Toru Motokura

Prospective Comparison of the Diagnostic Potential of Real-Time PCR, Double-Sandwich Enzyme-Linked Immunosorbent Assay for Galactomannan, and a (1→3)-β-d-Glucan Test in Weekly Screening for Invasive Aspergillosis in Patients with Hematological Disorders

ABSTRACT The establishment of an optimal noninvasive method for diagnosing invasive aspergillosis (IA) is needed to improve the management of this life-threatening infection in patients with hematological disorders, and a number of noninvasive tests for IA that target different fungal components, including galactomannan, (1→3)-β-d-glucan (BDG), and Aspergillus DNA, have been developed. In this study, we prospectively evaluated the diagnostic potential of three noninvasive tests for IA that were used in a weekly screening strategy: the double-sandwich enzyme-linked immunosorbent assay (ELISA) for galactomannan (Platelia Aspergillus), a real-time PCR assay for Aspergillus DNA (GeniQ-Asper), and an assay for BDG (β-glucan Wako). We analyzed 149 consecutive treatment episodes in 96 patients with hematological disorders who were at high risk for IA and diagnosed 9 proven IA cases, 2 probable IA cases, and 13 possible invasive fugal infections. In a receiver-operating characteristic (ROC) analysis, the area under the ROC curve was greatest for ELISA, using two consecutive positive results (0.97; P = 0.036 for ELISA versus PCR, P = 0.055 for ELISA versus BDG). Based on the ROC curve, the cutoff for the ELISA could be reduced to an optical density index (O.D.I.) of 0.6. With the use of this cutoff for ELISA and cutoffs for PCR and BDG that give a comparable level of specificity, the sensitivity/specificity/positive predictive value/negative predictive value of the ELISA and the PCR and BDG tests were 1.00/0.93/0.55/1.00, 0.55/0.93/0.40/0.96, and 0.55/0.93/0.40/0.96, respectively. In conclusion, among these weekly screening tests for IA, the double-sandwich ELISA test was the most sensitive at predicting the diagnosis of IA in high-risk patients with hematological disorders, using a reduced cutoff of 0.6 O.D.I.

Parathyroid hormone-related protein in adult T-cell leukemia-lymphoma

OBJECTIVE To determine whether parathyroid hormone-related protein is synthesized and secreted by the tumor cells of patients with adult T-cell leukemia-lymphoma. DESIGN AND PATIENTS Convenience sample of three patients with adult T-cell leukemia-lymphoma. Two patients developed hypercalcemia, and one patient was normocalcemic. SETTING Inpatient facilities at two university-affiliated medical centers. INTERVENTION All patients had a lymph node biopsy. In addition, samples of ascitic or pleural fluid, or both, were obtained from these patients. MEASUREMENTS AND RESULTS Using RNA blot analysis, we showed that parathyroid hormone-related protein (PTHrP) messenger RNAs (mRNAs) were constitutively expressed in the tumor cells from all patients. Cyclic adenosine monophosphate (cAMP) production-stimulating activity, assessed using osteoblast-like UMR106 cells, was demonstrated in the pleural and ascitic fluids from the two patients who developed hypercalcemia. The elution profiles of the cAMP production-stimulating activity in the ascitic fluid extracts were very similar to those of the tumor extracts from hypercalcemic nude rats that had been implanted with a human cancer tumor. CONCLUSIONS Parathyroid hormone-related protein is produced by tumor cells in adult T-cell leukemia-lymphoma, which may be an important factor in the development of hypercalcemia in the patients with this disease. However, the development of hypercalcemia may depend on other factors such as the number of tumor cells, access of the protein into systemic circulation, and the presence of some additional substances.

Long‐term ultra‐low‐dose acyclovir against varicella‐zoster virus reactivation after allogeneic hematopoietic stem cell transplantation

To evaluate the efficacy of long‐term prophylaxis with ultra‐low‐dose acyclovir against varicella‐zoster virus (VZV) reactivation, we analyzed the records of 242 Japanese adult patients who underwent allogeneic hematopoietic stem cell transplantation for the first time from 1995 to 2006 at our hospital. We started long‐term oral acyclovir at 200 mg/day in July 2001. Acyclovir was continued until the end of immunosuppressive therapy and at least 1 year after transplantation. Sixty‐six patients developed VZV reactivation at a median of 248 days after HSCT, with a cumulative incidence of 34.7%. Only one breakthrough reactivation occurred during long‐term acyclovir, which responded well to therapeutic dose of valacyclovir. The use of long‐term acyclovir was the only independent determinant that significantly decreased the overall incidence of VZV reactivation (20% vs. 50%, P < 0.0001). With this prophylaxis, visceral dissemination and serious complications other than post‐herpetic neuralgia was completely eliminated, and thereby need for hospitalization was significantly reduced (21% vs. 71%, P = 0.0034). Fifteen of the 57 patients who discontinued acyclovir developed VZV reactivation, with a cumulative incidence of 32.1%. VZV reactivation following discontinuation tended to occur in patients who were receiving immunosuppressive therapy at the cessation of acyclovir. These findings suggested that long‐term prophylaxis of ultra‐low‐dose acyclovir resulted in a successful prevention of severe VZV‐related symptoms and death, with a significantly decreased overall incidence of VZV reactivation. Prolongation of prophylactic acyclovir on profound immunosuppression might be important for thorough suppression of VZV reactivation. Am. J. Hematol., 2008.

Expression of parathyroid hormone-related protein in a human T cell lymphotrophic virus type I-infected T cell line

We analyzed human T cell lymphotrophic virus type I (HTLV-I)-infected T cells for the presence of mRNA coding for parathyroid hormone-related protein (PTHrP) by Northern blotting using synthetic DNA probes. We report here that PTHrP mRNAs were detected in a HTLV-I-infected T cell line, MT-2, but not in uninfected T cell or B cell lines, and that PTH-like bioactivity was detected only in the conditioned medium of MT-2 cells. Our study suggests that the pathophysiology of hypercalcemia in patients with adult T cell leukemia/lymphoma may resemble that which occurs with solid tumors.

Expression of p16INK4A and p14ARF in hematological malignancies

The INK4A/ARF locus yields two tumor suppressors, p16INK4A and p14ARF, and is frequently deleted in human tumors. We studied their mRNA expressions in 41 hematopoietic cell lines and in 137 patients with hematological malignancies; we used a quantitative reverse transcription-PCR assay. Normal peripheral bloods, bone marrow and lymph nodes expressed little or undetectable p16INK4A and p14ARF mRNAs, which were readily detected in 12 and 17 of 41 cell lines, respectively. Patients with hematological malignancies frequently lacked p16INK4A expression (60/137) and lost p14ARF expression less frequently (19/137, 13.9%). Almost all patients without p14ARF expression lacked p16INK4A expression, which may correspond to deletions of the INK4A/ARF locus. Undetectable p16INK4A expression with p14ARFexpression in 41 patients may correspond to p16INK4A promoter methylation or to normal expression status of the p16INK4A gene. All patients with follicular lymphoma (FL), myeloma or acute myeloid leukemia (AML) expressed p14ARF while nine of 23 patients with diffuse large B cell lymphoma (DLBCL) lost p14ARF expression. Patients with ALL, AML or blast crisis of chronic myelogenous leukemia expressed abundant p16INK4A mRNAs more frequently than patients with other diseases (12/33 vs 6/104, P < 0.01). patients with fl and high p14ARF expression had a significantly shorter survival time while survival for patients with DLBCL and increased p14ARFexpression tended to be longer. These observations indicate that p16INK4A and p14ARF expression is differentially affected among hemato- logical malignancies and that not only inactivation but also increased expression may have clinical significance.

Reverse seroconversion of Hepatitis B virus after hematopoietic stem cell transplantation

Hepatitis B virus (HBV) reactivation in patients previously positive for hepatitis B surface antibody (HBsAb), so-called reverse seroconversion, has been considered to be a rare complication after hematopoietic stem cell transplantation (HSCT). We experienced two patients who developed reverse seroconversion among nine who were HBsAb positive and Hepatitis B core antibody (HBcAb) positive before HSCT; one after autologous bone marrow transplantation (BMT) and another after allogeneic peripheral blood stem cell transplantation (PBSCT). We reviewed the literature and considered that reverse seroconversion of HBV after HSCT is not uncommon among HBsAb positive recipients. The use of corticosteroids, the lack of HBsAb in donor, and a decrease in serum HBsAb and HBcAb levels may predict reverse seroconversion after HSCT.

Male predominance among Japanese adult patients with late-onset hemorrhagic cystitis after hematopoietic stem cell transplantation.

Summary:Late-onset hemorrhagic cystitis (LHC) after hematopoietic stem cell transplantation (HSCT) is mainly caused by viral infections. We retrospectively analyzed the records of 141 Japanese adult patients who underwent a first allogeneic HSCT from 1995 to 2002. In all, 19 patients developed LHC a median of 51 days after HSCT. Adenovirus (AdV) was detected in the urine of 10 LHC patients, of whom eight had AdV type 11. Five of the six available serum samples from these patients were also positive for AdV type 11, but the detection of AdV in serum was not associated with a worse outcome. Male sex and the development of grade II–IV acute graft-versus-host disease were identified as independent significant risk factors for LHC. Male predominance was detected in LHC after HSCT, as has been previously shown in children with AdV-induced acute HC. The detection of AdV DNA in serum did not predict a poor outcome.

Predictors for severe cardiac complications after hematopoietic stem cell transplantation

Summary:The value of pre-transplant factors for predicting the development of cardiac complications after transplantation has been inconsistent among studies. We analyzed the impact of pre-transplant factors on the incidence of severe cardiac complications in 164 hematopoietic stem cell transplant recipients. We identified eight patients (4.8%) who experienced grade III or IV cardiac complications according to the Bearman criteria. Seven died of cardiac causes a median of 3 days after the onset of cardiac complications. On univariate analysis, both the cumulative dose of anthracyclines and the use of anthracyclines within 60 days before transplantation affected the incidence of severe cardiac complications (P=0.0091 and 0.011). The dissociation of heart rate and body temperature, which reflects ‘relative tachycardia’, was also associated with a higher incidence of cardiac complications (P=0.024). None of the variables obtained by electrocardiography or echocardiography were useful for predicting cardiac complications after transplantation, although the statistical power might not be sufficient to detect the usefulness of ejection fraction. On a multivariate analysis, the cumulative dose of anthracyclines was the only independent significant risk factor for severe cardiac complications. We conclude that the cumulative dose of anthracyclines is the most potent predictor of cardiac complications and the administration of anthracyclines should be avoided within two months before transplantation.

High-grade cytomegalovirus antigenemia after hematopoietic stem cell transplantation

Summary:Clinical impact of high-grade (HG) cytomegalovirus (CMV) antigenemia after hematopoietic stem cell transplantation has not been clarified. Therefore, in order to investigate the risk factors and outcome for HG-CMV antigenemia, we retrospectively analyzed the records of 154 Japanese adult patients who underwent allogeneic hematopoietic stem cell transplantation for the first time from 1995 to 2002 at the University of Tokyo Hospital. Among 107 patients who developed positive CMV antigenemia at any level, 74 received risk-adapted preemptive therapy with ganciclovir (GCV), and 17 of these developed HG-antigenemia defined as ⩾50 positive cells per two slides. The use of systemic corticosteroids at ⩾0.5 mg/kg/day at the initiation of GCV was identified as an independent significant risk factor for HG-antigenemia. Seven of the 17 HG-antigenemia patients developed CMV disease, with a cumulative incidence of 49.5%, which was significantly higher than that in the low-grade antigenemia patients (4%, P<0.001). However, overall survival was almost equivalent in the two groups. In conclusion, the development of HG-antigenemia appeared to depend on the profound immune suppression of the recipient. Although CMV disease frequently developed in HG-antigenemia patients, antiviral therapy could prevent a fatal outcome.

Graft-versus-tumor effect against advanced pancreatic cancer after allogeneic reduced-intensity stem cell transplantation.

Background. The prognosis of advanced pancreatic cancer is extremely poor and therefore a novel treatment strategy is desired. The authors thus started a prospective study of allogeneic reduced-intensity hematopoietic stem cell transplantation (RIST) for patients with advanced pancreatic cancer to evaluate the feasibility and efficacy of this approach for such patients. Methods. Only patients with pathologically proven pancreatic cancer that was locally advanced or metastatic and not amenable to curative resection were included. The conditioning regimen consisted of gemcitabine, fludarabine, and busulfan. Results. In the first stage of this study, the authors treated seven patients. Treatment-related mortality before day 100 was observed in one patient. The median survival after RIST was 229 days. An objective response on computed tomographic scan was observed in two patients and another had a tumor marker response. Marked tumor shrinkage was observed in one of the remaining patients after donor lymphocyte infusion. These antitumor effects appeared after the effect of the conditioning regimen had disappeared. In addition, some of these responses were associated with an increase in the serum anticarcinoembryonic antigen antibody level. Conclusions. Pancreatic cancer appeared to be sensitive to a graft-versus-tumor effect; therefore, a larger clinical study with a refined strategy is warranted.