Improved Outcome of Allogeneic Transplantation in Older Patients Treated for Myeloid Malignancies Using Post-Transplantation Cyclophosphamide and Reduced Duration of Immune Suppression.

Allogeneic stem cell transplantation (alloSCT) offers curative potential for older patients with myeloid malignancies. We evaluated the efficacy and safety of alloSCT using post-transplantation cyclophosphamide (PTCy) in combination with a very short duration of immune suppression (IS) in this population. We retrospectively analyzed 92 consecutive patients aged 65 years and older who underwent an alloSCT for myeloid malignancies between February 2018 and December 2022 at our institution. Data on patient characteristics, treatment modalities, and outcomes were collected. Ninety-two patients received an alloSCT with PTCy-based graft versus host disease (GVHD) prophylaxis. The majority had minimal comorbidities and were diagnosed with acute myeloid leukemia. Patients mostly received conditioning regimens with low to intermediate transplant conditioning intensity scores. In 43% of patients, IS could be permanently stopped at day +90, resulting in a median time of IS of 2.93 months in high-risk patients. At a median follow-up of 21.3 months, the 1- and 2-year overall survival rates were 89% and 87%, respectively. Relapse-free survival rates were 88% and 84% at 1 and 2 years, respectively. The 1- and 2-year cumulative incidences of relapse were 8% and 13%, while treatment-related mortality (TRM) estimates were 9% at both time points. Acute GVHD grade 3 to 4 occurred in 7% within the first 180 days and severe chronic GVHD in 6% of patients. This all resulted in a 1- and 2-year graft versus host and relapse-free survival of 74% and 70%, respectively. AlloSCT using PTCy in combination with a short duration of IS in older patients with myeloid malignancies demonstrates favorable survival outcomes due to low relapse rates and a low TRM. The low incidence of relapse and acceptable rates of graft-versus-host disease suggest the efficacy and safety of this approach. Further studies are warranted to validate these findings and optimize transplant strategies for older patients with myeloid malignancies.

Clonal hematopoiesis in patients with stem cell mobilization failure: a nested case-control study.

Inadequate mobilization of peripheral blood progenitor cells (PBPCs) is a limiting factor to proceeding with autologous hematopoietic cell transplantation (auto-HCT). To assess the impact of clonal hematopoiesis (CH) on mobilization failure of PBPC for auto-HCT, we investigated the characteristics of poor mobilizers (with a total PBPC collection <2 × 106 CD34+ cells per kg) in a consecutive single-center cohort of 776 patients. Targeted error-corrected next-generation sequencing of 28 genes was performed in a nested case-control cohort of 90 poor mobilizers and 89 matched controls. CH was detected in 48 out of 179 patients (27%), with most patients carrying a single mutation. The presence of CH (detected at variant allele frequency [VAF] ≥ 1%) did not associate with poor mobilization potential (31% vs 22% in controls, odds ratio, 1.55; 95% confidence interval, 0.76-3.23; P = .238). PPM1D mutations were detected more often in poor mobilizers (P = .005). In addition, TP53 mutations in this cohort were detected exclusively in patients with poor mobilization potential (P = .06). The incidence of therapy-related myeloid neoplasms (t-MN) was higher among patients with mobilization failure (P = .014). Although poor mobilizers experienced worse overall survival (P = .019), this was not affected by the presence of CH. We conclude that CH at low VAF (1%-10%) is common at the time of stem cell mobilization. TP53 mutations and PPM1D mutations are associated with poor mobilization potential and their role in subsequent development of t-MN in these individuals should be established.

Posttransplant cyclophosphamide for prevention of graft-versus-host disease: results of the prospective randomized HOVON-96 trial.

Graft-versus-host disease (GVHD) is the most important complication of allogeneic hematopoietic stem cell transplantation (alloHSCT). We performed a prospective randomized, multicenter, phase 3 trial to study whether posttransplant cyclophosphamide (PT-Cy) combined with a short course of cyclosporine A (CsA) would result in a reduction of severe GVHD and improvement of GVHD-free, relapse-free survival (GRFS) as compared with the combination of CsA and mycophenolic acid (MPA) after nonmyeloablative (NMA) matched related and unrelated peripheral blood alloHSCT. Between October 2013 and June 2018, 160 patients diagnosed with a high-risk hematological malignancy and having a matched related or at least 8 out of 8 HLA-matched unrelated donor were randomized and allocated in a 1:2 ratio to CsA/MPA or PT-Cy/CsA; a total of 151 patients were transplanted (52 vs 99 patients, respectively). The cumulative incidence of grade 2 to 4 acute GVHD at 6 months was 48% in recipients of CsA/MPA vs 30% following PT-Cy/CsA (hazard ratio [HR], 0.48; 95% confidence interval [CI], 0.29-0.82; P = .007). The 2-year cumulative incidence of extensive chronic GVHD was 48% vs 16% (HR, 0.36; 95% CI, 0.21-0.64; P < .001). The 1-year estimate of GRFS was 21% (11% to 32%) vs 45% (35% to 55%), P < .001. With a median follow-up of 56.4 months, relapse incidence, progression-free survival, and overall survival were not significantly different between the 2 treatment arms. PT-Cy combined with a short course of CsA after NMA matched alloHSCT significantly improves GRFS due to a significant reduction in severe acute and chronic GVHD.

Effect of ruxolitinib on the oral mucosa of patients with steroid-refractory chronic Graft-versus-Host disease and oral involvement.

BACKGROUND: Chronic Graft-versus-Host Disease (cGVHD) can impact quality of life, especially in patients with oral involvement. Half of the patients with cGVHD do not respond to first-line therapy with corticosteroids and calcineurin inhibitors. Ruxolitinib is effective in steroid-refractory (SR)-cGVHD cases, but the long-term effects of ruxolitinib on the oral mucosa are unknown. OBJECTIVE(S): This study aims to assess the effect of ruxolitinib on the oral mucosa of SR-cGVHD patients with oral involvement. MATERIALS AND METHODS: An observational longitudinal patient study was conducted in 53 patients with SR-cGVHD and oral involvement who were treated with ruxolitinib. The baseline condition of the oral mucosa was compared to its condition at 4 and 12 weeks after starting ruxolitinib. RESULTS: The overall response was 81% (43/53), with a complete response in 53% (28/53) and partial response in 28% (15/53) after 12 weeks (p < 0.001). Men and patients concurrently using immunosuppressive therapy responded better than women (p = 0.005) and patients with ruxolitinib monotherapy (p = 0.02), respectively. At a longer follow-up (median 20 months), oral symptoms were comparable to the 12-week symptoms (p = 0.78), regardless of ruxolitinib use (p = 0.83). CONCLUSION: Ruxolitinib treatment of SR-cGVHD patients with oral involvement was associated with a significant response of the oral manifestations at 12 weeks. CLINICAL RELEVANCE: The oral mucosa of SR-cGVHD patients is likely to improve after 4 and 12 weeks of ruxolitinib treatment. Symptom severity at baseline does not affect the response of the oral mucosa.

Eltrombopag Added to Immunosuppression in Severe Aplastic Anemia.

BACKGROUND: A single-group, phase 1-2 study indicated that eltrombopag improved the efficacy of standard immunosuppressive therapy that entailed horse antithymocyte globulin (ATG) plus cyclosporine in patients with severe aplastic anemia. METHODS: In this prospective, investigator-led, open-label, multicenter, randomized, phase 3 trial, we compared the efficacy and safety of horse ATG plus cyclosporine with or without eltrombopag as front-line therapy in previously untreated patients with severe aplastic anemia. The primary end point was a hematologic complete response at 3 months. RESULTS: Patients were assigned to receive immunosuppressive therapy (Group A, 101 patients) or immunosuppressive therapy plus eltrombopag (Group B, 96 patients). The percentage of patients who had a complete response at 3 months was 10% in Group A and 22% in Group B (odds ratio, 3.2; 95% confidence interval [CI], 1.3 to 7.8; P = 0.01). At 6 months, the overall response rate (the percentage of patients who had a complete or partial response) was 41% in Group A and 68% in Group B. The median times to the first response were 8.8 months (Group A) and 3.0 months (Group B). The incidence of severe adverse events was similar in the two groups. With a median follow-up of 24 months, a karyotypic abnormality that was classified as myelodysplastic syndrome developed in 1 patient (Group A) and 2 patients (Group B); event-free survival was 34% and 46%, respectively. Somatic mutations were detected in 29% (Group A) and 31% (Group Β) of the patients at baseline; these percentages increased to 66% and 55%, respectively, at 6 months, without affecting the hematologic response and 2-year outcome. CONCLUSIONS: The addition of eltrombopag to standard immunosuppressive therapy improved the rate, rapidity, and strength of hematologic response among previously untreated patients with severe aplastic anemia, without additional toxic effects. (Funded by Novartis and others; RACE ClinicalTrials.gov number, NCT02099747; EudraCT number, 2014-000363-40.).

Immune checkpoint inhibitor treatment induces colitis with heavy infiltration of CD8 + T cells and an infiltration pattern that resembles ulcerative colitis.

Colitis is a common, but poorly understood, adverse event of immune checkpoint inhibitors that are standard-of-care for an expanding range of cancer types. This explorative study aimed to describe the immune infiltrates in the colon from individuals developing checkpoint inhibitor colitis and compare them to well-known immunophenotypes of acute graft-versus-host disease, ulcerative colitis, and Crohn's disease. Colon biopsies (n = 20 per group) of patients with checkpoint inhibitor colitis, acute graft-versus-host disease, ulcerative colitis and Crohn's disease, all colitis treatment-naïve, and of individuals with a normal colon were analyzed using immunohistochemistry: CD8 for cytotoxic T cells, CD4 for T helper cells, and CD68 to identify cells of macrophage lineage. CD8 + T cell, CD4 + T cell, and CD68 + cell counts were performed. Cell infiltration was scored as scattered/patchy or band-like in the superficial and deep gut mucosa. Checkpoint inhibitor colitis was found to be heavily infiltrated by CD8 + T cells. Comparative analysis between groups showed that both CD8 + T cell counts (P < 0.01) and immune cell infiltration patterns in checkpoint inhibitor colitis were most similar to those observed in ulcerative colitis, with a deep band-like CD4 + T cell infiltration pattern and a superficial band-like CD68 + cell infiltration pattern in both. In conclusion, this is the first immunohistopathological study comparing infiltrate characteristics of checkpoint inhibitor colitis, acute graft-versus-host disease, ulcerative colitis, and Crohn's disease. Checkpoint inhibitor colitis samples are heterogeneous, heavily infiltrated by CD8 + T cells, and show an immune cell infiltration pattern that is more similar to ulcerative colitis than to colonic acute graft-versus-host disease or colonic Crohn's disease.

Low relapse risk in poor risk AML after conditioning with 10-day decitabine, fludarabine and 2 Gray TBI prior to allogeneic hematopoietic cell transplantation.

Patients with poor risk acute myeloid leukemia (AML) have a dismal outcome. We hypothesized that combining decitabine with a standard non-myeloablative (NMA) conditioning regimen prior to allogeneic hematopoietic cell transplantation (allo HCT), might decrease the relapse incidence. We conducted a multicenter prospective phase II study (NCT02252107) with 10-day decitabine (20 mg/m2/day) integrated in a standard non-myeloablative conditioning regimen (3 days fludarabine 30 mg/m2 with 2 Gray total body irradiation (TBI)). Patients with AML ≥ 18 years in 1st (in)complete remission (CR/CRi) with a poor or very poor risk profile, as defined by the HOVON-132 protocol, were eligible. Results: Forty-six patients (median age 60; range 23-74) were included. Median follow up time was 44 months (range 31-65 months). The cumulative 1-year incidence of relapse and NRM were respectively 23% and 11%. Incidence of grade III-IV acute graft-vs-host-disease (GVHD) and severe chronic GVHD were 13% and 20%, respectively. One-year OS was 70%. Application of ELN 2017 risk classification to the study cohort revealed a cumulative one-year relapse rate of respectively 31% and 13% for the adverse and intermediate risk patients. To conclude, the 10-day DEC/FLU/TBI conditioning regimen prior to allo HCT in poor risk AML patients is effective and feasible.

Exploratory Study of Predicted Indirectly ReCognizable HLA Epitopes in Mismatched Hematopoietic Cell Transplantations.

HLA-mismatches in hematopoietic stem-cell transplantation are associated with an impaired overall survival (OS). The aim of this study is to explore whether the Predicted Indirectly ReCognizable HLA-Epitopes (PIRCHE) algorithm can be used to identify HLA-mismatches that are related to an impaired transplant outcome. PIRCHE are computationally predicted peptides derived from the patient's mismatched-HLA molecules that can be presented by donor-patient shared HLA. We retrospectively scored PIRCHE numbers either presented on HLA class-I (PIRCHE-I) or class-II (PIRCHE-II) for a Dutch multicenter cohort of 103 patients who received a single HLA-mismatched (9/10) unrelated donor transplant in an early phase of their disease. These patients were divided into low and high PIRCHE-I and PIRCHE-II groups, based on their PIRCHE scores, and compared using multivariate statistical analysis methods. The high PIRCHE-II group had a significantly impaired OS compared to the low PIRCHE-II group and the 10/10 reference group (HR: 1.86, 95%-CI: 1.02-3.40; and HR: 2.65, 95%-CI: 1.53-4.60, respectively). Overall, PIRCHE-II seem to have a more prominent effect on OS than PIRCHE-I. This impaired OS is probably due to an increased risk for severe acute graft-vs.-host disease. These data suggest that high PIRCHE-II scores may be used to identify non-permissible HLA mismatches within single HLA-mismatched hematopoietic stem-cell transplantations.

Clinical application of a dried blood spot assay for sirolimus and everolimus in transplant patients.

Background Monitoring of immunosuppressive drugs such as everolimus and sirolimus is important in allograft rejection prevention in transplant patients. Dried blood spots (DBS) sampling gives patients the opportunity to sample a drop of blood from a fingerprick at home, which can be sent to the laboratory by mail. Methods A total of 39 sirolimus and 44 everolimus paired fingerprick DBS and whole blood (WB) samples were obtained from 60 adult transplant patients for method comparison using Passing-Bablok regression. Bias was assessed using Bland-Altman. Two validation limits were pre-defined: limits of analytical acceptance were set at >67% of all paired samples within 20% of the mean of both samples and limits of clinical relevance were set in a multidisciplinary team at >80% of all paired samples within 15% of the mean of both samples. Results For both sirolimus and everolimus, Passing-Bablok regression showed no differences between WB and DBS with slopes of 0.86 (95% CI slope, 0.72-1.02) and 0.96 (95% CI 0.84-1.06), respectively. Only everolimus showed a significant constant bias of 4%. For both sirolimus and everolimus, limits of analytical acceptance were met (76.9% and 81.8%, respectively), but limits or clinical relevance were not met (77.3% and 61.5%, respectively). Conclusions Because pre-defined limits of clinical relevance were not met, this DBS sampling method for sirolimus and everolimus cannot replace WB sampling in our center at this time. However, if the clinical setting is compatible with less strict limits for clinical relevance, this DBS method is suitable for clinical application.

Impact of Donor Type in Patients with AML Given Allogeneic Hematopoietic Cell Transplantation After Low-Dose TBI-Based Regimen.

Purpose: We assessed the impact of donor type in acute myeloid leukemia (AML) patients transplanted with 2 Gy total body irradiation (TBI)-based nonmyeloablative conditioning regimen.Patients and Methods: Data from 1,715 adult patients, with AML in CR1 or CR2 were included in this retrospective survey.Results: Donors consisted either of HLA-matched sibling donors (MSD, n = 701), 10/10 HLA-matched unrelated donors (MUD, n = 611), HLA-haploidentical donors (haplo, n = 112) or single or double umbilical cord bloods (CBT, n = 291). Chronic graft-versus-host disease (GVHD) was less frequent in CBT (28%) and in haplo (30%) patients than in MSD (50%) and MUD (51%) recipients (P < 0.001). Two-year incidence of relapse was 32%, 30%, 34%, and 34% in MSD, MUD, CBT and haplo patients, respectively (P = 0.7). Two-year overall (OS) and GVHD-free relapse-free survival (GRFS) were 59% and 29% in MSD patients, 56% and 39% in CBT recipients, 53% and 23% in MUD recipients, and 43% and 37% in haplo patients, respectively. In multivariate analyses, MUD patients had lower GRFS than MSD patients beyond day 100 (HR 1.3, P = 0.001) while CBT was associated with a better GRFS than MSD beyond day 100 (HR 0.6, P = 0.002).Conclusions: In this large cohort of AML patients transplanted following low-dose TBI-based conditioning, the relapse incidence was not affected by donor type suggesting that the intensity of GVL effects might be comparable with these four transplant approaches. Furthermore, CBT was associated with better GRFS beyond day 100 than MSD while the opposite was observed for MUD. Clin Cancer Res; 24(12); 2794-803. ©2018 AACR.

Pooled Analysis of the Prognostic Relevance of Circulating Tumor Cells in Primary Breast Cancer.

PURPOSE: Although unequivocal evidence has shown the prognostic relevance of circulating tumor cells (CTC) in the peripheral blood of patients with metastatic breast cancer, less evidence is available for the prognostic relevance of CTCs at the time of primary diagnosis. EXPERIMENTAL DESIGN: We conducted a pooled analysis of individual data from 3,173 patients with nonmetastatic (stage I-III) breast cancer from five breast cancer institutions. The prevalence and numbers of CTCs were assessed at the time of primary diagnosis with the FDA-cleared CellSearch System (Janssen Diagnostics, LLC). Patient outcomes were analyzed using meta-analytic procedures, univariate log-rank tests, and multivariate Cox proportional hazard regression analyses. The median follow-up duration was 62.8 months. RESULTS: One or more CTCs were detected in 20.2% of the patients. CTC-positive patients had larger tumors, increased lymph node involvement, and a higher histologic tumor grade than did CTC-negative patients (all P < 0.002). Multivariate Cox regressions, which included tumor size, nodal status, histologic tumor grade, and hormone receptor and HER2 status, confirmed that the presence of CTCs was an independent prognostic factor for disease-free survival [HR, 1.82; 95% confidence interval (CI), 1.47-2.26], distant disease-free survival (HR, 1.89; 95% CI, 1.49-2.40), breast cancer-specific survival (HR, 2.04; 95% CI, 1.52-2.75), and overall survival (HR, 1.97; 95% CI, 1.51-2.59). CONCLUSIONS: In patients with primary breast cancer, the presence of CTCs was an independent predictor of poor disease-free, overall, breast cancer-specific, and distant disease-free survival. Clin Cancer Res; 22(10); 2583-93. ©2016 AACR.

Importance of circulating tumor cells in newly diagnosed colorectal cancer.

Presence of circulating tumor cells (CTC) is associated with poor prognosis in patients with metastatic colorectal cancer (CRC). The present study was conducted to determine if the presence of CTC prior to surgery and during follow­up in patients with newly diagnosed non-metastatic CRC can identify patients at risk for disease recurrence. In a prospective single center study 183 patients with newly diagnosed non-disseminated CRC, scheduled for surgery, were enrolled and followed-up for a median of 5.1 years. CTC were enumerated with the CellSearch system in 4 aliquots of 7.5 ml of blood before surgery and at several time-points during follow-up after surgery. The results showed that ≥1 CTC/30 ml of blood were detected in 44 (24%) patients before surgery. Patients with CTC before surgery had a significant decrease in recurrence-free survival (RFS, log-rank test p=0.014) and colon cancer related survival (CCRS, p=0.002). The 5-year RFS dropped from 75 to 61% and the 5-year CCRS from 83 to 69% for patients with CTC before surgery. The presence of CTC and positive lymph nodes remained significant factors in multivariate analysis for recurrence-free survival (RFS). Surprisingly, the presence of CTC weeks after surgery was not significantly associated with RFS and CCRD whereas CTC 2-3 years after surgery was again significantly associated with RFS and CCRD. The presence of CTC in patients with stage I-III CRC before surgery is associated with a significant reduction in RFS and CCRS. These findings suggest a role of CTC detection to assess which patients need adjuvant treatment.

Circulating tumor cells before and during follow-up after breast cancer surgery.

The presence of circulating tumor cells (CTC) is an independent prognostic factor for progression-free and overall survival for patients with metastatic and newly diagnosed breast cancer. The present study was undertaken to explore whether the presence of CTC before and during follow-up after surgery is associated with recurrence free survival (RFS) and overall survival (OS). In a prospective single center study, CTC were enumerated with the CellSearch system in 30 ml of peripheral blood of 403 stage I-III patients before undergoing surgery for breast cancer (A) and if available 1 week after surgery (B), after adjuvant chemo- and/or radiotherapy or before start of long-term hormonal therapy (C), one (D), two (E) and three (F) years after surgery. Patients were stratified into unfavorable (CTC≥1) and favorable (CTC=0) prognostic groups. >1 CTC in 30 ml blood was detected in 75/403 (19%) at A, 66/367 (18%) at B, 40/263 (15%) at C, 30/235 (12%) at D, 18/144 (11%) at E and 11/83 (13%) at F. RFS and OS was significantly lower for unfavorable CTC as compared to favorable CTC before surgery (p=0.022 and p=0.006), after adjuvant therapy (p<0.001 and p=0.018) and one (p=0.006 and p=0.013) and two (p<0.001 and p=0.045) years after surgery, but not 1 week post-surgery. The presence of CTC in blood drawn pre and one and two years after surgery, but not post-surgery is associated with shorter RFS and OS for stage I-III breast cancer.

Circulating Tumor Cells Count and Morphological Features in Breast, Colorectal and Prostate Cancer.

BACKGROUND: Presence of circulating tumor cells (CTC) in patients with metastatic breast, colorectal and prostate cancer is indicative for poor prognosis. An automated CTC (aCTC) algorithm developed previously to eliminate the variability in manual counting of CTC (mCTC) was used to extract morphological features. Here we validated the aCTC algorithm on CTC images from prostate, breast and colorectal cancer patients and investigated the role of quantitative morphological parameters. METHODOLOGY: Stored images of samples from patients with prostate, breast and colorectal cancer, healthy controls, benign breast and colorectal tumors were obtained using the CellSearch system. Images were analyzed for the presence of aCTC and their morphological parameters measured and correlated with survival. RESULTS: Overall survival hazard ratio was not significantly different for aCTC and mCTC. The number of CTC correlated strongest with survival, whereas CTC size, roundness and apoptosis features reached significance in univariate analysis, but not in multivariate analysis. One aCTC/7.5 ml of blood was found in 7 of 204 healthy controls and 9 of 694 benign tumors. In one patient with benign tumor 2 and another 9 aCTC were detected. SIGNIFICANCE OF THE STUDY: CTC can be identified and morphological features extracted by an algorithm on images stored by the CellSearch system and strongly correlate with clinical outcome in metastatic breast, colorectal and prostate cancer.

Circulating tumor cells, disease recurrence and survival in newly diagnosed breast cancer.

INTRODUCTION: The presence of circulating tumor cells (CTC) is an independent prognostic factor for progression-free survival and breast cancer-related death (BRD) for patients with metastatic breast cancer beginning a new line of systemic therapy. The current study was undertaken to explore whether the presence of CTC at the time of diagnosis was associated with recurrence-free survival (RFS) and BRD. METHODS: In a prospective single center study, CTC were enumerated with the CellSearch system in 30 ml of peripheral blood of 602 patients before undergoing surgery for breast cancer. There were 97 patients with a benign tumor, 101 did not meet the inclusion criteria of which there were 48 patients with DCIS, leaving 404 stage I to III patients. Patients were stratified into unfavorable (CTC ≥1) and favorable (CTC = 0) prognostic groups. RESULTS: ≥1 CTC in 30 ml blood was detected in 15 (15%) benign tumors, in 9 DCIS (19%), in 28 (16%) stage I, 32 (18%) stage II and in 16 (31%) patients with stage III. In stage I to III patients 76 (19%) had ≥1 CTC of whom 16 (21.1%) developed a recurrence. In 328 patients with 0 CTC 38 (11.6%) developed a recurrence. Four-year RFS was 88.4% for favorable CTC and 78.9% for unfavorable CTC (P = 0.038). A total of 25 patients died of breast cancer-related causes and 11 (44%) had ≥1 CTC. BRD was 4.3% for favorable and 14.5% for unfavorable CTC (P = 0.001). In multivariate analysis ≥1 CTC was associated with distant disease-free survival, but not for overall recurrence-free survival. CTC, progesterone receptor and N-stage were independent predictors of BRD in multivariate analysis. CONCLUSIONS: Presence of CTC in breast cancer patients before undergoing surgery with curative intent is associated with an increased risk for breast cancer-related death.

Heavy chain myosin 9-related disease (MYH9 -RD): neutrophil inclusions of myosin-9 as a pathognomonic sign of the disorder.

MYH9-related disease ( MYH9-RD) is an autosomal dominant thrombocytopenia with giant platelets variably associated with young-adult onset of progressive sensorineural hearing loss, presenile cataract, and renal damage. MYH9-RD is caused by mutations of MYH9 , the gene encoding for non-muscle heavy-chain myosin-9. Wild-type and mutant myosin-9 aggregate as cytoplasmic inclusions in patients' leukocytes, the identification of which by immunofluorescence has been proposed as a suitable tool for the diagnosis of MYH9-RD. Since the predictive value of this assay, in terms of sensitivity and specificity, is unknown, we investigated 118 consecutive unrelated patients with a clinical presentation strongly consistent with MYH9-RD. All patients prospectively underwent both the immunofluorescence assay for myosin-9 aggregate detection and molecular genetic analysis of the MYH9 gene. Myosin-9 aggregates were identified in 82 patients, 80 of which (98%) had also a MYH9 mutation. In the remaining 36 patients neither myosin-9 aggregates nor MYH9 mutations were found. Sensitivity and specificity of the immunofluorescence assay was evaluated to be 100% and 95%, respectively. Except for the presence of aggregates, we did not find any other significant difference between patients with or without aggregates, demonstrating that the myosin-9 inclusions in neutrophils are a pathognomonic sign of the disease. However, the identification of the specific MYH9 mutation is still of importance for prognostic aspects of MYH9-RD.