The challenging task of enumerating blasts in the bone marrow.

Enumeration of blasts in the bone marrow is critical for diagnostic, prognostic, and therapeutic response evaluation in myelodysplastic syndromes, myeloproliferative neoplasms and acute leukemias. However, few studies have examined the accuracy and precision of marrow blast counting using standard microscopic procedures. In our study, 4 experienced hematopathologists evaluated blast percentages in marrow using either differential counts on aspirate smears or visual estimates on CD34-stained trephine biopsies. Results of an independent observer's manual counts of individual labeled and unlabeled cells performed on high resolution digital images of CD34-stained trephine biopsies were designated as the "Digital Reference." Hematopathologists' blast counts showed excellent interobserver reproducibility, but the counts in smears and trephine biopsies correlated poorly with each other. Compared to the Digital Reference, both smear and trephine evaluations showed positive bias and high variability. The biopsy showed less variability but higher positive bias relative to the smears, indicating that counts were overestimated more in the hematopathologists' biopsy evaluation. Flow cytometric counts correlated well with the Digital Reference, and cases with high blast count generally showed worse cytogenetic findings. Our results demonstrate the need for better counting methods if significant decisions are made based on microscopic enumeration of blasts. Further efforts should be made to develop markers to better define blast cells and perhaps incorporate automated digital imaging technologies to enumerate them. Also, consideration should be given to quantifying blasts per marrow area in biopsies instead of per nucleated cells.

Validation of rapid polymerase chain reaction-based detection of all length polymorphisms in the UGT 1A1 gene promoter.

UDP glucuronosyltransferase (UGT) 1A1 gene promoter polymorphism can affect the expression level of the UGT 1A1 enzyme. The polymorphism consists of an insertion of a TA nucleotide sequence into a (TA)6TAA sequence in the gene promoter resulting in (TA)7TAA (UGT1A1*28). This results in a reduced UGT 1A1 expression with 70% less glucuronidation capacity for bilirubin and other UGT1A1 substrates. Other polymorphisms include (TA)8TAA (UGT1A1*37) and (TA)5TAA (UGT1A1*36). The longer the TA repeats the lower the enzyme expression level. The anticancer agent irinotecan is metabolized to the active SN-38, which is further glucuronidated and detoxified by UGT 1A1. Decreased glucuronidation leads to SN-38 accumulation with severe neutropenia and diarrhea. We have developed a rapid polymerase chain reaction (PCR)-based detection of all length polymorphisms in the UGT 1A1 gene promoter. It uses PCR and DNA fragment analysis using an ABI Genetic Analyzer. Thirty-two blood samples were analyzed for UGT 1A1 promoter polymorphism. We found 2 (TA)(5)TAA/(TA)(5)TAA, 4 (TA)(5)TAA/(TA)(6)TAA, 2 (TA)(5)TAA/(TA)(7)TAA, 9 (TA)(6)TAA/(TA)(6)TAA, 11 (TA)(6)TAA/(TA)(7)TAA, 2 (TA)(7)TAA/(TA)(7)TAA, and 2 (TA)(7)TAA/(TA)(8)TAA in our sample group. To confirm the results, 6 samples with different repeats were also analyzed by DNA sequencing method. This is a rapid and reliable method for analysis of the promoter length polymorphisms of UGT 1A1 gene.

Germinal center and activated b-cell profiles separate Burkitt lymphoma and diffuse large B-cell lymphoma in AIDS and non-AIDS cases.

Morphologic features of Burkitt lymphoma (BL) and diffuse large B-cell lymphoma (DLBCL) overlap. No single phenotypic marker or molecular abnormality is pathognomonic. We tested a panel of 8 germinal center (GC) and activated B-cell (ABC) markers for their ability to separate BL and DLBCL. We diagnosed 16 BL and 39 DLBCL cases from 21 patients with AIDS and 34 without AIDS based on traditional morphologic criteria, Ki-67 proliferative index, and c-myc rearrangement (fluorescence in situ hybridization). After immunohistochemically staining tissue microarrays of BL and DLBCL for markers of GC (bcl-6, CD10, cyclin H) and ABC (MUM1, CD138, PAK1, CD44, bcl-2), we scored each case for the percentage of positive cells. Hierarchical clustering yielded 2 major clusters significantly associated with morphologic diagnosis (P < .001). For comparison, we plotted the sum of the GC scores and ABC scores for each case as x and y data points. This revealed a high-GC/low-ABC group and a low-GC/high-ABC group that were associated significantly with morphologic diagnosis (P < .001). Protein expression of multiple GC and ABC markers can separate BL and DLBCL.

Panniculitis, infection, and dermatomyositis: case and literature review.

The most common cause of panniculitis (inflammation of the subcutaneous adipose tissue) is infection. Clinical panniculitis in dermatomyositis is rare. We found in the English literature 24 cases, including ours. Six cases involved children. Buttocks or thighs and arms were involved more frequently. Lobular panniculitis with lymphoplasmacytic infiltration was the usual pathology. Calcifications of the panniculus were found in 6/24 (25%) of the cases. Membrano-cystic changes were associated with worse prognosis. Sixteen of 18 cases (89%) without associated infection, responded to increased immunosuppression. Eighty-nine percent responded to steroids alone. Intravenous immune globulin (IVIG) was effective in steroid-resistant cases. No spontaneous improvement was reported. Three cases were associated with infection; Staphylococcus aureus in two and Mycobacterium chelonae in one. All three responded to antibiotics with simultaneous decrease of the immunosuppressive therapy. Concomitant infection may play a role in the worsening of panniculitis and needs to be aggressively identified and treated.

AIDS and non-AIDS diffuse large B-cell lymphomas express different antigen profiles.

Based on gene expression profiling, diffuse large B-cell lymphomas arising in immunocompetent patients can be divided into germinal center and activated B-cell types. Since little is known about acquired immunodeficiency syndrome associated diffuse large B-cell lymphomas, we tested whether the protein expression of germinal center and activated B-cell markers differed between acquired immunodeficiency syndrome (AIDS) vs non-AIDS diffuse large B-cell lymphomas. We immunohistochemically stained tissue microarrays of 39 de novo diffuse large B-cell lymphomas: 12 AIDS associated and 27 non-AIDS, with germinal center (BCL6, CD10, CyclinH) and activated B-cell markers (MUM1, CD138, PAK1, CD44, BCL2). We scored each case for percent positive cells (0-19%=0; 20-49%=1; 50-100%=2). The activated B-cell and germinal center summation scores of each case were used as (x, y) coordinate data points to construct two-dimensional contour-frequency plots. The contour plot of non-AIDS diffuse large B-cell lymphomas showed two distinct clusters: a cluster with a high germinal center phenotype (cluster 1) and a cluster with a high activated B-cell phenotype (cluster 3). In contrast, the AIDS-related diffuse large B-cell lymphomas formed a single aggregate (cluster 2) (P=0.02, Fisher exact test). When the contour plots of the AIDS-related and the non-AIDS cases were superimposed, cluster 2 of the AIDS cases expressed an intermediate germinal center/activated B-cell phenotype compared to clusters 1 and 3 of the non-AIDS diffuse large B-cell lymphomas. Our results confirm that non-AIDS diffuse large B-cell lymphomas segregate into two groups with either germinal center or activated B-cell phenotype. We report the new finding that the AIDS status of the patient predicts the immunophenotype of the diffuse large B-cell lymphomas.