Manabu Fujisawa

Quantification of bone marrow plasma cell infiltration in multiple myeloma: Usefulness of bone marrow aspirate clot with CD138 immunohistochemistry

Accurate quantification of plasma cells (PCs) in bone marrow (BM) is critical for diagnosis and assessment of treatment response in patients with multiple myeloma (MM). We compared the % of BM PC quantified by 250 cell differential count on May–Giemsa‐stained BM smears, by counting 500 – 2500 cells in 2 – 5 representative microscopy fields in CD138‐immunostained BM clot and biopsy sections, and CD38/CD45/CD138 gated BM PCs on flow cytometry (FCM) in 150 sets of BM samples from 120 patients. Percentages of PC were significantly correlated between BM biopsy and clot, and between smear and FCM (r = 0.96, 0.93, respectively). However, quantification by smear and FCM significantly underestimated the PC compared to biopsy or clot, and the degree of underestimation increased with blood dilution. FCM consistently showed lower % of PC compared to aspirate smears. Fifty‐nine of 103 patients with M‐protein level < 3000 mg/dL in serum or 500 mg/24 h in urine and diagnosed with monoclonal gammopathy of undetermined significance (MGUS) based on smear alone were reclassified as smoldering MM when reassessed using CD138‐stained biopsy/clot sections. Among the 72 patients with sMM diagnosed by BM biopsy and/clot, three patients (4.2%) had extensive BM infiltration of PC (≥ 60%) and required treatment. Our data clearly showed the necessity of CD138 immunostaining of BM biopsy/clot specimens for correct diagnosis of MM and related disorders. Copyright

oligoclonal bands in patients with multiple myeloma: its emergence per se could not be translated to improved survival.

The emergence of oligoclonal bands (OB) has been reported in patients with multiple myeloma (MM) after stem cell transplantation (SCT) or successful chemotherapy. However, their clinical relevance remains unclear. We reviewed the clinical records of MM patients from January 2006 to May 2014. Treatment response was evaluated by International Working Group (IMWG) criteria. Serum immunofixation tests were performed at least every 3 months if the patient achieved more than very good partial response (VGPR). Free light chain (FLC) and minimal residual disease measurement by multicolor flow cytometry (MFC) were performed to evaluate the response to treatment. Among the 163 patients included in the study, 40 developed OB. Detection rates of OB in patients with complete response (CR), VGPR and partial response (PR) or less were 51.8, 36.3 and 0%, respectively. Patients with OB showed better progression‐free survival (PFS) and overall survival (OS) rates than those without OB (P = 0.028 and P < 0.001, respectively). However, if the patients were limited to ≥VGPR or CR, development of OB did not affect PFS (P = 0.621 and P = 0.646, respectively) or OS (P = 0.189 and P = 0.766, respectively). OB was observed in 60% of patients after SCT, and in 36.6% of patients with more than VGPR without SCT (P < 0.001). Patients with OB tended to have less minimal residual disease than those without OB (P = 0.054) and its presence may affect the stringent CR criteria. In conclusion, the emergence of OB was seen exclusively in patients with favorable responses, but its emergence per se could not be translated to improved survival.

Abnormal heavy/light chain ratio after treatment is associated with shorter survival in patients with IgA myeloma

Immunoglobulin (Ig) heavy/light chain (HLC) assays enable the separate quantification of the different light chain types of each Ig class. We retrospectively analyzed the correlation of heavy/light chain ratio (HLCR) with clinical status and its impact on outcome in 120 patients with multiple myeloma (MM). Abnormal HLCR was seen more frequently in patients with poorer myeloma response, and it appeared to be more sensitive for detecting clonality in IgA myeloma compared to IgG myeloma after treatment. Among the 85 patients who achieved ≥VGPR, the patients remained HLCR abnormal were showed significantly shorter overall survival (OS) compared to those achieving a normal HLCR (not reached vs 55.5 months, P = 0.032). This correlation was seen in IgA myeloma patients (not reached vs 30.1 months, P = 0.014), but not in IgG myeloma patients when patients were analyzed separately. Univariate and multivariate analysis of factors that may affect survival identified abnormal HLCR at the best response as the only independent risk factor (hazard ratio, 4.7; 95% confidence interval, 1.4 – 15.26; P = 0.012) for shorter OS in this subset of patients. This study highlighted the HLC assay as a prognostic predictor in patients with IgA myeloma.

Clinical features and treatment outcome of very elderly patients over 80 years old with multiple myeloma: comparison with patients in different age groups in the era of novel agents

We retrospectively analyzed the outcomes of 175 consecutive patients admitted to our hospital between April 2004 and June 2014, and identified 42 (24%), 80 (46%), and 53 (30%) patients ≥ 80, 66–79, and ≤ 65 years old, respectively. The median progression-free survival (PFS) and overall survival (OS) of the ≥ 80, 66–79, and ≤ 65 years old groups were 19.1, 26.3, and 54.3 months, and 31.9, 54.8, and 83.8 months, respectively. Patients ≥ 80 but not ≤ 79 years old with ECOG performance score (PS) ≥ 3 and/or Charlson comorbidity index (CCI) ≥ 5 showed significantly shorter survival. ECOG PS and CCI predicted the treatment outcome of patients ≥ 80 but did not predict ≤ 79 years old.

Automatic digital quantification of bone marrow myeloma volume in appendicular skeletons - clinical implications and prognostic significance

Multiple myeloma (MM) is a clonal plasma cell disorder originating in bone marrow. Whole body low-dose multidetector CT (MDCT) can depict bone marrow infiltration by myeloma cells into the adipose-rich fatty marrow of the appendicular skeleton. However, automated and objective volume measurement of bone marrow infiltration has not been established, and its clinical relevance remains unclear. We therefore developed novel CT post-processing software (MABLE software) and measured the total sum of CT values (cumulative CT value, cCTv) representing bone marrow infiltration, by combining volume and voxel-based CT values. The cCTv was greater in patients with symptomatic MM than in those with smouldering MM or monoclonal gammopathy of unknown significance. Patients with revised International Staging System (R-ISS) III had a higher cCTv than those with R-ISS I or II. Age, albumin, and M-protein levels independently predicted cCTv. Mixed graphical model analysis revealed direct relationships between cCTv and age or R-ISS. Tree-structured survival analysis and multivariate Cox analysis revealed that a cCTv greater than or equal to 4.4 was independently prognostic for overall survival. Anti-myeloma therapy reduced cCTv after treatment. These findings suggest that the automatically calculated cCTv reflects disease aggressiveness and is useful for accurate prognostic prediction in MM patients.

Recent Progress in the Understanding of Angioimmunoblastic T-cell Lymphoma

Angioimmunoblastic T-cell lymphoma (AITL) has been classified as a subtype of mature T-cell neoplasms. The recent revision of the WHO classification proposed a new category of nodal T-cell lymphoma with follicular helper T (TFH)-cell phenotype, which was classified into three diseases: AITL, follicular T-cell lymphoma, and nodal peripheral T-cell lymphoma with TFH phenotype. These lymphomas are defined by the expression of TFH-related antigens, CD279/PD-1, CD10, BCL6, CXCL13, ICOS, SAP, and CXCR5. Although recurrent mutations in TET2, IDH2, DNMT3A, RHOA, and CD28, as well as gene fusions, such as ITK-SYK and CTLA4-CD28, were not diagnostic criteria, they may be considered as novel criteria in the near future. Notably, premalignant mutations, tumor-specific mutations, and mutations specific to tumor-infiltrating B cells were identified in AITL. Thus, multi-step and multi-lineage genetic events may lead to the development of AITL.

Prognostic implication of appendicular skeleton bone marrow abnormalities detected using low-dose multidetector computed tomography in patients with myelodysplastic syndrome

Myelodysplastic syndrome (MDS) encompasses a series of heterogeneous hematologic conditions characterized by cytopenia, ineffective hematopoiesis, and an increased risk of developing acute myeloid leukemia (AML) [1]. A number of prognostic systems have been developed and validated in patients with MDS [2–4]. Among these, the Revised International Prognostic Scoring System (R-IPSS) integrates the number of known clinical features and stratifies the prognosis of MDS patients more precisely [5]. However, these prognostic systems are exclusively based on various clinical and demographic information derived mainly from the hematologic data of peripheral blood and bone marrow, as well as cytogenetic analyses. Low-dose whole-body multidetector computed tomography (MDCT) is a useful modality for depicting bone marrow medullary and lytic bone lesions in patients with multiple myeloma (MM) [6–8]. We previously reported the prognostic role of abnormal medullary lesion of appendicular skeletons (AS) in patients with MM and related diseases using the MDCT [8]. Here, we examined the medullary abnormality in AS among patients with MDS and analyzed its relationship with the clinical parameters, subsequent development of leukemic transformation, and survival. Between January 2009 and May 2017, 150 consecutive patients with MDS diagnosed at Kameda Medical Center, Kamogawa-shi, Japan were recruited. Diagnosis and classification of MDS were made per 2008 WHO criteria [1]. MDCT were performed within one month of the diagnosis of MDS and before the start of any therapy. Written informed consent was obtained from included patients. The study was conducted per the Declaration of Helsinki and was approved by the institutional review board of Kameda Medical Center. Baseline clinical characteristics and patterns of patients are summarized in supplemental Table 1S. The procedure for acquiring MDCT images has been previously reported [8], and categorization of the MDCT pattern of AS is shown in Fig. 1a (1–3). Of the 150 patients with MDS, there were 49 (32.6%) with fatty, 53 (35.3%) with focal/scattered, and 48 (32.0%) with diffuse marrow patterns. As expected, all of the normal adults (n= 50) exhibited fatty pattern. Fatty pattern was seen more among non-refractory anemia with excess of blast (RAEB) patients (77.6%), focal/scattered pattern was seen approximately equally, and diffuse pattern was seen more (64.6%) in RAEB patients. Regarding RIPSS risk groups, there were 11 (7.3%) very low risk, 42 (28.0%) low risk, 22 (14.7%) high risk, and 39 (26.0%) very high-risk patients. There were significant differences in hemoglobin concentration, with the lowest occurring in diffuse marrow pattern and the highest occuring in fatty marrow pattern. Moreover, diffuse pattern was associated with higher bone marrow blast percentage (P= 0.004), complex karyotypes (P < 0.001), del5/-5 (P= 0.009), del7q/-7 (P= 0.014), poorer cytogenetic abnormalities (CA) (P < 0.001), and higher risk R-IPSS categories compared to fatty or focal/ scattered MDCT patterns. * Kosei Matsue koseimatsue@gmail.com

Droplet digital polymerase chain reaction assay and peptide nucleic acid-locked nucleic acid clamp method for RHOA mutation detection in angioimmunoblastic T-cell lymphoma

Angioimmunoblastic T‐cell lymphoma (AITL) is a subtype of nodal peripheral T‐cell lymphoma (PTCL). Somatic RHOA mutations, most frequently found at the hotspot site c.50G > T, p.Gly17Val (G17V RHOA mutation) are a genetic hallmark of AITL. Detection of the G17V RHOA mutations assists prompt and appropriate diagnosis of AITL. However, an optimal detection method for the G17V RHOA mutation remains to be elucidated. We compared the sensitivity and concordance of next‐generation sequencing (NGS), droplet digital PCR (ddPCR) and peptide nucleic acid‐locked nucleic acid (PNA‐LNA) clamp method for detecting the G17V RHOA mutation. G17V RHOA mutations were identified in 27 of 67 (40.3%) PTCL samples using NGS. ddPCR and PNA‐LNA clamp method both detected G17V mutations in 4 samples in addition to those detected with NGS (31 of 67, 46.3%). Additionally, variant allele frequencies with ddPCR and those with NGS showed high concordance (P < .001). Three other RHOA mutations involving the p.Gly17 position (c.[49G > T;50G > T], p.Gly17Leu in PTCL198; c.[50G > T;51A > C], p.Gly17Val in PTCL216; and c.50G > A, p.Gly17Glu in PTCL223) were detected using NGS. These sequence changes could not appropriately be detected using the ddPCR assay and the PNA‐LNA clamp method although both indicated that the samples might have mutations. In total, 34 out of 67 PTCL samples (50.7%) had RHOA mutations at the p.Gly17 position. In conclusion, our results suggested that a combination of ddPCR/PNA‐LNA clamp methods and NGS are best method to assist the diagnosis of AITL by detecting RHOA mutations at the p.Gly17 position.