MYC Rearrangement Research Articles

PB2132: COPANLISIB PLUS RITUXIMAB-BENDAMUSTINE FOR RELAPSED-REFRACTORY DIFFUSE LARGE B-CELL LYMPHOMA: RECRUITMENT UPDATE ON AN ONGOING PHASE II TRIAL OF THE FONDAZIONE ITALIANA LINFOMI (FIL_COPA-RB)

Background: Diffuse large B cell lymphoma (DLBCL) is curable in 50-60% of cases with frontline RCHOP. Relapsed/refractory (R/R) DLBCL can be cured with salvage treatments in roughly 40-50% of cases with standard available treatments which include high dose-chemotherapy plus autologous stem cell transplant (ASCT) in fit patients, and chimeric antigen receptor T-cells (CAR-T), permitted as third or subsequent line in patients with no major comorbidities. The outcome of R/R DLBCL unresponsive or ineligible to ASCT or CAR-T is highly unfavorable and new treatment options are needed. Rituximab plus bendamustine (RB) is active in R/R DLBCL with overall response rates (ORR) of 40-60% but generally short-lasting. Copanlisib is an intravenous pan-class I PI3K inhibitor with predominant activity against PI3K-α and -δ isoforms, active as single agent in DLBCL with ORR 25% (NCT02391116). The combination of copanlisib plus RB (Copa-RB) has showed a safe toxicity profile in R/R indolent lymphomas (iNHL) (NCT02626455). Aims: To assess the safety and efficacy of Copa-RB followed by copanlisib maintenance in R/R DLBCL not eligible or relapsed after ASCT or CAR-T. Methods: FIL Copa-RB is a multicentric open-label single arm, phase II trial. An estimated 81 patients will be enrolled, from 30 centers in Italy. Main inclusion criteria are patients aged ≥ 18 affected by DLBCL (including de-novo DLBCL, DLBCL transformed by iNHL and high-grade lymphoma) or FL grade IIIb R/R after ≤ 3 previous lines of therapy, ineligible or R/R to ASCT or CART, ECOG PS ≤ 2. Patients with primary mediastinal B cell lymphoma histology, significant organ dysfunction, uncontrolled hypertension and hyperglycemia (HbA1c> 8.5%) are excluded. Treatment consists of an induction phase with six 28-day cycles of Copa-RB (copanlisib 60 mg intravenously on day (D) 1, D8 and D15, rituximab 375 mg/m2 on D1, bendamustine 90 mg/m2 on D1-D2) followed by a maintenance phase of twelve 28-day cycles of copanlisib 60 mg given on D1 and D15. The primary endpoint is progression free survival (PFS) defined as the time between the date of enrolment and the date of disease progression, relapse or death from any cause, whichever occurs earlier, with an expected improvement of 12-month PFS from 20% to 35%. Secondary endpoints include overall survival, ORR, complete response (CR) rate, duration of response and rate of response improvement during maintenance. Exploratory analysis will include association between outcome and cell of origin, MYC, BCL2 and BCL6 overexpression and rearrangements, and mutational analysis of selected genes possibly involved in predicting response to copanlisib. The expected study duration from first patient’s first visit to last patient’s last visit is approximately 4 years. Results: The recruitment started in November 2020; at March 1st, 27/81 patients have been enrolled. Results for this study are not yet available. Summary/Conclusion: The treatment of R/R DLBCL ineligible or R/R to ASCT or CAR-T is still an unmet clinical need and new therapeutic strategies are warranted. Copanlisib as single agent has been already shown a moderate activity in DLBCL and the association Copa-RB plus copanlisib maintenance could be a new treatment option to improve the outcome of these patients. Biomarkers studies may further elucidate patients who are more likely to respond to this treatment. Funding: The study is sponsored by the Fondazione Italiana Linfomi; Bayer partly supported the study and kindly provided Copanlisib; Sandoz kindly provided Rituximab.

P1280: TARGETED INHIBITION OF DHODH IS SYNERGISTIC WITH BCL2 BLOCKADE IN LYMPHOMA WITH CONCURRENT MYC AND BCL2 REARRANGEMENT

Background: Diffuse large B-cell lymphoma (DLBCL), the most common subtype of non-Hodgkin lymphoma, can be cured in approximately two-thirds of patients after initial therapy. The remaining one-third of patients have inferior survival outcomes due to relapse or refractoriness despite salvage chemotherapy with or without stem cell transplantation. The dual dysregulation of MYC and BCL2 is one of the most important pathogenic mechanisms. Thus, new therapies are needed to improve the prognosis of patients, and combined targeting of MYC and BCL2 appears to be a promising strategy. Dihydroorotate dehydrogenase (DHODH) is the fourth rate-limiting enzyme for the de novo biosynthesis of pyrimidine and has been shown to be a potential therapeutic target for multiple diseases. Recent data have demonstrated that DHODH inhibitors can target MYC in some tumors. Aims: To develop and assess new therapeutics, we investigated the effects of the DHODH inhibitor brequinar in DHL lymphoma cell lines and the synthetic effects of brequinar and venetoclax. Methods: We first investigated the activity of DHODH inhibition in genetically defined double-hit DLBCL(DHL) cell lines. We then investigated whether c-MYC could be repressed by brequinar in DHL cells. Then we tried to uncover the synergistic mechanism of brequinar and venetoclax. To understand the genome-wide effects and target genes of brequinar and the combination with venetoclax, we performed an RNA-seq of SU-DHL4 cells treated with brequinar, venetoclax, or the combination of both agents versus DMSO-treated cells. At last, we confirmed the synergistic effect of DHODH inhibition and BCL2 blockade in vivo. Results: DHODH inhibition could repress DLBCL cell lines with MYC and BCL2 abnormalities. The protein level of c-MYC was upregulated 6 hours after brequinar treatment and then downregulated gradually with prolonged treatment time. The gene levels of c-MYC decreased over time. The synergistic mechanism of brequinar and venetoclax is the complementarity of different pathways. RNA-seq of SU-DHL4 cells treated with different agents further implied the possible synergistic mechanism of the two agents. Finally, in vivo study confirmed targeted inhibition of DHODH is synergistic with BCL2 blockade in lymphoma with concurrent MYC and BCL2 rearrangement Summary/Conclusion: Our study demonstrates that combined blockade of DHODH and BCL2 could synergistically inhibit DLBCL with concurrent MYC and BCL2 abnormalities through multiple pathways. Thus, it can be reasonably expected that a clinical-grade DHODH inhibitor combined with venetoclax might improve outcomes for patients with DHL or double-expression lymphoma and other cancers with high expression of MYC and BCL2 or MYC and MCL-1. Our findings provide new insights into the molecular basis of this effect and offer opportunities to design new targeted treatments for lymphoma.

P1254: RESPONSE TO DA-EPOCH-R IN PATIENTS WITH HIGH GRADE B CELL LYMPHOMA IS ASSOCIATED WITH A REDUCTION OF PD1 POSITIVE EXHAUSTED CD8 T CELLS

Background: Patients with high grade B cell lymphoma (HGBL) who harbor a MYC rearrangement with BCL2 and/or BCL6 rearrangements (HGBL-DH/TH) have a poor prognosis upon treatment with R-CHOP [Rosenwald, JCO 2019]. Although disease free survival may be prolonged with intensification of chemotherapy with DA-EPOCH-R, improvement of overall survival has not been demonstrated [Dunleavy, Lancet Haematol 2018]. Aims: Preclinical data suggest that high MYC expression impairs anti-tumor immune responses [Casey, Science 2016]. Therefore, we designed the HOVON-152 trial to investigate whether survival can be improved by nivolumab consolidation after DA-EPOCH-R induction. We also investigated whether DA-EPOCH-R induction influences the immune system to have an impact on the response. Methods: In the HOVON-152 single arm, phase II trial (NCT03620578) HGBL-DH/TH patients received 1 cycle of R-CHOP followed by 5 cycles of DA-EPOCH-R induction treatment. Patients in complete metabolic remission (CMR) after induction (Deauville score (DS) 1-3 or a negative lymphoma biopsy in case of DS 4) proceeded with nivolumab consolidation (480 mg iv every 4 weeks) for one year. Peripheral blood was sampled after one cycle of R-CHOP (enroll), before start of the 3rd DA-EPOCH-R cycle (midterm) and after the last DA-EPOCH-R cycle (end-of-induction, EOI). As of January 2022, 55/90 enrolled patients completed DA-EPOCH-R induction, of whom 31 achieved PET-CT based CMR, 24 did not achieve CMR or went off study prematurely. Multiparameter flow cytometry of unseparated full blood was used to enumerate major immune cell subsets. Cryopreserved peripheral blood mononuclear cells were used to determine the frequencies of T cell subsets. Computational flow cytometry analyses (i.e UMAP and FlowSOM) and appropriate unpaired (Mann-Whitney U) or repeated (Friedman) statistical tests were performed in R version 4.0.3. Results: In full blood, CD3 T cell frequencies and numbers decreased midterm DA-EPOCH-R treatment and restored at EOI. Remarkably, the proportion of CD4 T cells decreased (p=0.048) and that of CD8 T cells increased (p=0.01), resulting in lower CD4/CD8 ratios at EOI. In depth analysis revealed a significant decrease of CD4 naïve cells (p=0.00002) and increase of CD4 central memory (CM) cells (p=0.0005). Over time, CD8 cells showed a more senescent phenotype (CD28-KLRG1+CD57+, p=0.013), but the proportion of PD-1 positive exhausted CD8 cells decreased (p=0.007). When stratified for response, it appeared that patients achieving CMR after DA-EPOCH-R had significantly higher CD3 T cells (p=0.04) at enroll than patients not achieving CMR. T cell differentiation states (CD4 or CD8 naïve cells, stem cell memory cells, CM, effector memory (EM) or effector memory RA (EMRA) cells) and the proportion of TIGIT and TIM3 positive cells did not significantly differ between patients achieving CMR or not achieving CMR. Most interestingly, the decrease in PD-1 positive exhausted CD8 T cells was most outspoken in the CMR group (p=0.0008) (Figure 1) in CD8 CM, EM and EMRA cells (all p<0.008). Thus, at EOI, a lower proportion of CD8 PD-1 cells was associated with achieving CMR (p=0.04). Image:Summary/Conclusion: Treatment of HGBL-DH/TH patients with DA-EPOCH-R results in a shift towards a lower CD4/CD8 ratio. Response to DA-EPOCH-R is associated with higher T cell frequencies at enroll and a significant decrease in PD-1 positive exhausted CD8 T cells during DA-EPOCH-R. Additional analyses on NK cells are ongoing. Future analyses of the nivolumab consolidation phase of this study will indicate whether additional blockade of PD-1 prolongs survival.

Abstract 2502: Unravelling the heterogenous molecular landscape of pediatric post-transplant lymphoproliferative disorders

Abstract The genetic landscape of post-transplant lymphoproliferative disorders (PTLD) in pediatric population has not been fully elucidated. This absence of information raises the question whether therapeutic strategies should be the same as for their counterparts in immunocompetent (IC) patients. The aim of this study was to characterize genetically and immunophenotypically pediatric monomorphic PTLD. Thirty-nine monomorphic PTLD ≤19 years-old (mean 10y, gender 25 male/14 female) were recruited and analyzed for germinal center markers, IRF4 and EBER expression. Presence of MYC, PAX5, IRF4, BCL2, BCL6 and 11q alterations was investigated by FISH. Additional molecular studies included clonality, copy number (CN) arrays, cell of origin-COO (Nanostring) and mutational analyses (Custom 167 lymphoma related genes panel, SureSelectXT, Agilent). Twenty-nine patients received solid organ transplantation and eight were hematopoietic stem cell transplant recipients. The mean time from transplant to PTLD diagnosis was 34 months (range 2-170) and the estimated 5-year overall survival (5y-OS) rate was 67%. Patients that received a solid organ had a better prognosis than hematopoietic stem cell transplant recipients (5y-OS 83% vs. 38%, p=0.03). Thirty-three cases were classified as diffuse large B-cell lymphoma (DLBCL) and six as Burkitt lymphoma (BL). Thirty-two cases had extranodal localization, 21 of which in the gastrointestinal tract. Among the DLBCL, 24/28 cases had an ABC/non-GC COO phenotype and the six BL were GCB. EBER was positive in 33/37 cases. Five out of six BL and one DLBCL had MYC rearrangements, while no 11q alterations or other rearrangements were observed. Ten out of the 23 pediatric monomorphic PTLD studied displayed CN alterations (mean 1.6 alt/case; range 0-12). Comparative analyses showed that pediatric PTLD had lower genetic complexity than BL (Scholtysik, 2010) and DLBCL (Ramis-Zaldivar, 2020) in IC patients and adult-PTLD (Ferreiro, 2016; Rinaldi, 2010) and lacked characteristic CN alterations of those groups. Regarding the mutational profile, all 6 PTLD-BL carried MYC mutations in addition to ID3 (4 cases), ARID1A (2 cases) or CCND3 (1 case) and a higher mutational burden than PTLD-DLBCL (12.3 vs 6.2, p=0.01). PTLD-DLBCL showed a very heterogeneous profile characterized by a lower number of mutations than their counterparts in IC patients (2.4 vs 6.5, p=0.01). Pathway enrichment analysis revealed that epigenetic modifiers and NOTCH pathway (4 cases each) were the most recurrently affected. Two out of 20 cases were classified as N1 according to LymphGen (Wright, 2020) algorithm while the rest remained undetermined. The mutational profile of pediatric PTLD-BL is similar to that observed in IC patients whereas PTLD-DLBCL are less complex than their counterpart in IC children and present a very heterogeneous mutational landscape with enrichment in NOTCH pathway mutations. Citation Format: Julia Salmeron, Natalia Castrejón-de-Anta, Pilar Guerra-Garcia, Joan Enric Ramis-Zaldivar, Mónica López-Guerra, Dolors Colomer, Francisco Diaz-Crespo, Marta Garrido, Javier Menarguez, Maria del Mar Andrés, Eugenia Garcia-Fernandez, Margarita Llavador, Noelia Garcia, Blanca Gonzalez-Farré, Idoia Martin-Guerrero, Carmen Garrido, Itziar Astigarraga, Alba Fernández, Jaime Verdú-Amorós, Soledad González-Muñíz, Berta Gonzalez, Verónica Celis, Elias Campo, Olga Balagué, Itziar Salaverria. Unravelling the heterogenous molecular landscape of pediatric post-transplant lymphoproliferative disorders [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2502.

Abstract 1859: VIP152, a selective CDK9 inhibitor, demonstrates sensitivity in gynecologic cell lines that are cisplatin sensitive or resistant and delivers in vivo antitumor efficacy

Abstract Background: The Myc transcription factor has a short half-life of 20-30 minutes (Ramsay et al. 1984) and oncogenic activation and transcriptional addiction leads to sustained levels of Myc, (Gabay et al. 2014). Mechanisms of activation include MYC gene amplification, mutation or rearrangement and have been reported to be prevalent in gynecologic malignancies, such as serous ovarian (39.2%), uterine (28.1%) and endometrial (19.5%) (https://www.cancer.gov/tcga). Since CDK9 phosphorylation of RNA polymerase II is required for transcription of Myc mRNA, we evaluate VIP152, a highly selective CDK9 inhibitor (Lücking et al 2021), to deliver antitumor responses in preclinical models of gynecologic malignancies. Methods: Gynecologic cell lines were treated with 9 dose levels of VIP152 with DMSO and cisplatin as controls. IC50 were calculated using CellTiter-Glo luminescent cell viability assay after 72 hours treatment. A preliminary analysis of 19 cell lines was undertaken to determine whether response to VIP152 is associated with baseline mutation, gene expression, or copy number variation data. Monotherapy efficacy of VIP152 was evaluated in a cisplatin sensitive A2780 in vivo xenograft mouse model. Results: Screening of gynecologic cell lines demonstrates a 3-log range of sensitivity with VIP152 IC50s ranging from 38-593nM. Simultaneous cisplatin screening identified sensitive and resistant cell lines in the same panel. Low VIP152 IC50s were observed in cell lines sensitive or resistant to cisplatin. Cell lines were assigned as sensitive and resistant based on the VIP152 IC50 values. Preliminary analysis suggests a gene signature could predict response to VIP152 in gynecologic malignancies. VIP152 was evaluated in an ovarian cancer A2780 in vivo xenograft model. Monotherapy treatment with 4 doses of VIP152 across 5 dose levels from 5- to 15-mg/kg weekly regimens provided increasing tumor growth inhibition compared with vehicle control. Conclusions: VIP152 demonstrates sensitivity in gynecologic cell lines independent of cisplatin sensitivity. An interim gene signature that is associated with VIP152 sensitivity was defined and we plan to optimize this signature with a larger cell line panel. Dose-dependent tumor growth inhibition in an in vivo xenograft model is demonstrated. VIP152 is currently being evaluated in the clinic (ClinicalTrials.gov Identifiers: NCT02635672 and NCT04978779).

Clinicopathologic and Molecular Characterization of Epstein-Barr Virus-positive Plasmacytoma.

Epstein-Barr virus (EBV)-positive plasmacytoma is a rare plasma cell neoplasm. It remains unclear whether EBV-positive plasmacytoma represents a distinct entity or a variant of plasmacytoma. It shares morphologic features with plasmablastic lymphoma (PBL) and may cause diagnostic uncertainty. To better understand EBV-positive plasmacytoma and explore diagnostic criteria, this study describes 19 cases of EBV-positive plasmacytoma, compared with 27 cases of EBV-negative plasmacytoma and 48 cases of EBV-positive PBL. We reviewed the clinicopathologic findings and performed immunohistochemistry, in situ hybridization for EBV, fluorescence in situ hybridization for MYC , and next-generation sequencing. We found that 63.2% of patients with EBV-positive plasmacytoma were immunocompromised. Anaplastic features were observed in 7/19 cases. MYC rearrangement was found in 25.0% of them, and extra copies of MYC in 81.3%. EBV-positive and EBV-negative plasmacytomas possessed similar clinicopathologic features, except more frequent cytologic atypia, bone involvement and MYC aberrations in the former group. The survival rate of patients with EBV-positive plasmacytoma was comparable to that of patients with EBV-negative plasmacytoma. In comparison to PBL, EBV-positive plasmacytoma is less commonly associated with a "starry-sky" appearance, necrosis, absence of light chain expression, and a high Ki67 index (&gt;75%). The most recurrently mutated genes/signaling pathways in EBV-positive plasmacytoma are epigenetic regulators, MAPK pathway, and DNA damage response, while the most frequently reported mutations in PBL are not observed. Collectively, EBV-positive plasmacytoma should be regarded as a biological variant of plasmacytoma. Thorough morphologic examination remains the cornerstone for distinguishing EBV-positive plasmacytoma and PBL, and molecular studies can be a valuable complementary tool.

Outcomes by BCL2 and MYC expression and rearrangements in untreated diffuse large B-cell lymphoma (DLBCL) from the POLARIX trial.

7517 Background: Overexpression of BCL2 and MYC, and translocation of MYC, BCL2, and BCL6, are associated with poorer outcomes in patients (pts) with DLBCL (Horn et al. Blood 2013). We previously reported progression-free survival (PFS) from POLARIX in subgroups of pts with DLBCL receiving Pola-R-CHP or R-CHOP, including pts with double expressor lymphoma (DEL; favoring Pola-R-CHP: hazard ratio [HR] 0.64, 95% confidence interval [CI] 0.42–0.97), and double- or triple-hit lymphoma (DHL/THL; favoring R-CHOP: HR 3.81, 95% CI 0.82–17.64) (Tilly et al. NEJM 2022). In this prespecified exploratory analysis, we further analyzed immunohistochemistry (IHC) expression status of BCL2, MYC, and rearrangements (R) of BCL2, BCL6 and MYC as independent prognostic markers. We also explored the prognostic impact of DEL within treatment arms. Methods: BCL2 and MYC protein expression were assessed by IHC and identified as IHC+ (≥50% [BCL2]/≥40% [MYC]) or IHC−; MYC-R, BCL2-R, and BCL6-R were detected by fluorescence in situ hybridization (Tilly et al. NEJM, 2022). Exploratory multivariate Cox regression models were adjusted for treatment, stratification factors (IPI, bulky disease, geographic region), age &gt;60 years, cell of origin, and biomarker evaluated, as appropriate. Results: The prevalence, univariate HR, and 2-year (yr) PFS estimates of BCL2+/MYC+ and BCL2-R/ MYC-R/ BCL6-R subgroups are presented (Table) except for BCL6-R, because all 4 PFS events in this subgroup were with Pola-R-CHP. Multivariate analyses results for Pola-R-CHP vs R-CHOP in pts with BCL2+ (HR 0.60, 95% CI 0.43–0.86) and in pts with MYC+ (HR 0.63, 95% CI 0.45–0.89) were similar to results of univariate analyses. Multivariate analyses of other subgroups were not performed due to the low pt number with BCL2-R/ MYC-R/ BCL6-R. While pts with DEL treated with Pola-R-CHP had improved PFS vs pts treated with R-CHOP (HR 0.64, 0.42–0.97), the prognostic impact of DEL vs non-DEL was more pronounced with R-CHOP (univariate HR 1.53, 95% CI 1.06–2.21; multivariate HR 1.29, 95% CI 0.88–1.91) vs Pola-R-CHP (univariate HR 1.10, 95% CI 0.72–1.69; multivariate HR 1.42, 95% CI 0.89–2.28). Conclusions: Multivariate analyses support the benefit of Pola-R-CHP in pts with BCL2+ and MYC+ DLBCL. The poor prognostic impact associated with DEL appears reduced in Pola-R-CHP- vs R-CHOP-treated pts. Clinical trial information: NCT03274492. [Table: see text]

Clinical laboratory mutation analysis performed on aggressive B cell non-Hodgkin lymphoma patient biopsies.

e19561 Background: Results from comprehensive genomic analysis performed on aggressive B cell non-Hodgkin lymphomas (aBNHL) patient (pt) biopsies (bx) have identified mutations (mut) which may be predictive of pt survival. However, results from mut analysis (MA) performed on aBNHL pt bx in clinical laboratories are not well-described. Methods: Bx diagnostic of non-Burkitt aBNHL obtained from 7/2015-2/2021 with MA performed in the Penn Center for Personalized Diagnostics clinical laboratory using 3 consecutive versions of lymphoma sequencing panels were retrospectively analyzed. Mut tested varied by panel. Mut detected with variant allele frequency (VAF) ≥10% were included. Results: MA was performed on 293 aBNHL pt bx with a successful assay for 270 (7.8% failure rate). Request for MA was made by interpreting hematopathologist 93% and treating clinician 7%. Median turnaround time (TAT) from receipt of bx to result report was 18 (range 8-53) days. Notable pt characteristics included prior/concurrent indolent lymphoma (IL) 31% and prior systemic therapy received for IL and/or aBNHL 38%. Notable bx characteristics included bx type paraffin-embedded tissue 85% and bone marrow 12%, histologic classification diffuse large B cell lymphoma 86% and high grade B cell lymphoma 13%, cell of origin (COO) by Hans algorithm non-germinal center B (non-GCB) 51% and GCB 44%, known double expressor lymphoma 30%, known MYC rearrangement 18%, known double hit lymphoma 9% and any mut detected with VAF ≥10% 82%. Number and percentage of mut occurring in ≥10% of bx (minimum tested bx for mut ≥92) with associated COO are listed in the table. Subset analysis of 147 bx obtained from pts with newly-diagnosed aBNHL (15% with previously-untreated IL) had similar bx characteristics to the entire population were analyzed for the presence of mut reported in poor-prognosis COO-associated classifications: Cluster 5 for non-GCB and Cluster 3 for GCB (PMID: 29713087), double-hit signature (DHITsig) for GCB (PMID: 30523716) and molecular high grade (MHG) for GCB (PMID: 30523719). For 77 non-GCB bx, 19% had Cluster 5 mut. For 59 GCB bx, 56% had at least one of Cluster 3, DHITsig or MHG mut. Conclusions: Clinical laboratory MA performed on nearly 300 aBNHL pt bx was highly successful with a short TAT and demonstrated recurrent gene mut associated with aBNHL development. In the subset of bx from previously-untreated pts, MA identified mut associated with poor-prognosis genetic subtypes of aBNHL.[Table: see text]

IgG kappa multiple myeloma with isolated central nervous system relapse.

A 76-year-old male presented with acute renal failure and cast nephropathy was diagnosed with IgG kappa multiple myeloma (MM). His paraprotein was 40 g/L, kappa-free light chain level was 17,058 mg/L, kappa: lambda light chain ratio was 1445. An automated full blood count showed hemoglobin 90 g/L, white blood cells 2.96 × 109/L, neutrophil 1.7 × 109/L, and platelet 179 × 109/L. Bone marrow trephine sample was hypercellular with nearly 100% plasma cells, which were positive for CD138, CD56, and cyclin D1, and showed kappa light chain restriction. Fluorescence in situ hybridization analysis with dual color/dual probes for immunoglobulin heavy chain (IGH) and MYC genes (Cytocell) and whole genome screening using 8 × 60K oligonucleotide arrays (Agilent) performed on the CD138 enriched cells (CD138 microbeads Miltenyi Biotech) from the liquid bone marrow sample showed no evidence of IGH rearrangement, MYC rearrangement, 1q gain, 1p loss, 17p loss, or any clinically significant imbalance. He was treated with eight cycles of bortezomib, cyclophosphamide, and dexamethasone (VCD) to very good partial response. He relapsed 30 months after diagnosis with paraprotein of 36 g/L, kappa light chains of 10,000 mg/L, and creatinine of 498 μmol/L. He was treated with eight cycles of bortezomib, thalidomide, and dexamethasone (VTD) to paraprotein undetectable and kappa light chain 29 mg/L. Seven months post completion of VTD (4 years after myeloma diagnosis), he presented with abnormal gait, falls, and a left-sided facial droop. His paraprotein was 2 g/L and serum-free kappa light chain was 81 mg/L. Magnetic resonance imaging (MRI) head and whole spine showed multifocal diseases in spinal canal and intracranially, involving the cranial nerves most prominent the fifth, seventh, eighth, and hypoglossal nerve on the left, anterior surface of the inferior pons medulla (Figure 1A). There were multiple lesions in the cervical spine as well as in the thoracic spine (Figure 1B). Computerized tomography (CT) chest abdomen pelvis did not show the evidence of other new diseases. Plasma cells were detected in cerebral spinal fluid (CSF) (Figure 1C, May–Grünwald–Giemsa stain x100 objective). He was treated with pomalidomide and dexamethasone. Weekly cytarabine intrathecal (IT) treatment was given until the clearance of plasma cells from CSF samples for three times. Four months later, he achieved the complete remission on MRI head and whole spine with undetectable paraprotein and normal light chain ratio. Serial MRI scans confirmed ongoing remission. A year later, he relapsed again with worsening mobility and reappearance of myeloma cells in CSF. By flow cytometry, these cells were positive for CD138, CD38, CD56, CD117, and negative for CD19. MRI head showed leptomeningeal disease with a new 2.2 cm frontal lobe lesion. His paraprotein remained undetectable and kappa light chain remained at below 100 mg/L. He continued systemic pomalidomide and dexamethasone chemotherapy and received palliative whole brain radiotherapy 20 Gy in 5 fractions for symptom control. His disease continued progressing and he passed away 3 months later (5.5 years after myeloma diagnosis). Here, we reported a myeloma patient with isolated CNS relapse, who achieved 1 year of remission with combination of intrathecal chemotherapy and systemic pomalidomide and dexamethasone treatment, but sadly passed away 4 months after second CNS relapse. CNS MM is a rare form of extramedullary disease, but carries a very poor prognosis. The optimal approach to treatment of CNS MM is not currently known. The current approach includes systemic therapy with agents known to cross the blood brain barrier, intrathecal therapy, and CNS irradiation. Innovative approach to treatment is urgently needed. The authors received no specific funding for this work. Ke Xu and Lucy Kamuriwo wrote the manuscript. Ke Xu, Lucy Kamuriwo, Claire Anderson and Parag Jasani critically revised the final version of the manuscript. The authors declare they have no conflicts of interest. No written or verbal consent obtained from patient. Patient has passed away.

Safety and efficacy of a dose-dense short-term therapy in patients with MYC-translocated aggressive lymphoma

AbstractPatients with aggressive B-cell lymphoma and MYC rearrangement at fluorescence in situ hybridization exhibit poor outcome after R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone). In the last decade, 68 patients with Burkitt lymphoma ([BL] n = 46) or high-grade B-cell lymphoma ([HGBCL] single, double, or triple hit; n = 22) were treated with a dose-dense, short-term therapy termed “CARMEN regimen” at 5 Italian centers. Forty-six (68%) patients were HIV+. CARMEN included a 36-day induction with sequential, single weekly doses of cyclophosphamide, vincristine, rituximab, methotrexate, etoposide, and doxorubicin plus intrathecal chemotherapy, followed by high-dose-cytarabine–based consolidation. Patients who did not achieve complete remission (CR) after induction received BEAM (carmustina, etoposide, cytarabine, melfalan)-conditioned autologous stem cell transplantation (ASCT) after consolidation. Sixty-one (90%) patients completed induction, and 59 (87%) completed consolidation. Seventeen patients received ASCT. Grade 4 hematological toxicity was common but did not cause treatment discontinuation; grade 4 nonhematological toxicity was recorded in 11 (16%) patients, with grade 4 infections in 6 (9%). Six (9%) patients died of toxicity (sepsis in 4, COVID-19, acute respiratory distress syndrome). CR rate after the whole treatment was 73% (95% confidence interval [CI], 55% to 91%) for patients with HGBCL and 78% (95% CI, 66% to 90%) for patients with BL. At a median follow-up of 65 (interquartile range, 40-109) months, 48 patients remain event free, with a 5-year progression-free survival of 63% (95% CI, 58% to 68%) for HGBCL and 72% (95% CI, 71% to 73%) for BL, with a 5-year overall survival (OS) of 63% (95% CI, 58% to 68%) and 76% (95% CI, 75% to 77%), respectively. HIV seropositivity did not have a detrimental effect on outcome. This retrospective study shows that CARMEN is a safe and active regimen both in HIV-negative and -positive patients with MYC-rearranged lymphomas. Encouraging survival figures, attained with a single dose of doxorubicin and cyclophosphamide, deserve further investigation in HGBCL and other aggressive lymphomas.

An artificial intelligence system applied to recurrent cytogenetic aberrations and genetic progression scores predicts MYC rearrangements in large B-cell lymphoma.

Diffuse large B‐cell lymphoma (DLBCL), the most common type of non‐Hodgkin lymphoma, is characterized by MYC rearrangements (MYC R) in up to 15% of cases, and these have unfavorable prognosis. Due to cryptic rearrangements and variations in MYC breakpoints, MYC R may be undetectable by conventional methods in up to 10%–15% of cases. In this study, a retrospective proof of concept study, we sought to identify recurrent cytogenetic aberrations (RCAs), generate genetic progression scores (GP) from RCAs and apply these to an artificial intelligence (AI) algorithm to predict MYC status in the karyotypes of published cases. The developed AI algorithm is validated for its performance on our institutional cases. In addition, cytogenetic evolution pattern and clinical impact of RCAs was performed. Chromosome losses were associated with MYC‐, while partial gain of chromosome 1 was significant in MYC R tumors. MYC R was the sole driver alteration in MYC‐rearranged tumors, and evolution patterns revealed RCAs associated with gene expression signatures. A higher GPS value was associated with MYC R tumors. A subsequent AI algorithm (composed of RCAs + GPS) obtained a sensitivity of 91.4 and specificity of 93.8 at predicting MYC R. Analysis of an additional 59 institutional cases with the AI algorithm showed a sensitivity and specificity of 100% and 87% each with positive predictive value of 92%, and a negative predictive value of 100%. Cases with a MYC R showed a shorter survival.

A Combined Biomarker of Bright CD38 and MYC ≥55% Is Highly Predictive of Double-/Triple-Hit High-Grade B-Cell Lymphoma.

Diagnosis of high-grade B-cell lymphoma with MYC and BCL2 or BCL6 rearrangements (double-/triple-hit lymphoma [DTHL]) appears to mandate fluorescence in situ hybridization (FISH) testing for all large B-cell lymphoma (LBCL). Given the low incidence of DTHL, we aimed to identify flow cytometry (FC) and immunohistochemistry (IHC) features of DTHL that could be used to develop an optimal screening strategy. This combined FC-IHC approach has not yet been studied. We compared features of 40 cases of DTHL and 39 cases of diffuse LBCL (DLBCL) without MYC rearrangement. Bright CD38 expression (CD38bright) by FC, high MYC expression (≥55%), and double-expressor phenotype by IHC were significantly associated with DTHL. The biomarker combining FC and IHC, CD38bright and/or MYC ≥55%, was superior to FC and IHC markers alone in predicting DTHL. Restricting FISH testing to approximately 25% of LBCL based on CD38brightand/or MYC ≥55% would detect approximately 95% of DTHL-BCL2 and approximately 75% of DHL-BCL6. Our study demonstrated that the novel biomarker of CD38bright and/or MYC ≥55% is highly predictive of DTHL. Awareness of the advantages and limitations of this screening strategy would facilitate development of a rational diagnostic workflow to provide high-quality patient care.

Molecular characterization and clinical outcome of B-cell precursor acute lymphoblastic leukemia with IG-MYC rearrangement.

Rarely, immunophenotypically immature B-cell precursor acute lymphoblastic leukemia (BCP-ALL) carries an immunoglobulin- MYC rearrangement (IG-MYC-r). This can result in diagnostic confusion with Burkitt lymphoma/leukemia and use of individualized treatment schedules of unproven efficacy. Here we compare the molecular characteristics of these conditions and investigate historic clinical outcome data. We identified 90 cases registered in a national BCP-ALL clinical trial/registry. When present, diagnostic material underwent cytogenetic, exome, methylome and transcriptome analyses. The outcomes analyzed were 3-year event-free survival and overall survival. IG-MYC-r was identified in diverse cytogenetic backgrounds, co-existing with either established BCP-ALL-specific abnormalities (high hyperdiploidy, n=3; KMT2A-rearrangement, n=6; iAMP21, n=1; BCR-ABL1, n=1); BCL2/BCL6-rearrangements (n=15); or, most commonly, as the only defining feature (n=64). Within this final group, precursor-like V(D)J breakpoints predominated (8/9) and KRAS mutations were common (5/11). DNA methylation identified a cluster of V(D)J-rearranged cases, clearly distinct from Burkitt leukemia/lymphoma. Children with IG-MYC-r within that subgroup had a 3-year event-free survival of 47% and overall survival of 60%, representing a high-risk BCP-ALL. To develop effective management strategies this group of patients must be allowed access to contemporary, minimal residual disease-adapted, prospective clinical trial protocols.

Impact of autograft-absolute lymphocyte count on survival in double/triple hit lymphomas post-autologous stem cell transplantation

The autograft absolute lymphocyte count (A-ALC) ≥0.5 × 109 cells/kg is a survival prognostic factor for lymphoma patients undergoing autologous peripheral blood hematopoietic stem cell transplantation (APBHSCT). However, the A-ALC has not be tested as prognostic factor against double hit/triple hit lymphomas (DHL/THL). Thus, we set up to investigate if A-ALC is a prognostic factor for overall survival (OS) and progression-free survival (PFS) for DHL/THL post-APBHSCT. From January 2012 until December 2020, we identified 77 DHL/THL patients treated with APBHSCT. All patients required to have the diagnosis of DHL/THL by FISH for rearrangements of MYC, BCL2, and BCL6. With a median follow-up of 20.4 months (range, 0.4-94.5 months), DHL/THL patients infused with A-ALC ≥0.5 x 109 cells/kg experienced superior OS (HR = 0.251, 95%CI 0.117–0.539, p < 0.0004) and PFS (HR = 0.347, 95%CI 0.160–0.753, p < 0.007). Multivariate analysis showed that A-ALC was an independent predictor for OS (HR =0.119, 95%CI 0.030–0.473, p < 0.003) and PFS (HR = 0.400, 95%CI 0.189–0.850, p < 0.02). Our study showed that A-ALC is a prognostic factor for survival in DHL/THL. Our current practice for lymphoma patients is to collect enough stem cell but also A-ALC to improve clinical outcomes post-APBHSCT.

Clinical and Pathological Features of Double-Hit and Triple-Hit High-Grade B-Cell Lymphomas: A Retrospective Study from Three Portuguese Tertiary Centers.

Background: High-grade B-cell lymphoma (HGBL) with rearrangements of MYC and BCL2 and/or BCL6, called double and triple-hit lymphomas (DTH-HGBL), are lymphoid malignancies with inferior outcomes when treated with standard chemotherapy. The identification of DTH-HGBL cases is challenging, considering their variable clinical, morphologic, and immunohistochemical features. Materials and Methods: Retrospective revision of medical data of patients diagnosed with DTH-HGBL confirmed by FISH, between January 2010 and January 2020, in three Tertiary Portuguese Hospitals (Coimbra Hospital and University Center, Portuguese Oncology Institute – Coimbra and Portuguese Oncology Institute – Porto). Pathological features, morphology, and immunohistochemical profile were evaluated by at least two experienced pathologists in hematopoietic and lymphoid neoplasms. Results: The cohort included 24 patients: 33.3% triple-hit, 58.3%, MYC/BCL2 double-hit and 8.3% MYC/BCL6 double-hit. There was no gender predominance, with a median age of 62.5±14.3y, 33.3% were diagnosed as nodal disease, and 66.7% as extranodal. Morphologic features of DLBCL were present in 50% of cases, morphological features of both DLBCL and Burkitt lymphoma (DLBCL/BL) in 45.8% and 4.2% of blastoid morphology. Immunohistochemical evaluation, regarding the Hans algorithm, revealed a Germinal center (GC)/GC-like subtype in 83.3% of cases and a non-GC/non-GC-like subtype in 16.7%. MYC was positive in 42.9% and the median proliferative index was 80±12.4%. Conclusion: DTH-HGBL has a very broad range of features. We consider that a cost-effective approach would be to perform cytogenetic analysis in DLBCL and DLBCL/BL cases with GC/GC-like subtype. MYC and BCL2 immunohistochemistry can be useful to identify patients who may benefit from more aggressive therapies, but not as tools for case selection for FISH.

A molecular reappraisal of matrix-producing breast metaplastic carcinoma highlighted by PLAG1 and MYC rearrangements.

Very little is currently known about molecular alteration of matrix-producing carcinoma of the breast. However, the morphological similarity with other neoplasm with a myxo-chondroid component is remarkable. In this pilot study we evaluated the molecular alterations involving PLAG1 and MYC genes in 12 cases of matrix producing carcinoma. We evaluated PLAG1 rearrangements as Break-Apart and Gene Copy Gain, and MYC as amplification and polysomy in 12 cases of matrix producing carcinoma using a FISH method. Among the 12 cases of matrix producing carcinomas we found that the three cases harboring MYC amplification were all negative for PLAG1 break-apart; four cases with MYC polysomy were associated to PLAG1 break-apart and high Gene Copy Number; among four cases wild type for MYC, three showed a PLAG1- break-apart signal and of them two died with disease. One of the deceased patients showed an amplification of MYC with PLAG1- wild-type and the other showed a PLAG1 break-apart (6%) and a MYC wild-type. This is the first report to the best of our knowledge that shows a possible correlation between a matrix producing carcinoma with PLAG1 and MYC involvement in the development and progression of this kind of tumor. We can suppose that MYC amplification behaves in an aggressive way together with PLAG1- break-apart in the cases of matrix producing carcinoma presented here. The gene copy gain is a useful diagnostic tool in the case of difficult diagnosis because an increase was observed in more than 50% of cases.

Clinicopathological analysis of follicular lymphoma with BCL2, BCL6, and MYC rearrangements.

Most follicular lymphomas (FL) show t(14;18)/IGH-BCL2 translocation, but rearrangement (R) negative cases exist. A series of 140 FL patients with a BCL2, BCL6, and MYC gene status examined by fluorescence in situ hybridization (FISH) were classified into five groups: (a) BCL2-R group (BCL2-R/BCL6-G/MYC-G) (G, germline), 77 cases; (b) BCL2/BCL6 double-R group (BCL2-R/BCL6-R/MYC-G), 16 cases; (c) BCL6-R group (BCL2-G/BCL6-R/MYC-G), 16 cases; (d) MYC-R group (BCL2-R or G/BCL6-R or G/MYC-R), three cases; (e) Triple-G group (BCL2-G/BCL6-G/MYC-G), 28 cases. The BCL6-R group had different clinicopathological characteristics. It showed lower rates of an advanced clinical stage and bone marrow invasion, less disease progression (p = 0.036), and a 'trend' toward a favorable progression-free survival (PFS) (p = 0.06). It also showed higher rates of grade 3A and MUM1-expression, and when analyzing the interfollicular spread pattern of CD20-positive cells, had fewer cases showing the IF3+ pattern (high interfollicular spread). Moreover, cases with BCL6-R and/or BCL6 gain (with cases of BCL2 rearrangement and/or of copy number gain excluded) correlated with favorable PFS (p = 0.014) and less IF3+ pattern (p = 0.007). We demonstrated that BCL6-R FLs showed unique clinicopathological findings, and FISH of BCL2, BCL6, and MYC is useful for FL diagnosis and clinical management.

Blastoid high-grade B-cell lymphoma initially presenting in bone marrow: a diagnostic challenge

The 2016 WHO classification introduced the category of high-grade B-cell lymphoma (HGBL), which includes one poorly understood subset, blastoid-HGBL. Establishing the diagnosis and distinguishing blastoid-HGBL from B-acute lymphoblastic leukemia (B-ALL) in bone marrow can be challenging. We assessed 31 cases of blastoid-HGBL diagnosed initially in bone marrow and compared this group to 36 cases of B-ALL using immunophenotyping, fluorescence in situ hybridization, and targeted next generation sequencing analysis. The 31 blastoid-HGBL cases included 14 HGBL with MYC and BCL2 and/or BCL6 rearrangements (double hit lymphoma, DHL), 13 HGBL, not otherwise specified (NOS), and four cases with TdT expression that were difficult to classify. Compared with B-ALL, blastoid-HGBL cases more often showed increased intensity/bright expression of CD20, CD38, CD45, BCL-6, and MYC, and less frequent bright expression of CD10 and TdT. Cases of blastoid-HGBL also more frequently had MYC rearrangement, a complex karyotype and TP53 mutation (p < 0.01). With the exception of CD34, no other single factor, including TdT, was sensitive or adequately specific to distinguish blastoid-HGBL from B-ALL. We developed a scoring system using six distinctive features between 16 cases of unequivocal blastoid HGBL and 22 cases of CD34-positive B-ALL, with a score of ≥3 defining blastoid-HGBL. The system was further validated by using 15 cases of surface light chain negative, and/or CD45 dim to negative blastoid-HGBL and 14 cases of CD34-negative B-ALL. The sensitivity, specificity, positive, and negative predictive value of this scoring system were 100%, 94%, 94%, and 100%, respectively. Using this system, the four cases with TdT expression were all classified as blastoid-HGBL: three were DHL and one was HGBL-NOS. In conclusion, blastoid-HGBL shows distinctive immunophenotypic, cytogenetic, and molecular features as compared with B-ALL. The proposed scoring system can be helpful for the classification of diagnostically challenging blastoid lymphoid tumors presenting initially in the bone marrow.

Cryptic MYC insertions in Burkitt lymphoma: New data and a review of the literature.

The occurrence of MYC-negative Burkitt lymphoma (BL) has been discussed for many years. The real frequency of the MYC insertion in MYC-negative BL is still unknown. Fine-needle aspiration biopsies of 108 consecutive patients with clinicopathologically suspected BL (suspBL) were evaluated by flow cytometry, classical cytogenetics, and fluorescence in situ hybridization (FISH). We found 12 cases (11%) without the MYC rearrangement by FISH with a MYC breakapart probe: two patients (1.9%) with cryptic MYC/IGH fusion (finally diagnosed as BL) and 10 patients (9.3%) with 11q gain/loss (finally diagnosed as Burkitt-like lymphoma with 11q aberration). The exact breakpoints of the cryptic MYC/IGH were investigated by next-generation sequencing. The MYC insertions' breakpoints were identified in PVT1 in the first case, and 42 kb upstream of 5'MYC in the second case. To date, a molecular characterization of the MYC insertion in BL has only been reported in one case. Detailed descriptions of our MYC insertions in a routinely and consecutively diagnosed suspBL cohort will contribute to resolving the issue of MYC negativity in BL. In our opinion, the presence of the MYC insertions in BL and other lymphomas might be underestimated, because routine genetic diagnostics are usually based on FISH only, without karyotyping.

HIV-associated DLBCL: Clinicopathological factors including dual-colour chromogenic in situ hybridisation to assess MYC gene copies

IntroductionDiffuse large B-cell lymphoma (DLBCL) comprises up to 43% of non-Hodgkin lymphomas in South Africa due to the high seroprevalence of human immunodeficiency virus (HIV) infection in the southern African region. We explored the prognostic influence of an array of clinicopathological factors, including MYC gene copy numbers, within HIV-associated DLBCL. MethodsThe retrospective inclusion of 123 tumours was followed by c-MYC immunohistochemistry and dual-colour MYC and centromere 8 (CEN8) chromogenic in situ hybridisation on formalin-fixed paraffin-embedded sections. Clinicopathological data were collected, interpreted and analysed. ResultsHIV seropositive patients comprised 81% (93/115), mean age 42 (SD 10.8) years, with 55% males, HIV negative patients comprised 19% (22/115), mean age 57 (SD 16.7) years (p = 0.001), with 59% males and the HIV status was unknown for 8 patients. The median CD4 count was 162 (IQR 215) cells/mm3, 33% of patients presented with CD4 counts <100 cells/mm3 and the median viral load was 217 (IQR 182 981) copies/mL. There was advanced stage at presentation (i.e., III-IV, 87%), with Ki-67 proliferation indices ≥90% in 85%- and c-MYC expression (i.e., ≥40%) in 58% of tumours. Double expression of c-MYC and BCL2 was associated with a non-germinal center immunophenotype (p < 0.01). Low-level increase of MYC gene copy numbers and MYC rearrangements occurred in 57% and 12%, respectively. C8 polysomy, MYC gene clusters and concurrent MYC rearrangement/increased MYC gene copies were also detected. Inferior median overall survival (OS) occurred when the CD4 counts were <100 cells/mm3 (149 days 95% CI 44-254, p 0,04) and when IPI scores were 3–5 [155 days (95% CI 37–273), p = 0.01]. Concomitant infections negatively impacted the survival outcome, multivariate regression analysis (HR 4.01, 95% CI 1.86–12.20, p = 0.02). Conclusionc-MYC protein expression, low-level increase in MYC gene copy numbers, rearrangement, C8 polysomy, MYC gene clusters and concurrent MYC rearrangement/low-level gains are present in HIV+ DLBCL. CD4 counts < 100 cells/mm3, IPI scores 3-5 and concomitant infections negatively impact the survival outcome.