Ex vivo expansion and activation of Vγ9Vδ2 T cells by CELMoDs in combination with zoledronic acid.

As multiple myeloma (MM) progresses, immune effector cells decrease in number and function and become exhausted. This remains an insurmountable clinical issue that must be addressed by development of novel modalities to revitalize anti-MM immunity. Human Vγ9Vδ2 T (Vδ2+ γδ T) cells serve as the first line of defense against pathogens as well as tumors and can be expanded ex vivo from peripheral blood mononuclear cells (PBMCs) upon treatment with amino-bisphosphonates in combination with IL-2. Here, we demonstrated that next-generation immunomodulators called cereblon E3 ligase modulators (CELMoDs), as well as lenalidomide and pomalidomide, expanded Th1-like Vδ2+ γδ T cells from PBMCs in the presence of zoledronic acid (ZA). However, the expansion of Th1-like Vδ2+ γδ T cells by these immunomodulatory drugs was abolished under IL-2 blockade, although IL-2 production was induced in PBMCs. BTN3A1 triggers phosphoantigen presentation to γδ T-cell receptors and is required for γδ T-cell expansion and activation. ZA but not these immunomodulatory drugs upregulated BTN3A1 in monocytes. These results suggest that immunomodulatory drugs and ZA have cooperative roles in expansion of Th1-like Vδ2+ γδ T cells, and provide the important knowledge for clinical application of human Vδ2+ γδ T cells as effector cells.

Toxoplasmic encephalitis with high 201Tl uptake and retention mimicking malignant lymphoma in a patient with human immunodeficiency virus infection.

Various opportunistic infections develop during immunodeficiency due to human immunodeficiency virus (HIV) infection. The treatment options for malignant lymphoma (ML) and toxoplasmic encephalitis (TE) are completely different; therefore, their discrimination is critical. A 25-year-old female of foreign nationality had been experiencing headaches for several weeks and suddenly developed convulsions. Brain computed tomography revealed multiple intracranial lesions; therefore, the patient was referred to the neurosurgery department. Brain magnetic resonance imaging (MRI) revealed multiple masses with surrounding edema, accompanied by enhanced contrast. The largest mass (2 cm) in the left occipital lobe exhibited ringed contrast enhancement. Her blood test results showed a CD4 count of 40/µL, positive HIV Ag/Ab, HIV-RNA level of 56 × 104 copies/mL, positive anti-Toxoplasma IgG (63 IU/mL), and negative anti-Toxoplasma IgM. 201Tl- single photon emission computed tomography (201Tl-SPECT) revealed abnormal accumulation only in the tumor in the left occipital lobe (early T/N ratio, 3.034; delayed T/N ratio, 2.738; retention index, 0.9), which was suspected to be a ML. Both tumors, with or without high accumulation of 201Tl, were subjected to craniotomy biopsy. Pathological examination revealed infiltration of small lymphocytes with a necrotic background. The patient was diagnosed with TE based on a positive result of a tissue polymerase chain reaction test for Toxoplasma gondii. Two weeks after sulfamethoxazole and trimethoprim combination therapy, MRI imaging showed dramatic improvement in multiple brain tumors. This case is atypical because ML was ruled out despite high 201Tl-SPECT uptake and retention. Careful diagnosis through pathological examination and DNA testing is important.

Skeletal muscle mass during chemotherapy for haematological malignancies: a retrospective study.

OBJECTIVE: This study investigated whether baseline or alteration in muscle mass affects complications during chemotherapy or overall survival (OS) in haematological malignancies. METHODS: Skeletal Muscle Index (SMI) was evaluated by bioimpedance analysis before and after chemotherapy in patients with haematological malignancies, and the association between muscle mass and clinical data was retrospectively analysed. RESULTS: Exactly 104 patients were enrolled, with a mean age of 62.2 years. SMI was 7.85 and 6.08 in male and female patients under 65 years and 7.10 and 5.92 over 65 years, before chemotherapy, respectively. Lower baseline SMI was not correlated with worse OS in total patients (p=0.915). After a median measurement interval of 30 days after chemotherapy (n=67), body weight and SMI decreased by 2.73% and 2.87% (mean), respectively. The decrease in body weight correlated with the loss of trunk muscle mass (R2=0.2107) but was more strongly associated with the loss of lower limbs muscle mass (R2=0.3985). The muscle mass of lower limbs significantly decreased in lymphoma patients who experienced febrile neutropenia (-0.42% vs -6.04%, p=0.040). OS significantly decreased in lymphoma patients with loss of lower limbs muscle ≥2.8% (p=0.0327). CONCLUSIONS: Muscle loss occurred following anticancer treatments, significantly contributing to worse outcomes. Body composition assessment and relevant multimodal prevention of muscle loss may be vital for patients receiving chemotherapy for haematological malignancies.

Acute accumulation of PIM2 and NRF2 and recovery of ß5 subunit activity mitigate multiple myeloma cell susceptibility to proteasome inhibitors.

Resistance to proteasome inhibitors (PIs) has emerged as an important clinical issue. We investigated the mechanisms underlying multiple myeloma (MM) cell resistance to PIs. To mimic their pharmacokinetic/pharmacodynamic (PK/PD) profiles, MM cells were treated with bortezomib and carfilzomib for 1 h at concentrations up to 400 and 1,000 nM, respectively. Susceptibility to these PIs markedly varied among MM cell lines. Pulsatile treatments with PIs suppressed translation, as demonstrated by incorporation of puromycin at 24 h in PI-susceptible MM.1S cells, but not PI-resistant KMS-11 cells. Inhibition of ß5 subunit activity decreased at 24 h in KMS-11 cells, even with the irreversible PI carfilzomib, but not under suppression of protein synthesis with cycloheximide. Furthermore, the proteasome-degradable pro-survival factors PIM2 and NRF2 acutely accumulated in MM cells subjected to pulsatile PI treatments. Accumulated NRF2 was trans-localized into the nucleus to induce the expression of its target gene, HMOX1, in MM cells. PIM and Akt inhibition restored the anti-MM effects of PIs, even against PI-resistant KMS-11 cells. Collectively, these results suggest that increased synthesis of ß5 proteasome subunit and acute accumulation of PIM2 and NRF2 reduce the anti-MM effects of PIs.

Acute suppression of translation by hyperthermia enhances anti-myeloma activity of carfilzomib.

Hyperthermia is a unique treatment option for cancers. Multiple myeloma (MM) remains incurable and innovative therapeutic options are needed. We investigated the efficacy of hyperthermia and carfilzomib in combination against MM cells. Although MM cell lines exhibited different susceptibilities to pulsatile carfilzomib treatment, mild hyperthermia at 43℃ induced MM cell death in all cell lines in a time-dependent manner. Hyperthermia and carfilzomib cooperatively induced MM cell death even under suboptimal conditions. The pro-survival mediators PIM2 and NRF2 accumulated in MM cells due to inhibition of their proteasomal degradation by carfilzomib; however, hyperthermia acutely suppressed translation in parallel with phosphorylation of eIF2α to reduce these proteins in MM cells. Recovery of ß5 subunit enzymatic activity from its immediate inhibition by carfilzomib was observed at 24 h in carfilzomib-insusceptible KMS-11, OPM-2, and RPMI8226 cells, but not in carfilzomib-sensitive MM.1S cells. However, heat treatment suppressed the recovery of ß5 subunit activity in these carfilzomib-insusceptible cells. Therefore, hyperthermia re-sensitized MM cells to carfilzomib. Our results support the treatment of MM with hyperthermia in combination with carfilzomib. Further research is warranted on hyperthermia for drug-resistant extramedullary plasmacytoma.

Therapeutic efficacy of the resorcylic acid lactone LL-Z1640-2 for adult T-cell leukaemia/lymphoma.

Adult T-cell leukaemia/lymphoma (ATL) remains incurable. The NF-κB and interferon regulatory factor 4 (IRF4) signalling pathways are among the critical survival pathways for the progression of ATL. TGF-ß-activated kinase 1 (TAK1), an IκB kinase-activating kinase, triggers the activation of NF-κB. The resorcylic acid lactone LL-Z1640-2 is a potent irreversible inhibitor of TAK1/extracellular signal-regulated kinase 2 (ERK2). We herein examined the therapeutic efficacy of LL-Z1640-2 against ATL. LL-Z1640-2 effectively suppressed the in vivo growth of ATL cells. It induced in vitro apoptosis and inhibited the nuclear translocation of p65/RelA in ATL cells. The knockdown of IRF4 strongly induced ATL cell death while downregulating MYC. LL-Z1640-2 as well as the NF-κB inhibitor BAY11-7082 decreased the expression of IRF4 and MYC at the protein and mRNA levels, indicating the suppression of the NF-κB-IRF4-MYC axis. The treatment with LL-Z1640-2 also mitigated the phosphorylation of p38 MAPK along with the expression of CC chemokine receptor 4. Furthermore, the inhibition of STAT3/5 potentiated the cytotoxic activity of LL-Z1640-2 against IL-2-responsive ATL cells in the presence of IL-2. Therefore, LL-Z1640-2 appears to be an effective treatment for ATL. Further studies are needed to develop more potent compounds that retain the active motifs of LL-Z1640-2.

The EGF Motif With CXDXXXXYXCXC Sequence Suppresses Fibrosis in a Mouse Skin Wound Model.

BACKGROUND/AIM: Fibrosis is an essential process for wound healing, but excessive fibrosis, such as keloids and hypertrophic scars, can cause cosmetic and functional problems. These lesions are caused by abnormal deposition and shrinkage of collagen fibers. The light chain of FIX, a plasma protein essential for hemostasis, has the amino acid sequence CXDXXXXYXCXC in the EGF domain. Peptides containing this sequence inhibited stromal growth in a mouse transplant tumor model. In this study, the effect of the FIX light chain on wound healing was studied. MATERIALS AND METHODS: A full-layer wound was made on the back of each mouse, and cDNA encoding the light chain of mouse FIX (F9-LC) in an expression vector was injected locally once each week using a non-viral vector. Histochemical analysis of the wound was then performed to assess the effects on wound healing. Moreover, the effect of F9-LC on fibroblasts was studied in vitro. RESULTS: Macroscopic observation showed that wounds with forced expression of F9-LC appeared flatter and had fewer wrinkles than control wounds. Tissue collagen staining and immunostaining revealed that administration of F9-LC suppressed collagen 1 and 3 deposition and decreased α-smooth muscle actin expression. Electron microscopy revealed sparse and disorganized collagen fibers in the F9-LC-treated mice. In experiments using fibroblasts, addition of a recombinant protein of the FIX light chain disrupted the typical spindle shape and alignment of fibroblasts. CONCLUSION: F9-LC is a new candidate for use in treatments to regulate excessive fibrosis and contraction in wound healing.

Novel antimyeloma therapeutic option with inhibition of the HDAC1-IRF4 axis and PIM kinase.

Multiple myeloma (MM) preferentially expands and acquires drug resistance in the bone marrow (BM). We herein examined the role of histone deacetylase 1 (HDAC1) in the constitutive activation of the master transcription factor IRF4 and the prosurvival mediator PIM2 kinase in MM cells. The knockdown or inhibition of HDAC1 by the class I HDAC inhibitor MS-275 reduced the basal expression of IRF4 and PIM2 in MM cells. Mechanistically, the inhibition of HDAC1 decreased IRF4 transcription through histone hyperacetylation and inhibiting the recruitment of RNA polymerase II at the IRF4 locus, thereby reducing IRF4-targeting genes, including PIM2. In addition to the transcriptional regulation of PIM2 by the HDAC1-IRF4 axis, PIM2 was markedly upregulated by external stimuli from BM stromal cells and interleukin-6 (IL-6). Upregulated PIM2 contributed to the attenuation of the cytotoxic effects of MS-275. Class I HDAC and PIM kinase inhibitors cooperatively suppressed MM cell growth in the presence of IL-6 and in vivo. Therefore, the present results demonstrate the potential of the simultaneous targeting of the intrinsic HDAC1-IRF4 axis plus externally activated PIM2 as an efficient therapeutic option for MM fostered in the BM.

[Primary cutaneous anaplastic large cell lymphoma with systemic progression responding to low-dose methotrexate therapy].

The standard therapies for primary cutaneous anaplastic large cell lymphoma (pcALCL) in an advanced stage remain undefined. A 71-year-old man presented with multiple erythema and nodules. He was diagnosed with lymphomatoid papulosis (LyP) through a skin biopsy from the left postauricular area. All skin lesions achieved complete response by electron beam irradiation. However, nodular lesions appeared in both inner canthi 5 months later. Histopathological evaluation of the lesional biopsy revealed dominant infiltration of CD30-positive large cells. Positron emission tomography/computed tomography revealed fluorodeoxyglucose-positive cervical and inguinal lymph node swelling and right tonsillitis, followed by the diagnosis of pcALCL and TNM classification T3bN3M0. Since the patient had severe chronic obstructive pulmonary disease and recurrent pneumonia, he received low-dose methotrexate (MTX) (15 mg/week) therapy. Low-dose MTX effectively debulked the lymphadenopathies over time without particular adverse effects. Although the standard therapies for pcALCL are not established, low-dose MTX was effective and considered safe for patients with frailty and compromised respiratory function. Further study is warranted on the pathophysiology of pcALCL after the development of LyP and mechanisms of action of low-dose MTX against LyP and pcALCL.

First reported case of Lachnoanaerobaculum gingivalis bacteremia in an acute myeloid leukemia patient with oral mucositis during high dose chemotherapy.

CASE DESCRIPTION: Lachnoanaerobaculum gingivalis is an obligate anaerobe identified in a human dental plaque in 2019. Here, we report the first case of L. gingivalis bacteremia in a patient with oral mucositis during chemotherapy. L. gingivalis was confirmed by 16S rRNA gene analysis but not by MALDI-TOF-MS. CONCLUSION: During chemotherapy in patients with oral mucositis, we should consider the possibility of L. gingivalis bacteremia.

The importance of retaining physical functions to prevent skeletal-related events in multiple myeloma patients with bone disease.

This study was undertaken to identify baseline conditions and triggering factors for skeletal-related events (SRE) in multiple myeloma (MM) patients treated with denosumab. During the median follow-up of 17 months, SRE occurred in 6 out of 52 newly diagnosed patients and in 5 out of 23 relapsed/refractory patients. Bone fractures occurred by falling down due to orthostatic hypotension and/or muscle weakness in three out of four cases with amyloid light-chain (AL) amyloidosis. A loss of balance and falling down appear to be triggering factors for SRE, especially in frail MM patients with AL amyloidosis, indicating the importance of retaining physical functions to prevent SRE.

Allogeneic haematopoietic stem cell transplantation and patient falls: impact of lower extremity muscle strength.

OBJECTIVES: Patients undergoing allogeneic haematopoietic stem cell transplantation (allo-HSCT) have a higher risk of falls than those receiving other therapies for haematological disorders. This study aimed to investigate the impact of pretransplant lower extremity muscle strength (LEMS) on post-transplant falls. METHODS: In this retrospective cohort study, patients aged ≥18 years who underwent allo-HSCT were included. All data were extracted from medical records. LEMS was defined as the knee extension force measured by a handheld dynamometer divided by the patient's weight. The receiver operating characteristic (ROC) curve was used to calculate the optimal LEMS cut-off value for prediction of falls. Patients were categorised into low and normal LEMS groups based on the cut-off value. The impact of pretransplant LEMS on post-transplant falls was analysed using a Cox proportional hazards model. RESULTS: In total, 101 patients were analysed. During the observation period, falls occurred in 32 patients (31.7%). The ROC curve analysis results showed that the optimal LEMS cut-off value for prediction of falls was 45.4% per body weight. In multivariate analysis, pretransplant low LEMS was a significant predictor of falls in model 1 with patient characteristics as a confounding factor and model 2 with medications-inducing falls as a confounding factor, respectively (model 1: HR 3.23, 95% CI 1.37 to 7.64; model 2: HR 2.82, 95% CI 1.20 to 6.59). CONCLUSIONS: Pretransplant LEMS was a significant predictor of post-transplant falls. The results of this study may help to prevent falls in patients undergoing allo-HSCT.

Intravenous busulfan-based conditioning with autologous stem cell transplantation for refractory B-cell lymphoma with central nervous system involvement.

The prognosis of relapsed or refractory lymphoma with central nervous system (CNS) involvement remains poor because of the lack of anticancer drugs with sufficient CNS penetration. [Case 1] A 65-year-old man was diagnosed with Stage IV mantle cell lymphoma. After two courses of chemotherapy and autologous hematopoietic stem cell (HSC) collection, urinary retention with fever developed. Cerebrospinal fluid analysis revealed leptomeningeal involvement, which was refractory to high-dose methotrexate therapy. Autologous peripheral blood stem cell transplantation (ASCT) was performed, followed by intravenous busulfan (ivBU), cyclophosphamide, and etoposide ; thereafter, no relapse has been detected for over six years. [Case 2] A 40-year-old woman with right lower hemiplegia was diagnosed with primary CNS lymphoma. Although four courses of high-dose methotrexate therapy were administered, the cerebral tumor increased in size. HSCs were collected after methotrexate therapy, and ASCT was performed in addition to conditioning using ivBU, cyclophosphamide, and etoposide, followed by whole-brain and local boost irradiation. She achieved complete remission, but relapsed two years after ASCT. High-dose ivBU-containing conditioning regimens with ASCT may be useful for refractory B-cell lymphoma with CNS involvement. J. Med. Invest. 68 : 196-201, February, 2021.

Acute Myeloid Leukemia Developing with Acute Pancreatitis Mimicking Autoimmune Pancreatitis.

A 33-year-old man was admitted to our hospital for fever and abdominal pain. A blood analysis revealed pancytopenia and increased serum pancreatic enzymes with disseminated intravascular coagulation. A detailed examination revealed acute pancreatitis, with diffuse swelling of the pancreas and diffuse beaded dilatation of the main pancreatic duct, which mimicked autoimmune pancreatitis complicated by acute myeloid leukemia. Systemic cytotoxic chemotherapy led to the remission of leukemia and pancreatitis. We hypothesized that the etiology of acute pancreatitis was invasion of leukemia cells. Acute pancreatitis is rare as a symptom of leukemia; however, we should consider the possibility of leukemia during the differential diagnosis of acute pancreatitis.

The Roles of ROS Generation in RANKL-Induced Osteoclastogenesis: Suppressive Effects of Febuxostat.

Receptor activator of NF-κB ligand (RANKL), a critical mediator of osteoclastogenesis, is upregulated in multiple myeloma (MM). The xanthine oxidase inhibitor febuxostat, clinically used for prevention of tumor lysis syndrome, has been demonstrated to effectively inhibit not only the generation of uric acid but also the formation of reactive oxygen species (ROS). ROS has been demonstrated to mediate RANKL-mediated osteoclastogenesis. In the present study, we therefore explored the role of cancer-treatment-induced ROS in RANKL-mediated osteoclastogenesis and the suppressive effects of febuxostat on ROS generation and osteoclastogenesis. RANKL dose-dependently induced ROS production in RAW264.7 preosteoclastic cells; however, febuxostat inhibited the RANKL-induced ROS production and osteoclast (OC) formation. Interestingly, doxorubicin (Dox) further enhanced RANKL-induced osteoclastogenesis through upregulation of ROS production, which was mostly abolished by addition of febuxostat. Febuxostat also inhibited osteoclastogenesis enhanced in cocultures of bone marrow cells with MM cells. Importantly, febuxostat rather suppressed MM cell viability and did not compromise Dox's anti-MM activity. In addition, febuxostat was able to alleviate pathological osteoclastic activity and bone loss in ovariectomized mice. Collectively, these results suggest that excessive ROS production by aberrant RANKL overexpression and/or anticancer treatment disadvantageously impacts bone, and that febuxostat can prevent the ROS-mediated osteoclastic bone damage.

Effective impairment of myeloma cells and their progenitors by hyperthermia.

Multiple myeloma (MM) remains incurable, and MM-initiating cells or MM progenitors are considered to contribute to disease relapse through their drug-resistant nature. In order to improve the therapeutic efficacy for MM, we recently developed novel superparamagnetic nanoparticles which selectively accumulate in MM tumors and extirpate them by heat generated with magnetic resonance. We here aimed to clarify the therapeutic effects on MM cells and their progenitors by hyperthermia. Heat treatment at 43°C time-dependently induced MM cell death. The treatment upregulated endoplasmic reticulum (ER) stress mediators, ATF4 and CHOP, while reducing the protein levels of Pim-2, IRF4, c-Myc and Mcl-1. Combination with the proteasome inhibitor bortezomib further enhanced ER stress to potentiate MM cell death. The Pim inhibitor SMI-16a also enhanced the reduction of the Pim-2-driven survival factors, IRF4 and c-Myc, in combination with the heat treatment. The heat treatment almost completely eradicated "side population" fractions in RPMI8226 and KMS-11 cells and suppressed their clonogenic capacity as determined by in vitro colony formation and tumorigenic capacity in SCID mice. These results collectively demonstrated that hyperthermia is able to impair clonogenic drug-resistant fractions of MM cells and enhance their susceptibility to chemotherapeutic drugs.

Unique anti-myeloma activity by thiazolidine-2,4-dione compounds with Pim inhibiting activity.

Proviral Integrations of Moloney virus 2 (PIM2) is overexpressed in multiple myeloma (MM) cells, and regarded as an important therapeutic target. Here, we aimed to validate the therapeutic efficacy of different types of PIM inhibitors against MM cells for their possible clinical application. Intriguingly, the thiazolidine-2,4-dione-family compounds SMI-16a and SMI-4a reduced PIM2 protein levels and impaired MM cell survival preferentially in acidic conditions, in contrast to other types of PIM inhibitors, including AZD1208, CX-6258 and PIM447. SMI-16a also suppressed the drug efflux function of breast cancer resistance protein, minimized the sizes of side populations and reduced in vitro colony-forming capacity and in vivo tumourigenic activity in MM cells, suggesting impairment of their clonogenic capacity. PIM2 is known to be subject to ubiquitination-independent proteasomal degradation. Consistent with this, the proteasome inhibitors bortezomib and carfilzomib increased PIM2 protein levels in MM cells without affecting its mRNA levels. However, SMI-16a mitigated the PIM2 protein increase and cooperatively enhanced anti-MM effects in combination with carfilzomib. Collectively, the thiazolidine-2,4-dione-family compounds SMI-16a and SMI-4a uniquely reduce PIM2 protein in MM cells, which may contribute to their profound efficacy in addition to their immediate kinase inhibition. Their combination with proteasome inhibitors is envisioned.

Cryptosporidiosis in a transplant recipient with severe intractable diarrhea: Detection of Cryptosporidium oocysts by intestinal biopsies.

Disseminated Cryptosporidium infection results in manifestations similar to those of graft-versus-host disease (GVHD), which hampers the detection of Cryptosporidium infection after allogeneic hematopoietic stem cell transplantation. Surveillance of oocysts on the surface of intestinal epithelial cells is needed for early and appropriate detection of Cryptosporidium infection in transplant recipients on immunosuppressants with severe intractable diarrhea. We present the first case of Cryptosporidium meleagridis infection in Japan after allogeneic cord blood transplantation.

[Analysis of long-term survivors with cardiac AL amyloidosis].

Cardiac AL amyloidosis (CA) is generally known as a severe disease with very poor prognosis. Here we retrospectively examined seven patients with CA in our cohort who achieved long-term survival. All six patients who underwent high-dose melphalan and autologous stem cell transplantation (ASCT) survived for >3 years, whereas four patients survived for >5 years. Patients who underwent ASCT had prompt hematological responses, and five patients showed organ responses. ASCT helps to achieve a quick and deep hematological response required for long-term survival in patients with CA. New agents have been implemented for the treatment of CA. However, the risks and benefits of each treatment modality should be considered according to patient condition, thus making the best use of ASCT in combination with new agents for the treatment of CA.