin. None of the anaplastic large cell lymphomas expressed synaptophysin. Synaptophysin showed weak but specific expression in the cytoplasm of the Hodgkin lymphoma tumor cells. Other background inflammatory cells (such as macrophages, B-, and T-lymphocytes) were all negative for synaptophysin expression. Chromogranin and CD56a were not expressed in the synaptophysin-positive classic Hodgkin lymphomas.ConclusionsSynaptophysin is an integral glycoprotein present in presynaptic vesicles of neurons and neuroendocrine cells. It is a diagnostic marker for neuroendocrine tumors. Aberrant synaptophysin expression has been reported in non-neuroendocrine tumors but not in lymphoma or leukemia. To the best of our knowledge, synaptophysin positivity has only been reported in a single case of precursor T-lymphoblastic leukemia/lymphoma to date. Our study showed that aberrant synaptophysin expression in classic Hodgkin lymphoma is an unexpectedly frequent finding. The mechanism underlying, and prognostic significance of, such aberrant expression is unclear. Thus, in a small biopsy, aberrant synaptophysin expression could be a diagnostic pitfall and should be carefully avoided.

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

Viral cfDNA Profiling Reveals Distinct EBV Subtypes and Stratifies Risk in Hodgkin Lymphomas

Introduction: Approximately 10-40% of classic Hodgkin Lymphoma (cHL) tumors are Epstein-Barr virus (EBV) positive, and immunodeficiency can predispose to cHL, suggesting that viral infections are important in cHL pathogenesis. We hypothesized that circulating viral genomic and fragmentomic features would inform pathogenicity and evolutionary selection of EBV in cHL and potentially allow for risk stratification of cHL subtypes. We developed Viral CAncer Personalized Profiling by deep Sequencing (VirCAPP-Seq), a novel capture-based method for sequencing viral cell-free DNA (cfDNA). Using VirCAPP-Seq, we surveyed the composition of the circulating virome in cHL patients, and identified EBV mutations under positive selection in cHL. Methods: 352 peripheral blood plasma specimens were collected from 315 cHL patients (mean age = 32, SD = 16) and 44 controls, including cord blood donors and healthy adults (mean age = 43, SD = 16). cfDNA was extracted from plasma and processed as previously described (Newman et al, 2016). Libraries were subjected to hybrid capture with a custom VirCAPP-Seq viral capture panel spanning 180 viral species, including EBV types 1 and 2, and other common oncotropic viruses. Human sequencing reads were subtracted before alignment to a composite reference genome of targeted viral species. Read coverage and plasma cfDNA concentration were used to calculate absolute viral abundance, and expressed as genome copies per mL plasma. Additional EBV cfDNA sequencing data were analyzed from 55 nasopharyngeal carcinoma patients and 24 EBV+ controls (Lo et al, PNAS 2018). EBV variants were called with GATK HaplotypeCaller (v4.1.2). Results: 48% of cHL plasma samples profiled by VirCAPP-Seq had detectable EBV cfDNA above the background level in healthy controls. Plasma EBV load (PEL) was significantly higher in cHL patients with Epstein-Barr-encoded small RNA (EBER) positive tumors (N = 24, mean = 3810, SD = 8083 copies / mL plasma) compared to healthy controls (N = 29, mean = 0.1, SD = 0.25) (p < 0.001, Wilcoxon T-test). PEL strongly predicted tumor EBER status with an accuracy of 94% at a threshold of 32 copies EBV / mL plasma. Unexpectedly, patients with EBER-neg tumors also demonstrated elevated detectable PEL (N = 137, mean = 11, SD = 81 copies / mL plasma). PEL was correlated with International Prognostic Score (IPS) (r = 0.42, p < 0.001), and patients with "high" pre-treatment PEL (≥ 10 copies / mL plasma) had a significantly lower 5-year PFS (73%) compared to patients with "low" pre-treatment PEL (90%) (p = 0.03, Figure A). In contrast, tumor EBER status showed no association with PFS. Hierarchical clustering of circulating viral load for 180 targeted viral species identified subclusters of cHL patients bearing distinct metaviromic signatures, including 18% of cHL patients with elevated plasma Anellovirus burden, an observation associated with compromised immune function (De Vlaminck et al, Cell 2013). Mutation analysis performed on 92 EBV isolates from cHL patients revealed non-synonymous variants in BARF0, BHLF1, and EBNA-1 under strong positive selection by dN / dS analysis. Fragment length analysis showed that EBV cfDNA molecules were, on average, longer in EBER-pos cHL patients (median = 157 bp, IQR = 154 - 158 bp) compared to healthy controls (median = 147 bp, IQR = 143 -149 bp) (Figure B). Strikingly, EBV cfDNA fragment length profiles were similar between EBER-pos and EBER-neg cHL patients (Median = 155 bp, IQR = 151 - 161 bp), suggesting that a significant proportion of EBV cfDNA may yet be tumor-derived in patients with EBER-neg tumors, reflecting either the limited sensitivity of clinical EBER ISH or, perhaps, "hit-and-run" viral oncogenesis. Conclusions: VirCAPP-Seq enables ultrasensitive detection, quantification, and genotyping of EBV cfDNA in cHL patients, and identifies viral variants under strong evolutionary selection. Our findings provide a proof-of-concept for viral cfDNA sequencing as a powerful tool for discovering prognostic biomarkers. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Plasma EBV DNA Methylation: Evidence of EBV Lytic Infection in HIV(+) EBV(+) Plasmablastic Lymphoma

Background: Among people living with HIV, plasmablastic lymphoma (PBL) and classical Hodgkin lymphoma (HL) are almost uniformly associated with Epstein-Barr virus (EBV). Immunohistochemical studies have demonstrated high levels of lytic gene expression in PL,1 whereas EBV(+) HL shows exclusively latent viral gene expression. EBV virion DNA is never CpG methylated, but tumor-derived plasma EBV is CpG methylated as previously shown by our group in EBV(+) HL and nasopharyngeal carcinoma (NPC).2 Others have also presented evidence that patterns of CpG methylation of plasma EBV DNA varies between people with NPC and non-malignant EBV(+) conditions.3 To better characterize the nature of plasma EBV in patients with lymphoma, we investigated EBV DNA copy number and EBV DNA methylation in HIV-associated PBL and HL. Methods: With appropriate approvals from the Human Research Ethics Committee of the University of the Witwatersrand and the Johns Hopkins Institutional Review Boards, we collected plasma from newly diagnosed untreated patients with HIV and PBL or HL in Johannesburg, South Africa. Diagnostic biopsy specimens were reviewed in Baltimore by a hematopathologist to confirm the diagnosis and Epstein-Barr encoding region (EBER) in situ hybridization was performed to determine EBV association. Plasma EBV copies were measured by standard qPCR targeting the BamW repeats. qPCR using the same primers was also performed after capture with methyl-DNA binding protein 2 (MBD2) magnetic beads, where the captured fraction represents methylated DNA and the flow-through represents unmethylated DNA. Results: We studied plasma from 7 patients with PBL and 12 patients with HL. Four of five PBL tumors were EBV (+) and eight of 10 HL tumors were EBV (+) by tissue EBER in situ hybridization. Analysis of plasma EBV DNA copy number showed overlapping levels of EBV DNA (median 2.7 Log10 copy/mL in PBL and 3.2 Log10 copy/mL in HL) (Fig. 1). Patients with at least 1.5 Log10 copy/mL of EBV underwent CpG methylation evaluation, which showed very different EBV CpG methylation indices. The EBV methylation index was highly variable in PBL (median of 51%) and was uniformly high in HL (median of 97%). Conclusions: Although plasma from plasmablastic lymphoma patients show high EBV copy number, the characteristic of that EBV DNA is very different from the plasma EBV detected in Hodgkin lymphoma patients. This difference is hypothesized to be related to evidence of viral lytic gene expression seen in PBL. The degree of plasma EBV DNA methylation may have broad implications for lymphoma diagnosis and subtyping.

Abdominal tumors in children: A comprehensive study on the diagnosis, management, and clinical outcome of pediatric patients with lymphoma in Kashmir

Background: Although primary abdominal tumors are rare but their diagnosis, high-cost treatment and psychological instability among both patients and parents are challenging. There are several types of abdominal tumors in pediatric age group which include neuroblastoma, Wilm’s tumor, hepatoblastoma, lymphomas, soft-tissue sarcomas, and germ cell tumors. Aims and Objectives: The present study was objectively conducted to identify Hodgkin lymphoma (HL) and non-Hodgkin lymphoma (NHL) tumors among all patients presented with abdominal tumors. In addition to this, histopatological profile, clinical profile and disease prognosis among such patients has also been analyzed. Materials and Methods: A total of 64 pediatric patients presented with primary abdominal tumors were included in the study but only 20 patients with lymphoma were identified. The study was conducted at the Department of Pediatric Division, SMHS Hospital Srinagar with effect from 2005 to 2008. Results: The Majority of NHL patients were found to have Stage II and Stage IV. Moreover, all the four cases of Hodkins lymphoma had Stage IV. Out of the 20 patients with lymphoma six died, of them five had NHL and one patient had HL, thus placing the overall survival rate as 70%. Conclusion: The present study demonstrated that both NHL and HL tumors may develop to advanced stage and even patient may die if the timely diagnosis and proper management is not there. We recommend collective team work of medicos from various departments for the precise diagnosis and optimal management of such patients.

Tislelizumab, a PD-1 inhibitor for relapsed/refractory mature T/NK-cell neoplasms: Results from a phase 2 study.

7552 Background: Effective treatment choices for patients (pts) with relapsed/refractory (R/R) mature T/NK-cell neoplasms after failure of standard therapies are limited. Tislelizumab (TIS), a humanized anti-PD-1 mAb, demonstrated outstanding efficacy and favorable safety in pts with R/R classical Hodgkin lymphoma or solid tumors. We present safety and efficacy of the phase 2 study of TIS in pts with R/R mature T/NK-cell neoplasms. Methods: Pts were enrolled into 3 cohorts stratified by the type of T/NK-cell neoplasm to receive TIS 200 mg intravenously every 3 weeks until disease progression or intolerable toxicity. Eligible pts had ≥1 prior systemic therapy, disease progression during/after most recent therapy completion or refractory disease, ECOG ≤2, and life expectancy ≥6 mo. Primary endpoint was investigator-assessed overall response rate (ORR). Secondary endpoints included duration of response (DoR), complete response (CR) rate, progression-free survival (PFS), overall survival (OS) in cohorts 1 and 2, and safety. Results: 77 pts were treated. Cohort 1: R/R extranodal NK/T-cell lymphoma (n = 22); cohort 2: R/R mature T-cell neoplasms (n = 44; 21 peripheral T-cell lymphoma not otherwise specified; 11 angioimmunoblastic T-cell lymphoma; 12 anaplastic large cell lymphoma); cohort 3: R/R cutaneous T-cell lymphomas (CTCL; stage ≥1B; n = 11; 8 mycosis fungoides and 3 Sézary syndrome). Of all pts, 76.6% had advanced-stage disease, 51.9% had refractory disease, and 49.4% had ≥3 prior systemic regimens. Median treatment cycles for cohorts 1, 2, and 3 were 5 (range, 1-37), 4.5 (range, 1-38), and 17 (range, 3-25), respectively. Cohort 3 had promising efficacy (median follow-up [FU] 16.6 mo): ORR 45.5%; CR 9.1%; median DoR 11.3 mo (95% CI: 2.76-11.30); median PFS 16.8 mo; median OS not reached (NR). Modest efficacy was reported in cohort 1 (median FU 8.4 mo): ORR 31.8%; CR 18.2%; median DoR NR (95% CI: 2.66-not estimable [NE]); median PFS 2.7 mo; median OS 8.8 mo and also in cohort 2 (median FU 9.3 mo): ORR 20.5%; CR 9.1%; median DoR 8.2 mo (95% CI: 2.50-NE); median PFS 2.7 mo; median OS 13.3 mo. Most frequent adverse events (AEs) were pyrexia (32.5%), anemia (18.2%), arthralgia (18.2%), and diarrhea (15.6%); most frequent grade ≥3 AEs were anemia (7.8%), pneumonia (6.5%), and neutropenia (5.2%). Any grade immune-mediated AEs occurred in 22 (28.6%) pts, most frequently hypothyroidism (10.4%), hyperglycemia (5.2%), and rash (5.2%); and grade ≥3 in 4 (5.2%) pts (blood creatine phosphokinase increased, hepatitis, hypothyroidism, rash, and urticaria [1 pt each]). No treatment-related AEs led to death. Conclusions: TIS was well tolerated, achieving modest efficacy in R/R mature T/NK-cell neoplasms, with some long-lasting remissions particularly in CTCL. Further studies are warranted to determine the biologic features associated with response and explore optimal combination therapies. Clinical trial information: NCT03493451.

Checkpoint CD47 expression in classical Hodgkin lymphoma.

SummaryThe glycoprotein CD47 regulates antiphagocytic activity via signal regulatory protein alpha (SIRPa). This study investigated CD47 expression on Hodgkin and Reed–Sternberg (HRS) cells in the classical Hodgkin lymphoma (cHL) tumour microenvironment and its correlation with prognosis, programmed‐death (PD) immune markers, and SIRPa+ leukocytes. We conducted immunohistochemistry with CD47 and SIRPa antibodies on diagnostic biopsies (tissue microarrays) from cHL patients from two cohorts (n = 178). In cohort I (n = 136) patients with high expression of CD47 on HRS cells (n = 48) had a significantly inferior event‐free survival [hazard ratio (HR) = 5.57; 95% confidence interval (CI), 2.78–11.20; p < 0.001] and overall survival (OS) (HR = 8.54; 95% CI, 3.19–22.90; p < 0.001) compared with patients with low expression (n = 88). The survival results remained statistically significant in multivariable Cox regression adjusted for known prognostic factors. In cohort II (n = 42) high HRS cell CD47 expression also carried shorter event‐free survival (EFS) (HR = 5.96; 95% CI, 1.20–29.59; p = 0.029) and OS (HR = 5.61; 95% CI, 0.58–54.15; p = 0.136), although it did not retain statistical significance in the multivariable analysis. Further, high CD47 expression did not correlate with SIRPa+ leukocytes or PD‐1, PD‐L1 and PD‐L2 expression. This study provides a deeper understanding of the role of CD47 in cHL during an era of emerging CD47 therapies.

The Hodgkin Lymphoma Immune Microenvironment: Turning Bad News into Good.

Simple SummaryClassic Hodgkin lymphoma (cHL) is one of the most enigmatic study models of the tumor microenvironment (TME), in which the Hodgkin–Reed Sternberg (HRS) cells are distributed throughout an abundant but ineffective immune ecosystem. The hyperactivation of HRS cells due to somatic mutations leads to complex interactions with the different subsets of immune cell populations, modeling the pathophysiology of the disease. There are remaining loose ends regarding the identification of the immune functional states in the cHL microenvironment and their influence on tumor cell survival. Here, we review the most relevant immune populations identified in the cHL context, focusing on integrative functional signatures.The classic Hodgkin lymphoma (cHL) tumor microenvironment (TME) is by far the most abundant component of tumors and is responsible for most of their biological and clinical characteristics. Recent advances in our knowledge of these networks in cellular interactions allow us to understand that the neoplastic Hodgkin and Reed Sternberg (HRS) cells, although they are in the minority, are the main architects of this dysregulated immune milieu. Here, we review the major changes that have happened in recent years: from TME as a helpless bystander, reflecting an ineffective immune response, to a dynamic tumor-promoting and immunosuppressive element. The HRS cells promote survival through interconnected intrinsic and extrinsic alterations, boosting pro-tumoral signaling pathways through genetic aberrations and autocrine growth signals, in parallel with abnormal cytokine secretion for the recruitment and selection of the best cell partners for this immunosuppressive TME. In turn, cHL is already proving to be the perfect model with which to address an immune checkpoint blockade. Preliminary data demonstrate the utility of druggable key signaling pathways in this ensemble, such as JAK-STAT, NF-κB, and others. In addition, myriad biomarkers predicting a response await validation by new in situ multiplex analytical methods, single-cell gene expression, and other techniques. Together, these components will define the functional phenotypes with which we will elucidate the molecular pathogenesis of the disease and improve the survival of patients who are refractory to conventional therapies.

CD8+ T Lymphocytes Immune Depletion and LAG-3 Overexpression in Hodgkin Lymphoma Tumor Microenvironment Exposed to Anti-PD-1 Immunotherapy.

Background: Resistance to anti-PD-1 remains a considerable clinical challenge for the treatment of patients with classical Hodgkin lymphoma (cHL), and mechanisms of anti-PD-1 resistance remain unknown. This pilot study aims to investigate the tumor microenvironment in patients with cHL relapsing after anti-PD-1. Methods: This study investigated tumor samples of eight patients with cHL, including four patients exposed to anti-PD-1 with a paired longitudinal histological analysis before and after anti-PD-1, and four patients not exposed to anti-PD-1 who served as control for the cellular biological investigations. Fresh cells tumor microenvironment analysis included phenotypic characterization of their T cell surfaces immune checkpoint markers PD-1, PD-L1, ICOS, TIM-3, LAG-3, 41-BB and BTLA. Tumor tissues immunohistochemistry staining included CD30, CD4, CD8, CD68, CD163, PD-L1, PD-1, LAG-3 and TIM-3. Findings: Paired longitudinal tumor tissues analysis in the tumor microenvironment found a CD8+ lymphocytes tumor depletion and an increase of LAG-3 staining after anti-PD-1 exposure. The fresh cells analysis of the tumor microenvironment in patients exposed to anti-PD-1 found CD8+ lymphocyte depletion, with an elevated CD4+/CD8+ lymphocytes ratio (median ratio 9.77 in exposed anti-PD-1 versus 2.39 in not-exposed anti-PD-1 patients; p = 0.0943). On the cell surfaces of CD4+ lymphocytes, the median positive expression of LAG-3 was significantly higher in the samples exposed to anti-PD-1 compared to the controls (15.05 [IQR:17.91-10.65] versus 3.84 [IQR 1.87-6.57]; p = 0.0376). Interpretation: This pilot study proposes hypotheses for understanding the resistance to immunotherapies in patients with Hodgkin lymphoma. Hodgkin lymphoma exposed to anti-PD-1 correlated in tumor microenvironment with an immune depletion of CD8+ T lymphocytes and overexpression of LAG-3 on CD4+ helper T lymphocytes.