We have developed a robust algorithm for copy number analysis of the human genome using high-density oligonucleotide microarrays containing 116,204 single-nucleotide polymorphisms. The advantages of this algorithm include the improvement of signal-to-noise (S/N) ratios and the use of an optimized reference. The raw S/N ratios were improved by accounting for the length and GC content of the PCR products using quadratic regressions. The use of constitutional DNA, when available, gives the lowest SD values (0.16 F 0.03) and also enables allele-based copy number detection in cancer genomes, which can unmask otherwise concealed allelic imbalances. In

Loss of heterozygosity (LOH), either with or without accompanying copy-number loss, is a cardinal feature of cancer genomes that is tightly linked to cancer development. However, detection of LOH is frequently hampered by the presence of normal cell components within tumor specimens and the limitation in availability of constitutive DNA. Here, we describe a simple but highly sensitive method for genomewide detection of allelic composition, based on the Affymetrix single-nucleotide-polymorphism genotyping microarray platform, without dependence on the availability of constitutive DNA. By sensing subtle distortions in allele-specific signals caused by allelic imbalance with the use of anonymous controls, sensitive detection of LOH is enabled with accurate determination of allele-specific copy numbers, even in the presence of up to 70%-80% normal cell contamination. The performance of the new algorithm, called "AsCNAR" (allele-specific copy-number analysis using anonymous references), was demonstrated by detecting the copy-number neutral LOH, or uniparental disomy (UPD), in a large number of acute leukemia samples. We next applied this technique to detection of UPD involving the 9p arm in myeloproliferative disorders (MPDs), which is tightly associated with a homozygous JAK2 mutation. It revealed an unexpectedly high frequency of 9p UPD that otherwise would have been undetected and also disclosed the existence of multiple subpopulations having distinct 9p UPD within the same MPD specimen. In conclusion, AsCNAR should substantially improve our ability to dissect the complexity of cancer genomes and should contribute to our understanding of the genetic basis of human cancers.

The Smad family proteins are critical components of the transforming growth factor (TGF)-b signaling pathway. TGF-b is a multipotent cytokine that elicits many biological functions. In particular, TGF-b exhibits e ects on the cell cycle manifested by G1-phase arrest, di erentiation, or apoptosis of several target cells, suggesting that disruption of TGF-b signaling pathway could be involved in cancer formation. Here we show one missense mutation of the Smad4 gene in the MH1 domain (P102L) and one frame shift mutation resulting in termination in the MH2 domain (D(483 ± 552)) in acute myelogeneous leukemia. Both of the mutated Smad4 proteins lack transcriptional activities. Concomitant expression of the P102L mutant with wild-type Smad4 inactivates wild-type Smad4 through inhibiting its DNA-binding ability. The D(483 ± 552) mutant blocks nuclear translocation of wild-type Smad4 and thus disrupts TGF-b signaling. This is the ®rst report showing that mutations in the Smad4 gene are associated with the pathogenesis of acute myelogeneous leukemia and the obtained results should provide useful insights into the mechanism whereby disruption of TGF-b signaling pathway could lead to acute myelogeneous leukemia. Oncogene (2001) 20, 88 ± 96.

Iron overload might be an important contributor to nonrelapse mortality (NRM) in hematopoietic stem cell transplantation (HSCT). We studied 264 patients undergoing allogeneic HSCT for hematologic malignancies between 1996 and 2006, using pretransplantation serum ferritin as a surrogate marker of iron overload. At 5 years, patients in the high ferritin group (>or= 599 ng/mL) had a lower overall survival (OS; 33.0% versus 63.5%; P< .001) and a higher NRM (34.9% versus 13.7%; P< .001) than those in the low ferritin group (<599 ng/mL). Multivariate analyses showed that high pretransplantation serum ferritin was a significant risk factor for worse survival (relative risk [RR]=1.68; P= .05) and increased NRM (RR=2.47; P= .01). There was no significant difference in the cumulative incidence of relapse, and acute and chronic graft-versus-host disease (aGVHD, cGVHD) between the 2 groups. Patients in the high ferritin group were more likely to die of infection (P< .010) and organ failure (P< .019). Similar results were observed after dividing the patients according to the intensity of conditioning regimens. These findings emphasize the prognostic impact of pretransplantation serum ferritin in HSCT recipients.

We investigated the role of therapeutic dose monitoring (TDM) in the treatment of fungal infections with voriconazole through 49 analyses of 34 patients who received treatment for hematologic diseases. Voriconazole concentration was highly variable among patients regardless of renal, liver functions, or age, and the effect of dose enhancement was not constant. This indicates the difficulty of predicting voriconazole concentration without TDM. We evaluated the outcome with the composite assessment system where patients were assumed non-responders when they failed to show improvement in at least 2 of the following 3 criteria: clinical, radiologic, and mycologic. We showed that concentration-response relationship depended on the status of underlying hematologic diseases; this relationship was observed only in cases without refractory hematologic diseases, but not in those with refractory diseases. In the former group, cases with >2 mg/L of concentration were associated with good response to voriconazole. On the other hand, elevation of hepatic enzyme was frequently observed when voriconazole concentration was >6 mg/L. From these results, we concluded that TDM should be executed and targeted to 2-6 mg/L to improve efficacy and to avoid side effects.