3D CNN-based Deep Learning Model-based Explanatory Prognostication in Patients with Multiple Myeloma using Whole-body MRI.

Although magnetic resonance imaging (MRI) data of patients with multiple myeloma (MM) are used to predict prognosis, few reports have applied artificial intelligence (AI) techniques for this purpose. We aimed to analyze whole-body diffusion-weighted MRI data using three-dimensional (3D) convolutional neural networks (CNNs) and Gradient-weighted Class Activation Mapping (Grad-CAM), an explainable AI, to predict prognosis and explore the factors involved in prediction. We retrospectively analyzed the MRI data of a total of 142 patients with MM obtained from two medical centers. We defined the occurrence of progressive disease after MRI evaluation within 12 months as a poor prognosis and constructed a 3D CNN-based deep learning model to predict prognosis. Images from 111 cases were used as the training and internal validation data; images from 31 cases were used as the external validation data. Internal validation of the AI model with stratified 5-fold cross-validation resulted in a significant difference in progression-free survival (PFS) between good and poor prognostic cases (2-year PFS, 91.2% versus [vs.] 61.1%, P = 0.0002). The AI model clearly stratified good and poor prognostic cases in the external validation cohort (2-year PFS, 92.9% vs. 55.6%, P = 0.004), with an area under the receiver operating characteristic curve of 0.804. According to Grad-CAM, the MRI signals of the spleen and bones of the vertebrae and pelvis contributed to prognosis prediction. This study is the first to show that image analysis of whole-body MRI using a 3D CNN without any other clinical data is effective in predicting the prognosis of patients with MM.

HLA Class I Allele Loss and Bone Marrow Transplantation Outcomes in Immune Aplastic Anemia.

In patients with immune-mediated acquired aplastic anemia (AA), HLA class I alleles often disappear from the surface of hematopoietic progenitor cells, potentially enabling evasion from cytotoxic T lymphocyte-mediated pathogenesis. Although HLA class I allele loss has been studied in AA patients treated with immunosuppressive therapy (IST), its impact on allogeneic bone marrow transplantation (BMT) has not been thoroughly investigated. The purpose of this study was to evaluate the clinical implications of HLA class I allele loss in patients with acquired AA undergoing allogeneic BMT. The study enrolled acquired AA patients who underwent initial BMT from unrelated donors through the Japan Marrow Donor Program between 1993 and 2011. The presence of HLA class I allele loss due to loss of heterozygosity (HLA-LOH) was assessed using pretransplantation blood DNA and correlated with clinical data obtained from the Japanese Transplant Registry Unified Management Program. A total of 432 patients with acquired AA were included in the study, and HLA-LOH was detected in 20 of the 178 patients (11%) available for analysis. Patients with HLA-LOH typically presented with more severe AA at diagnosis (P = .017) and underwent BMT earlier (P < .0001) compared to those without HLA-LOH. They also showed a slight but significant recovery in platelet count from the time of diagnosis to BMT (P = .00085). However, HLA-LOH status had no significant effect on survival, engraftment, graft failure, chimerism status, graft-versus-host disease, or other complications following BMT, even when the 20 HLA-LOH+ patients were compared with the 40 propensity score-matched HLA-LOH- patients. Nevertheless, patients lacking HLA-A*02:06 or HLA-B*40:02, the alleles most frequently lost and associated with a better IST response, showed higher survival rates compared to those lacking other alleles, with estimated 5-year overall survival (OS) rates of 100% and 44%, respectively (P = .0042). In addition, in a specific subset of HLA-LOH- patients showing clinical features similar to HLA-LOH+ patients, the HLA-A*02:06 and HLA-B*40:02 allele genotypes correlated with better survival rates compared with other allele genotypes, with estimated 5-year OS rates of 100% and 43%, respectively (P = .0096). However, this genotype correlation did not extend to all patients, suggesting that immunopathogenic mechanisms linked to the loss of certain HLA alleles, rather than the HLA genotypes themselves, influence survival outcomes. The survival benefit associated with the loss of these two alleles was confirmed in a multivariable Cox regression model. The observed correlations between HLA loss and the pretransplantation clinical manifestations and between loss of specific HLA class I alleles and survival outcomes in AA patients may improve patient selection for unrelated BMT and facilitate further investigations into the immune pathophysiology of the disease.

Familial immune-mediated aplastic anaemia in six different families.

We studied the pathophysiology of aplastic anaemia (AA) in six different pairs of relatives without a family history of hematologic disorders or congenital AA. Five and four of the six pairs shared the HLA-DRB1*15:01 and B*40:02 alleles, respectively. Glycosylphosphatidylinositol-anchored protein-deficient blood cells were detected in eight of the 10 patients evaluated. In a mother-daughter pair from one family, flow cytometry detected leukocytes lacking HLA-A2 due to loss of heterogeneity in chromosome 6p. Whole-exome sequencing of the family pair revealed a missense mutation in MYSM1. These results suggest that genetic inheritance of immune traits might underlie familial AA in some patients.

SMARCB1/INI1-deficient intrathoracic neoplasm with rhabdoid/plasmacytoid cytomorphology in a patient with plasma cell myeloma: A case report.

SMARCB1 (INI1) is one of the switch/sucrose nonfermentable (SWI/SNF) complexes whose loss is associated with several tumors. SMARCB1 (INI1)-deficient intrathoracic neoplasms are extremely rare and known to be highly malignant and lethal. This report presents the case of a patient diagnosed with SMARCB1 (INI1)-deficient intrathoracic neoplasm during chemotherapy for plasma cell myeloma. A 77-year-old male patient complained of cough, bloody sputum, and fever with an enlarged right lung mass and pleural effusion. His cytological examination revealed undifferentiated epithelioid and rhabdoid/plasmacytoid cells with bi- or multinucleation, vacuolization, mitosis, and pleomorphism. However, it was difficult to distinguish the relapse of plasma cell myeloma as atypical plasmacytoid cells were detected. Immunohistochemically, the neoplastic cells showed a loss of SMARCB1 (INI1) expression in the cell block of pleural fluid and in the right lung of the autopsy specimen. Further, the patient was diagnosed with SMARCB1 (INI1)-deficient intrathoracic neoplasm of the right lung based on histological and autopsy findings. To the best of our knowledge, this is the first report of cytomorphology in a SMARCB1 (INI1)-deficient intrathoracic neoplasm.

Clinical significance of the increased expression of the WT1 gene in peripheral blood of patients with acquired aplastic anemia.

To determine the significance of increased Wilms tumor 1 (WT1) gene expression in the peripheral blood of patients with acquired aplastic anemia (AA), we analyzed serial changes in WT1 mRNA copy number (WT1cn) in 63 patients with AA as well as in five patients with myelodysplastic syndromes (MDS) and seven patients with paroxysmal nocturnal hemoglobinuria (PNH). WT1cn was higher than the cut-off (≥50 copies/µg RNA) at the time of the first measurement in 41% of untreated (60-190 copies/µg RNA [median 130]) and 59% of treated (59-520 copies/µg RNA [median 150]) AA patients. Although WT1cns gradually increased in most AA patients during the 2-105 months follow-up period, they did not lead to clonal evolution except in three patients in whom the maximum change ratio of WT1cn (WT1cn-change max), defined as the ratio of WT1cn at the first examination to that of the maximum value, exceeded 20.0 and who developed MDS at 2, 46, and 105 months. Increased WT1 gene expression was enriched in granulocytes rather than in mononuclear cells in most WT1-positive AA patients and did not correlate with mutations of genes associated with myeloid malignancy. WT1cns were high at 690-5700 (median 2000) in MDS patients and remained high thereafter, while WT1cns in PNH patients (77-200; median 96) were similar to those in AA. Thus, moderate increases in WT1cns up to 600 are common in AA patients in stable remission. An increase in the WT1cn-change max over 20.0 may portend transformation from AA to MDS.

Hematopoietic stem progenitor cells with malignancy-related gene mutations in patients with acquired aplastic anemia are characterized by the increased expression of CXCR4.

The phenotypic changes in hematopoietic stem progenitor cells (HSPCs) with somatic mutations of malignancy-related genes in patients with acquired aplastic anemia (AA) are poorly understood. As our initial study showed increased CXCR4 expression on HLA allele-lacking (HLA[-]) HSPCs that solely support hematopoiesis in comparison to redundant HLA(+) HSPCs in AA patients, we screened the HSPCs of patients with various types of bone marrow (BM) failure to investigate their CXCR4 expression. In comparison to healthy individuals (n = 15, 12.3%-49.9%, median 43.2%), the median CXCR4+ cell percentages in the HSPCs of patients without somatic mutations were low: 29.3% (14.3%-37.3%) in the eight patients without HLA(-) granulocytes, 8.8% (4.1%-9.8%) in the five patients with HLA(-) cells accounting for >90% of granulocytes, and 7.8 (2.1%-8.7%) in the six patients with paroxysmal nocturnal hemoglobinuria. In contrast, the median percentage was much higher (78% [61.4%-88.7%]) in the five AA patients without HLA(-) granulocytes possessing somatic mutations (c-kit, t[8;21], monosomy 7 [one for each], ASXL1 [n = 2]), findings that were comparable to those (66.5%, 63.1%-88.9%) in the four patients with advanced myelodysplastic syndromes. The increased expression of CXCR4 may therefore reflect intrinsic abnormalities of HSPCs caused by somatic mutations that allow them to evade restriction by BM stromal cells.

The effectiveness of immunosuppressive therapy in patients with aplastic anaemia secondary to chemoradiotherapy for cancers.

The outcome of immunosuppressive therapy (IST) and prognosis in patients with aplastic anaemia (AA) secondary to chemotherapy or radiotherapy for cancers remains unknown. A total of 43 of 2559 patients with AA referred to our hospital had previously received chemoradiotherapy for various types of solid tumours (n = 25) or haematological malignancies (n = 18). Their cancer status was complete remission (CR) in 27, non-CR in 13, and unknown in three. Small populations of glycosylphosphatidylinositol-anchored protein-deficient [GPI(-)] granulocytes were detected in 16 patients (37·2%). Of 18 patients who were treated with IST, 50% improved regardless of the presence of GPI(-) cells. The overall survival (OS) rate was significantly higher in patients with a history of solid tumours patients than in those of haematological malignancies (median OS, 87 vs. 11 months, P = 0·0003), and in patients treated with IST than in those of untreated patients (median OS, 115 vs. 20 months, P = 0·028). Cancer aggravation occurred in two of four patients who were treated with IST while in non-CR of their original cancers. Progression to myelodysplastic syndromes was observed in two patients not possessing GPI(-) cells. IST should thus be considered for patients with AA secondary to chemoradiotherapy for cancers, particularly when their original solid tumours are in CR.

Hematopoietic stem progenitor cells lacking HLA differ from those lacking GPI-anchored proteins in the hierarchical stage and sensitivity to immune attack in patients with acquired aplastic anemia.

To characterize glycosylphosphatidylinositol-anchored protein-deficient (GPI[-]) and HLA-class I allele-lacking (HLA[-]) hematopoietic stem progenitor cells (HSPCs) in acquired aplastic anemia (AA), we studied the peripheral blood (PB) of 56 AA patients in remission who possessed both (n = 13, Group A) or either GPI(-) (n = 34, Group B) and HLA(-) (n = 9, Group C) cell populations. Seventy-seven percent (10/13) of Group A had HLA(-) cells in all lineages of PB cells, including platelets, while only 23% (3/13) had GPI(-) cells in all lineages, and the median percentage of HLA(-) granulocytes in the total granulocytes (21.2%) was significantly higher than that of GPI(-) granulocytes (0.28%, P < 0.05). The greater lineage diversity in HLA(-) cells than in GPI(-) cells was also seen when Group B and Group C were compared. Longitudinal studies of seven patients in Group A showed a gradual decrease in the percentage of HLA(-) granulocytes, with a reciprocal increase in the GPI(-) granulocytes in four patients responding to cyclosporine (CsA) and an increase in the HLA(-) granulocytes with a stable or declining GPI(-) granulocytes in three patients in sustained remission off CsA therapy. These findings suggest that HLA(-) HSPCs differ from GPI(-) HSPCs in the hierarchical stage and sensitivity to immune attack in AA.

Effectiveness of hyperbaric oxygen therapy for virus-associated hemorrhagic cystitis after allogeneic hematopoietic stem cell transplantation.

Although some studies have suggested the effectiveness of hyperbaric oxygen (HBO) therapy for hemorrhagic cystitis (HC) after allogeneic hematopoietic stem cell transplantation (HSCT), the role of HBO has not been established. We compared the treatment outcomes of 8 patients with viral HC (adenovirus [ADV], n = 2; BK virus [BKV], n = 6) treated with HBO (HBO[+]) and 8 patients (ADV, n = 2; BKV, n = 6) treated with conventional therapy (HBO[-]), such as urinary catheterization and intravenous cidofovir. HBO therapy was performed at 2.1 atmospheres for 90 min/day until clinical improvement was achieved. The median number of HBO treatments was 10 (range 8-12). The median duration of HBO treatment was 19.5 days (range 10-23 days). All 8 HBO(+) patients achieved complete remission (CR) at a median of 14.5 days (range 5-25 days). Of the 8 HBO(-) patients, 5 (62.5%) obtained CR and 3 remained symptomatic for 2-6 months. The cumulative incidence of transplant-related mortality at day 100 after allogeneic HSCT was significantly higher in the HBO(-) patients than in the HBO(+) patients (14.2 vs. 0%, P < 0.05). No severe HBO-related adverse effects were observed. In conclusion, HBO is a feasible option for treating viral HC after allogeneic HSCT.

A frequent nonsense mutation in exon 1 across certain HLA-A and -B alleles in leukocytes of patients with acquired aplastic anemia.

Leukocytes that lack HLA allelic expression are frequently detected in patients with acquired aplastic anemia (AA) who respond to immunosuppressive therapy (IST), although the exact mechanisms underlying the HLA loss and HLA allele repertoire likely to acquire loss-of-function mutations are unknown. We identified a common nonsense mutation at position 19 (c.19C>T, p.R7X) in exon 1 (Exon1mut) of different HLA-A and -B alleles in HLA-lacking granulocytes from AA patients. A droplet digital PCR (ddPCR) assay capable of detecting as few as 0.07% Exon1mut HLA alleles in total DNA revealed the mutation was present in 29% (101/353) of AA patients, with a median allele frequency of 0.42% (range, 0.071% to 21.3%). Exon1mut occurred in only 12 different HLA-A (n=4) and HLA-B (n=8) alleles, including B*40:02 (n=31) and A*02:06 (n=15), which correspond to 4 HLA supertypes (A02, A03, B07, and B44). The percentages of patients who possessed at least one of these 12 HLA alleles were significantly higher in the 353 AA patients (92%, P.

Paroxysmal nocturnal hemoglobinuria-phenotype cells predict a good response to eltrombopag in patients with refractory aplastic anemia.

To identify factors affecting responsiveness to eltrombopag (EPAG), we retrospectively analyzed 38 aplastic anemia patients treated with EPAG who were refractory (n = 29) or showed an inadequate response (n = 9) to conventional therapies. The efficacy was evaluated at 16 weeks after starting EPAG and at any given time when the best response was achieved. Hematologic responses were observed in 15 patients (39%) at week 16 and in 25 (66%) at any given time. Ten of 19 (53%) achieved transfusion independence. A univariate analysis revealed the presence of PNH-phenotype cells and the relatively higher platelet counts as associated with a good response to EPAG.

Frequent STAT3 mutations in CD8+ T cells from patients with pure red cell aplasia.

Dysregulation of T-cell-mediated immunity is responsible for acquired pure red cell aplasia (PRCA). Although STAT3 mutations are frequently detected in patients with T-cell large granular lymphocytic leukemia (T-LGLL), which is often complicated by PRCA and which is also reported to be associated with acquired aplastic anemia (AA) and myelodysplastic syndrome (MDS), whether STAT3-mutated T cells are involved in the pathophysiology of PRCA and other types of bone marrow failure remains unknown. We performed STAT3 mutation analyses of the peripheral blood mononuclear cells from PRCA patients (n = 42), AA (n = 54), AA-paroxysmal nocturnal hemoglobinuria (AA-PNH; n = 7), and MDS (n = 21) using an allele-specific polymerase chain reaction and amplicon sequencing. STAT3 mutations were not detected in any of the 82 patients with AA/PNH/MDS but were detected in 43% of the 42 PRCA patients. In all 7 STAT3-mutation-positive patients who were studied, the STAT3 mutations were restricted to sorted CD8+ T cells. The prevalence of STAT3 mutation in idiopathic, thymoma-associated, autoimmune disorder-associated, and T-LGLL-associated PRCA was 33% (5 of 15), 29% (2 of 7), 20% (1 of 5), and 77% (10 of 13), respectively. The STAT3-mutation-positive patients were younger (median age, 63 vs 73 years; P= .026) and less responsive to cyclosporine (46% [6 of 13] vs 100% [8 of 8]; P= .0092) in comparison with STAT3-mutation-negative patients. The data suggest that STAT3-mutated CD8+ T cells may be closely involved in the selective inhibition of erythroid progenitors in PRCA patients.

Sustained clonal hematopoiesis by HLA-lacking hematopoietic stem cells without driver mutations in aplastic anemia.

Clonal hematopoiesis by hematopoietic stem progenitor cells (HSPCs) that lack an HLA class I allele (HLA- HSPCs) is common in patients with acquired aplastic anemia (AA); however, it remains unknown whether the cytotoxic T lymphocyte (CTL) attack that allows for survival of HLA- HSPCs is directed at nonmutated HSPCs or HSPCs with somatic mutations or how escaped HLA- HSPC clones support sustained hematopoiesis. We investigated the presence of somatic mutations in HLA- granulocytes obtained from 15 AA patients in long-term remission (median, 13 years; range, 2-30 years). Targeted sequencing of HLA- granulocytes revealed somatic mutations (DNMT3A, n = 2; TET2, ZRSR2, and CBL, n = 1) in 3 elderly patients between 79 and 92 years of age, but not in 12 other patients aged 27 to 74 years (median, 51.5 years). The chronological and clonogenic analyses of the 3 cases revealed that ZRSR2 mutation in 1 case, which occurred in an HLA- HSPC with a DNMT3A mutation, was the only mutation associated with expansion of the HSPC clone. Whole-exome sequencing of the sorted HLA- granulocytes confirmed the absence of any driver mutations in 5 patients who had a particularly large loss of heterozygosity in chromosome 6p (6pLOH) clone size. Flow-fluorescence in situ hybridization analyses of sorted HLA+ and HLA- granulocytes showed no telomere attrition in HLA- granulocytes. The findings suggest that HLA- HSPC clones that escape CTL attack are essentially free from somatic mutations related to myeloid malignancies and are able to support long-term clonal hematopoiesis without developing driver mutations in AA patients unless HLA loss occurs in HSPCs with somatic mutations.

Hematopoiesis by iPSC-derived hematopoietic stem cells of aplastic anemia that escape cytotoxic T-cell attack.

Hematopoietic stem cells (HSCs) that lack HLA-class I alleles as a result of copy-number neutral loss of heterozygosity of the short arm of chromosome 6 (6pLOH) or HLA allelic mutations often constitute hematopoiesis in patients with acquired aplastic anemia (AA), but the precise mechanisms underlying clonal hematopoiesis induced by these HLA-lacking (HLA-) HSCs remain unknown. To address this issue, we generated induced pluripotent stem cells (iPSCs) from an AA patient who possessed HLA-B4002-lacking (B4002-) leukocytes. Three different iPSC clones (wild-type [WT], 6pLOH+, and B*40:02-mutant) were established from the patient's monocytes. Three-week cultures of the iPSCs in the presence of various growth factors produced hematopoietic cells that make up 50% to 70% of the CD34+ cells of each phenotype. When 106 iPSC-derived CD34+ (iCD34+) cells with the 3 different genotypes were injected into the femoral bone of C57BL/6.Rag2 mice, 2.1% to 7.3% human multilineage CD45+ cells of each HLA phenotype were detected in the bone marrow, spleen, and peripheral blood of the mice at 9 to 12 weeks after the injection, with no significant difference in the human:mouse chimerism ratio among the 3 groups. Stimulation of the patient's CD8+ T cells with the WT iCD34+ cells generated a cytotoxic T lymphocyte (CTL) line capable of killing WT iCD34+ cells but not B4002- iCD34+ cells. These data suggest that B4002- iCD34+ cells show a repopulating ability similar to that of WT iCD34+ cells when autologous T cells are absent and CTL precursors capable of selectively killing WT HSCs are present in the patient's peripheral blood.

Identification of an HLA class I allele closely involved in the autoantigen presentation in acquired aplastic anemia.

To identify HLA alleles closely involved in the autoantigen presentation in acquired aplastic anemia (AA), we studied the HLA allelic loss frequencies of 312 AA patients, including 43 patients with loss of heterozygosity of 6p chromosome (6pLOH). An analysis of the HLA alleles contained in the lost haplotype revealed HLA-B*40:02 to be the most frequently lost allele. When we examined 28 AA (12 6pLOH[+] and 16 6pLOH[-]) patients with HLA-B*40:02 for the presence of leukocytes lacking HLA-B4002 (B4002-) using a new monoclonal antibody specific to this allele, B4002- granulocytes were detected not only in all 6pLOH(+) patients but also in 9 (56%) of the 16 6pLOH(-) patients. Furthermore, 10 (83%) of the 12 6pLOH(+) patients possessed 1.0% to 78% B4002- granulocytes that retained the HLA-A allele on the same haplotype (B4002-A+), suggesting the frequent coexistence of granulocytes that underwent mutations restricted to HLA-B*40:02 with 6pLOH(+) (B4002-A-) granulocytes. Deep sequencing of the HLA-B*40:02 of sorted B4002-A+ granulocytes revealed various somatic mutations, such as frameshift, nonsense, and splice site mutations, in all 15 patients studied. Surprisingly, missense mutations in the α-3 domain of HLA-B*40:02 that are not involved in the antigen presentation were detected exclusively in the B4002+ granulocytes of 3 patients possessing B4002- granulocytes. The markedly high prevalence of leukocytes lacking HLA-B4002 as a result of either 6pLOH or structural gene mutations, or both, suggests that antigen presentation by hematopoietic stem/progenitor cells to cytotoxic T cells via the HLA-B allele plays a critical role in the pathogenesis of AA.