Ofer Shpilberg’s research while affiliated with Ariel University and other places

Background Frailty has been shown to have a major impact on treatment outcomes in myeloma patients (pts), yet prospective data on the effects of functional and cognitive assessments on treatment outcomes and their dynamics throughout therapy course, are scarce. We aimed to investigate the clinical utility of functional geriatric assessment and quality of life (QoL) measures in comparison with the commonly used International Myeloma Working Group (IMWG) frailty score, in a sub-study of the Kydar phase 2 prospective clinical trial, which investigated salvage of functional high risk (FHR) pts with a quadruple salvage regimen. Methods Forty-one FHR myeloma pts who failed to respond or experienced early (<18 months [mo] from treatment start) relapse after a bortezomib-containing induction enrolled to receive second line treatment in eighteen 28-day (d) cycles (C) or until disease progression/ unacceptable toxicity. Carfilzomib: 56 mg/m2 IV d 1,8,15 (C 1-9), d 1,15 (C 10-18). Lenalidomide (LEN): 25 mg (Frail: 15mg) d1-21; Dexamethasone (DEX) 40mg (Frail: 20mg) weekly; Daratumumab (DARA): IV 16mg/Kg weekly (C 1-2), q14d (C 3-6), q28d. After 18 cycles, pts continued to receive DARA/LEN/DEX. Here we focus on functional and geriatric sub-study of the above, evaluating IMWG frailty score and functional geriatric assessment examination at baseline and C4, including: European Organization for Research and Treatment of Cancer [EORTC] QoL Questionnaire Core Module [QLQ-C30] and multiple myeloma module [QLQ-MY20], frailty status (Activities of daily living [ADL], Instrumental activities of daily living [IADL], Charlson comorbidity index), cognition (MINI-COG), depression screening (Patient health questionnaire-2 [PHQ-2]), and physical assessment associated with frailty (Handgrip test [HG], 4-meter walking speed test [4MW]). Scales for analysis of test results were based on reported thresholds. Tests dynamics were assessed over time as were their effects on QoL, and their correlation with hematological response and the occurrence of significant toxicity (defined as experiencing ≥2 adverse events [AEs] graded 3-5 or serious adverse event). Results Forty-one pts, with a median age of 70, were enrolled across 14 medical centers in Israel between June 2018-August 2019. Durable and deep responses were achieved with an ORR of 90% (37/41): near CR-stringent CR 22% / very good partial response 42% / PR 27%. AEs graded 3-5 included mostly infections (43%). Pts were relatively well-preserved in terms of functional and cognitive status at screening point and maintained stable during treatment (C4). No significant differences were observed between these 2 time points, except for a moderate increase in frailty and depression indices at the fourth cycle. Achieving a deep hematological response (≥VGPR) was not associated with significant improvements in functional (ADL, IADL, HG, 4MW), cognitive, or frailty indices. Across all functional tests, pts with pathological scores experienced considerably more toxicity during treatment compared to those with normal range function and compared to the overall study population. A total functional score, per patient, was defined as pathological if more than 2/5 functional tests [ADL, IADL, 4MW, HG, frailty score] showed pathological results, this occurred in 35% of pts (10/28 with available functional score). Significant toxicity occurred in 100% vs 55% among pts with pathological vs normal total functional score (p=0.02), and in 77% vs 58% of frail vs non-frail pts (p NS). When compared to QoL indices, which are subjective and reflect patient self-reporting, there was a significant discrepancy between pts responses to questionnaires and the formal objective test results, in favor of the former. Conclusions Contrary to expectations, hematologic responses were not associated with enhancements in functional and cognitive indices, while pts reports indicated beneficial effects, highlighting importance of assessing both objective and subjective parameters. Possibly longer follow-up is needed to capture functional improvements in elderly FHR pts receiving a quadruple second line. Toxicity rate was associated with pathological functional (particularly HG, ADL, IADL) and depression (PHQ-2) scores; This can inform the development of a predictive model that complements the existing frailty score for predicting treatment-related toxicity and QoL improvements.

On behalf of the European MCL Network, ML and EH contributed equally Introduction: In younger patients with mantle cell lymphoma (MCL), the addition of ibrutinib during induction immuno-chemotherapy and as 2-years maintenance with and without autologous stem cell transplantation (ASCT) has shown high efficacy in the 3-arm randomized TRIANGLE trial (Dreyling et al., Lancet 2024), establishing a new standard of care induction treatment and maintenance. However, the efficacy comparison of the two ibrutinib-containing treatment arms with and without ASCT was still ongoing. With prolonged follow-up, we now aim to clarify the role of ASCT in the context of ibrutinib-containing treatment, to confirm the previously observed treatment effects and to perform overall survival (OS) comparisons. Patients and methods: In 2016, the European MCL Network initiated the randomized, open-label, 3-arm TRIANGLE trial to evaluate the addition of ibrutinib to standard treatment with ASCT (arm A+I) in comparison to the previous standard treatment (arm A) and an ibrutinib-containing treatment without ASCT (arm I). Patients with previously untreated, advanced stage II-IV MCL, up to 65 years, and suitable for high-dose cytarabine and ASCT, were randomized 1:1:1 to the 3 trial arms in 13 European countries and Israel. Study treatment consisted of 6 alternating cycles of R-CHOP and R-DHAP without (arm A) or with ibrutinib added to R-CHOP cycles and as 2 years maintenance (arms A+I, I). ASCT was planned for patients of arms A and A+I responding to induction therapy. Rituximab maintenance was recommended to be applied according to national guidelines in responding patients of each trial arm. For the primary outcome, failure-free survival (FFS), stable disease at the end of induction, progression, or death were counted as events. Three pairwise log-rank tests for FFS were monitored with regular pre-planned interim analyses, each maintaining a one-sided 1.67% significance level. A pre-defined decision criterion based on the statistical significance of the treatment comparisons for FFS was established to determine the future treatment recommendation. In 2022, arm A had failed to show FFS-superiority over I and FFS in arm A+I was shown to be superior to A. OS was a secondary outcome and formal pairwise OS comparisons between treatment arms were pre-planned with interim analyses based on O'Brien-Fleming boundaries to maintain pairwise two-sided 5% significance levels. Results: Between July 2016 and December 2020, 870 patients were randomized to arms A (n=288), A+I (n=292), and I (n=290). Median age was 57 years (range 27-68), 76% were male, 87% had stage IV, and 58%/27%/15% had low/intermediate/high risk MIPI. After a median follow-up of 53 months, A+I failed to show FFS-superiority over I (3-year FFS A+I: 86% vs. I: 85%; one-sided p=0.28, hazard ratio: 0.87). FFS-superiority of A over I was again not confirmed with 3-year FFS 75% (A) vs. 85% (I; one-sided p=0.9942, hazard ratio: 1.38). In contrast, the retrospectively calculated two-sided p-value on an overall 5% significance level was consistent with FFS-superiority of I over A (p=0.0102). FFS-superiority of A+I over A was again confirmed with 3-year FFS 86% (A+I) vs. 75% (A; one-sided p=0.0034, hazard ratio: 0.64). Compared with arm A (3-year OS 85%), OS was prolonged in arms A+I and I with 3-year OS of 90% in A+I (p=0.0069, hazard ratio 0.61), and 91% in I (p=0.0041, hazard ratio 0.59). Conclusions: The results confirm superiority of ibrutinib-containing treatment without ASCT (arm I) over ASCT-containing treatment without ibrutinib (arm A) in terms of both, FFS and OS. In the context of ibrutinib- and high-dose cytarabine-containing induction immuno-chemotherapy and ibrutinib maintenance, the addition of ASCT failed to show FFS superiority, while increasing toxicity during maintenance/follow-up. According to the pre-defined decision strategy, ibrutinib+R-CHOP/R-DHAP induction followed by 2 years of ibrutinib maintenance should be the new standard of care in younger MCL patients, thus ending the era of ASCT for MCL patients.

Background: Systemic light chain (AL) amyloidosis is a clonal plasma cell disorder characterized by the deposition of misfolded immunoglobulin light chain products in vital organs, leading to significant organ dysfunction. The molecular mechanisms underlying its pathogenesis remain incompletely elucidated. Identifying the molecular mechanisms and novel therapeutic targets is critical for improving treatment strategies and patient outcomes. Our previous research work identified that the PI3K/AKT signaling pathway is overexpressed in AL amyloidosis (Zvida et al., Blood, 2022, Fishov et al., Cancer Med., 2023). This pathway plays a critical role in regulating cellular metabolism. Specifically activated AKT can phosphorylate and regulate various target proteins involved in metabolic processes. One of the significant targets is fatty acid synthase (FASN), an enzyme responsible for the synthesis of fatty acids. AKT can enhance FASN expression and activity, thereby promoting lipid biosynthesis. Additionally, elevated FASN levels can drive inflammation through increased fatty acid synthesis, which activates inflammatory signaling pathways and enhances cytokine production (Acosta-Martinez et al.,IJMC, 2022). Elevated levels of FASN have been associated with hematological malignancies and cell survival (Vanauberg, et al., Oncogenesis, 2023). However, its role in AL amyloidosis is yet to be elucidated. Aim: To determine the role of FASN in the pathology and inflammatory response in AL amyloidosis, and to evaluate the potential of targeting FASN and the PI3K/AKT pathway for therapeutic intervention. Methods: We profiled FASN expression in CD138+ bone marrow (BM) samples from patients with AL amyloidosis (n=20), Multiple Myeloma (MM) (n=20), Monoclonal Gammopathy of Undetermined (MGUS) (n=8) and Healthy Controls (HC) (n=6), using qRT-PCR. To further explore the relationship between the PI3K/AKT pathway and FASN, we treated ALMC-1 cells with Capivasertib (an AKT inhibitor) and assessed FASN protein levels, cell proliferation by WST-1 assay, cell cycle distribution by flow cytometry, light chain secretion by ELISA, and expression of AKT and its downstream targets via western blot analysis. Furthermore, TVB-2640 (FASN inhibitor) was used to evaluate the cell response to FASN inhibition. Results: Our analysis revealed a significant upregulation of FASN in AL amyloidosis BM samples compared to MM, MGUS, and HCs. Capivasertib treatment resulted in a dose-dependent reduction in FASN protein levels. Additionally, Capivasertib treatment inhibited cell proliferation, induced G1 phase cell cycle arrest, reduced light chain secretion, and decreased AKT protein expression and its downstream targets p4EBP pS6 and pERK. Elevated levels of proinflammatory cytokines, including TNF and IP-10 (CXCL-10), were observed in AL amyloidosis serum samples, suggesting the involvement of immune mechanisms and the PI3K/AKT pathway, potentially induced by FASN. Finally, treatment with TVB-2640 reduced cell viability and decreased the protein expression of LIPIN-1, which is one of the key target genes regulated by FASN. Conclusions: Our findings highlight the significant upregulation of FASN in AL amyloidosis and its regulation by the PI3K/AKT pathway. The observed effects of Capivasertib on FASN expression and cell proliferation, along with the upregulation of inflammatory cytokines, underscore the potential for targeting the PI3K/AKT signaling pathway and FASN in developing more specific therapeutic strategies for AL amyloidosis.

Histiocytic neoplasms are diverse clonal haematopoietic disorders, and clinical disease is mediated by tumorous infiltration as well as uncontrolled systemic inflammation. Individual subtypes include Langerhans cell histiocytosis (LCH), Rosai–Dorfman–Destombes disease (RDD) and Erdheim–Chester disease (ECD), and these have been characterized with respect to clinical phenotypes, driver mutations and treatment paradigms. Less is known about patients with mixed histiocytic neoplasms (MXH), that is two or more coexisting disorders. This international collaboration examined patients with biopsy‐proven MXH with respect to component disease subtypes, oncogenic driver mutations and responses to conventional (chemotherapeutic or immunosuppressive) versus targeted (BRAF or MEK inhibitor) therapies. Twenty‐seven patients were studied with ECD/LCH (19/27), ECD/RDD (6/27), RDD/LCH (1/27) and ECD/RDD/LCH (1/27). Mutations previously undescribed in MXH were identified, including KRAS, MAP2K2, MAPK3, non‐V600‐BRAF, RAF1 and a BICD2‐BRAF fusion. A repeated‐measure generalized estimating equation demonstrated that targeted treatment was statistically significantly (1) more likely to result in a complete response (CR), partial response (PR) or stable disease (SD) (odds ratio [OR]: 17.34, 95% CI: 2.19–137.00, p = 0.007), and (2) less likely to result in progression (OR: 0.08, 95% CI: 0.03–0.23, p < 0.0001). Histiocytic neoplasms represent an entity with underappreciated clinical and molecular diversity, poor responsiveness to conventional therapy and exquisite sensitivity to targeted therapy.

Background: Histiocytic neoplasms are driven by mutations activating the MAPK/ERK pathway; however, their epigenetic landscape remains poorly understood. MicroRNAs (miRNAs) are epigenetic regulators implicated in cancer development. Moreover, DNA methylation of miRNAs affects hematological malignancies development. Our previous study identified a unique miRNA expression signature in Erdheim-Chester Disease (ECD) patients. To further understand the biological mechanisms of ECD, we investigated the involvement of miRNAs, genes, and their methylation status in ECD, Langerhans cell histiocytosis (LCH), and Rosai-Dorfman disease (RDD). Methods: Methylation profiles of 14 LCH, 6 ECD, and 10 RDD patients were determined from histyocitic infiltrated tissues (Illumina EPIC-methylation array). These were compared to controls, suture granulomas (n=4), localized inflammatory reactions characterized by non-neoplastic histiocytic infiltrates. miRNAome data was identified by NanoString analysis and qRT-PCR. Results: We identified significant alterations in 23,322 methylation sites (FDR<0.05, |Δß|≧20%) between controls and the histiocytosis group, indicating differential epigenetic regulation. ECD and LCH exhibited a shared methylation signature distinct from RDD. Pathway analyses revealed that the differentially methylated genes (DMGs) were involved in inflammatory response, RAS, TNF, and PI3K-mTOR signaling pathways, linked to histiocytic neoplasms. Specifically, we found DMG involvement in regulation and generation of non-coding RNAs. Notably, we observed significant downregulation of the let7-miRNA family, particularly let7b, in ECD and LCH patients. Subsequent analysis revealed hypermethylation of specific CpG islands in the let7b DNA sequence, potentially silencing let7b expression in these diseases. Given the let7-miRNA family's role in regulating the MAPK pathway, this suggests a novel mechanism in the pathogenesis of these neoplasms. Conclusions: We provide valuable insights into differential methylation patterns in histiocytosis neoplasms, highlighting the involvement of epigenetic modifications in their development and progression. This enhances our understanding of histiocytosis development and may contribute to the identification of novel diagnostic and therapeutic approaches for these patients.

Langerhans cell histiocytosis (LCH) is an inflammatory myeloid neoplasm characterized by the accumulation of clonal mononuclear phagocyte system cells expressing CD1a and CD207. In the past decade, molecular profiling of LCH, as well as other histiocytic neoplasms demonstrated that these diseases are driven by MAP kinase (MAPK) activating alterations, with somatic BRAFV600E mutations in >50% of LCH patients, and clinical inhibition of MAPK signaling has demonstrated remarkable clinical efficacy. At the same time, activating alterations in kinase-encoding genes such as PIK3CA, ALK, RET, and CSF1R which can activate mitogenic pathways independent from the MAPK pathway have been reported in a subset of histiocytic neoplasms with anecdotal evidence of successful targeted treatment of histiocytoses harboring driver alterations in RET, ALK, and CSF1R. However, evidence supporting the biological consequences of expression of PIK3CA mutations in hematopoietic cells has been lacking, and whether targeted inhibition of PI3K is clinically efficacious in histiocytic neoplasms is unknown. Here, we provide evidence that activating mutations in PIK3CA can drive histiocytic neoplasms in vivo using a conditional knock-in mouse expressing mutant PIK3CAH1047R in monocyte/dendritic cell progenitors. In parallel, we demonstrate successful treatment of PIK3CA-mutated, multisystemic LCH using alpelisib, an inhibitor of the alpha catalytic subunit of PI3K. Alpelisib demonstrated a tolerable safety profile at a dose of 750mg/week and clinical and metabolic complete remission in a PIK3CA-mutated LCH patient. These data demonstrate PIK3CA as a targetable non-canonical driver of LCH and underscore the importance of mutational analysis-based personalized treatment in histiocytic neoplasms.

UNFOLDER (NCT00278408, EUDRACT 2005-005218-19) is a phase-3 trial in patients with aggressive B-cell lymphoma and intermediate prognosis, including primary mediastinal B-cell lymphoma (PMBCL). In a 2 × 2 factorial design, patients were randomized to 6× R-CHOP-14 or R-CHOP-21 (rituximab, cyclophosphamide, doxorubicin, vincristine, and prediso(lo)ne) and to consolidation radiotherapy to extralymphatic/bulky disease or observation. Response was assessed according to the standardized criteria from 1999, which did not include F-18 fluordesoxyglucose positron emission tomography/computed tomography (FDG-PET) scans. Primary end point was event-free survival (EFS). A subgroup of 131 patients with PMBCLs was included (median age, 34 y; 54% female, 79% elevated lactate dehydrogenase (LDH), 20% LDH >2× upper limit of normal [ULN], and 24% extralymphatic involvement). Eighty-two (R-CHOP-21: 43 and R-CHOP-14: 39) patients were assigned to radiotherapy and 49 (R-CHOP-21: 27, R-CHOP-14: 22) to observation. The 3-year EFS was superior in radiotherapy arm (94% [95% confidence interval (CI), 89-99] versus 78% [95% CI, 66-89]; P = 0.0069), due to a lower rate of partial responses (PRs) (2% versus 10%). PR triggered additional treatment, mostly radiotherapy (n = 5; PR: 4; complete response/unconfirmed complete response: 1). No significant differences were observed in progression-free survival (PFS) (95% [95% CI, 90-100] versus 90% [95% CI, 81-98]; P = 0.25) nor in overall survival (OS) (98% [95% CI, 94-100] versus 96% [95% CI, 90-100]; P = 0.64). Comparing R-CHOP-14 and R-CHOP-21, EFS, PFS, and OS were not different. A prognostic marker for adverse outcome was elevated LDH >2× ULN (EFS: P = 0.016; PFS: P = 0.0049; OS: P = 0.0014). With the limitation of a pre-PET-era trial, the results suggest a benefit of radiotherapy only for patients responding to R-CHOP with PR. PMBCL treated with R-CHOP have a favorable prognosis with a 3-year OS of 97%.

UNFOLDER (Unfavorable Young Low-Risk Densification of R-Chemo Regimens) is an international phase-3 trial in patients 18–60 years with aggressive B-cell lymphoma and intermediate prognosis defined by age-adjusted International Prognostic Index (aaIPI) of 0 and bulky disease (≥7.5 cm) or aaIPI of 1. In a 2 × 2 factorial design patients were randomized to 6× R-CHOP-14 or 6× R-CHOP-21 (rituximab, cyclophosphamide, doxorubicin, vincristine, and prediso[lo]ne) and to consolidation radiotherapy to extralymphatic and bulky disease or observation. Response was assessed according to the standardized response criteria published in 1999, not including F-18 fluordesoxyglucose positron emission tomography/computed tomography (FDG-PET). Primary endpoint was event-free survival (EFS). A total of 695 of 700 patients were eligible for the intention-to-treat analysis. Totally 467 patients qualified for radiotherapy of whom 305 patients were randomized to receive radiotherapy (R-CHOP-21: 155; R-CHOP-14: 150) and 162 to observation (R-CHOP-21: 81, R-CHOP-14: 81). Two hundred twenty-eight patients not qualifying for radiotherapy were randomized for R-CHOP-14 versus R-CHOP-21. After a median observation of 66 months 3-year EFS was superior in the radiotherapy-arm versus observation-arm (84% versus 68%; P = 0.0012), due to a lower rate of partial responses (PR) (2% versus 11%). PR often triggered additional treatment, mostly radiotherapy. No significant difference was observed in progression-free survival (PFS) (89% versus 81%; P = 0.22) and overall survival (OS) (93% versus 93%; P = 0.51). Comparing R-CHOP-14 and R-CHOP-21 EFS, PFS and OS were not different. Patients randomized to radiotherapy had a superior EFS, largely due to a lower PR rate requiring less additional treatment (NCT00278408, EUDRACT 2005-005218-19).