Postrenal transplant non-EBV multiple myeloma of donor origin.

Multiple myeloma occurring after solid organ transplantation is a rare condition, with only a few case reports found in the literature. We report a case of Epstein-Barr virus-negative, posttransplant multiple myeloma in a 67-year-old female, presenting 18 months after renal transplantation. Interestingly, fluorescence in situ hybridization analysis of the tumor revealed a Y chromosome in the majority of the cells, indicating that the neoplasm was derived from the donor kidney. To our knowledge, this represents the first reported case with these features.

An evaluation of the cell-dyn 3200 for counting cells in cerebrospinal and other body fluids.

This study compared the white blood cell (WBC) and red blood cell (RBC) counts obtained with the Cell-Dyn 3200 (CD 3200) with results obtained by hemocytometer, the reference method for counting cerebrospinal fluid (CSF) and other body fluid specimens. Ninety-six CSF and 65 body fluid specimens were evaluated. Background counts were maintained on the CD 3200 at 0.001 x 10(9)/L and 0.00 x 10(12)/L for WBC and RBC counts, respectively. Linearity and precision were acceptable for both the total nucleated cell (TNC) count and the RBC count. The CD 3200 WBC optical count was correlated with the TNC count obtained by the manual reference method for CSF specimens across the range of 0 x 10(9 )/L to 7.863 x 10(9)/L (r2 = 0.9867) and for body fluid specimens across the range of 0 x 10(9)/L to 14.0 x 10(9)/L (r2 = 0.9955). An r2 value of 0.9016 was obtained for the 82 CSF specimens with manual TNC counts of <0.200 x 10(9)/L. Analysis of the CSF and body fluid specimens indicated that automated RBC counts could be reported at > or = 0.003 x 10(12)/L. In this study, 7 CSF and 30 body fluid specimens had RBC counts of >0.003 x 10(12)/L, and there was good agreement with manual RBC counts, with r2 values of 0.9893 and 0.9960 obtained for CSF and other body fluids, respectively. The CD 3200 in our experience has a lower reportable range than the ranges of most automated cell counters reported in the literature. In contrast to the only other instrument with comparable reportable ranges, the CD 3200 requires a smaller sample volume without any special sample preparation, reagents, or software. By using the CD 3200 with our laboratory-specific rules for agreement between duplicate counts, we would be able to reduce our manual CSF specimen counts from 192 TNC and 192 RBC counts to 2 TNC and 178 RBC counts. For body fluid specimens, our manual counts would be reduced from 130 TNC and 130 RBC counts to 10 TNC and 4 RBC counts.

A distinctive nuclear morphology in acute myeloid leukemia is strongly associated with loss of HLA-DR expression and FLT3 internal tandem duplication.

In a 5-year survey of nonpromyelocytic/nonmonocytic acute myeloid leukemias (AMLs) diagnosed in the University of Washington Hematopathology Laboratory, we identified 19 cases containing distinctive, cup-like nuclear indentation in 10% or more of the blasts ('AML-cuplike'). Fourteen of these cases (74%) demonstrated near-complete loss of HLA-DR expression, while the other five cases showed partial loss of HLA-DR. A total of 16 of the cases (84%) demonstrated internal tandem duplication (ITD) of the Flt3 gene. When compared to a selected set of AMLs lacking this nuclear morphology, AML-cuplike was significantly more likely to lack HLA-DR and CD34 expression, to express CD123 without CD133, to have a normal karyotype, and to harbor the Flt3 ITD. To characterize AML-cuplike in an unselected series of AMLs, we analyzed 42 consecutive nonpromyelocytic/nonmonocytic AMLs diagnosed in our laboratory during a 6-month period in 2002. Strikingly, in this unselected series, there was a statistically significant coincidence of invaginated nuclear morphology, loss of HLA-DR, and presence of the Flt3 ITD beyond that expected if these three features were unrelated, suggesting that AMLs with these three features may represent a distinct AML subset.

Identification of genes whose expression patterns differ in benign lymphoid tissue and follicular, mantle cell, and small lymphocytic lymphoma.

Improved methods for diagnosing small B-cell lymphomas (SBCLs) and predicting patient response to therapy are likely to result from the ongoing discovery of molecular markers that better define these malignancies. In this report, we identify 120 genes whose expression patterns differed between reactive lymph node tissue and three types of SBCL: follicular lymphoma, mantle cell lymphoma, and chronic lymphocytic leukemia/small lymphocytic lymphoma. Whereas previously published studies have generally analyzed the gene expression profiles of one type of SBCL, work presented in this paper was intended to identify genes that are differentially expressed between three SBCL subtypes. This analysis was performed using mRNA pooled from multiple specimens representing each tissue type. Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) was used to validate the differential expression of 23 of these genes. Among the 23 validated genes were cyclin D1 (CCND1) and B-cell CLL/lymphoma 2, which have well-known roles in lymphoma pathogenesis. The remaining 21 genes have no currently established role in lymphoma development. Using qRT-PCR, the expression of CCND1 and seven additional genes was further studied in a panel of individual specimens. Genes identified in this study are of biological interest and represent candidate diagnostic markers.

Assessment of proliferative activity in breast cancer: MIB-1 immunohistochemistry versus mitotic figure count.

The proliferative activity is one of the most important single prognostic parameters in breast cancer diagnosis and the time-honored measure of proliferative activity, the mitotic figure count, is an integral component of most combined prognostic scores. The detection of the cell cycle-specific antigens Ki-67, and the development of anti-Ki-67 antibodies, including the paraffin-reactive antibody MIB-1, have established immunohistochemical detection of cell cycle-specific antigens as a measure of proliferative activity in breast cancer diagnosis. The current study was performed to correlate mitotic figure counts with the proliferative activity as assessed by MIB-1 immunohistochemistry, taking into consideration the interobserver reliability of 5 pathologists in estimating mitotic figure counts. In 32 consecutive invasive ductal breast carcinomas, mitotic figure counts were performed independently by 5 pathologists. Mitotic activity was expressed as number of mitotic figures per 10 high-power fields and in a 3-tier score according to the Scarff Bloom Richardson system. Immunohistochemistry was performed using MIB-1 antibody, heat-induced epitope retrieval, and the standard avidin-biotin-immunoperoxidase method. MIB-1 immunohistochemistry was assessed in 3 representative 20x fields by semiquantitative estimation (% of tumor cells positive) and by image analysis (number of MIB-1-positive cells/mm2). We found a high degree of interobserver correlation among 4 experienced pathologists, in both mitotic figures per 10 high-power fields and the 3-tier scoring system. We observed significant, albeit weak, correlations between semiquantitative and quantitative MIB-1 immunohistochemistry and the number of mitotic figures per 10 high-power fields (r between .36 and .53), but this significance was lost in 3 of the 5 observers when mitotic activity was expressed in the 3-tier scoring system. This study confirms mitotic figure counting in the hands of experienced pathologists as a valid, reproducible means of assessing proliferative activity in routine breast cancer diagnosis. The statistically significant, albeit only weak, correlation with MIB-1 immunohistochemistry is in agreement with results obtained by others and suggests that MIB-1 immunohistochemistry cannot be translated by a simple conversion factor into combined prognostic scores to replace the time-honored mitotic figure counts.

Leukemic HRX fusion proteins inhibit GADD34-induced apoptosis and associate with the GADD34 and hSNF5/INI1 proteins.

One of the most common chromosomal abnormalities in acute leukemia is a reciprocal translocation involving the HRX gene (also called MLL, ALL-1, or HTRX) at chromosomal locus 11q23, resulting in the formation of HRX fusion proteins. Using the yeast two-hybrid system and human cell culture coimmunoprecipitation experiments, we show here that HRX proteins interact directly with the GADD34 protein. We have found that transfected cells overexpressing GADD34 display a significant increase in apoptosis after treatment with ionizing radiation, indicating that GADD34 expression not only correlates with apoptosis but also can enhance apoptosis. The amino-terminal third of the GADD34 protein was necessary for this observed increase in apoptosis. Furthermore, coexpression of three different HRX fusion proteins (HRX-ENL, HRX-AF9, and HRX-ELL) had an anti-apoptotic effect, abrogating GADD34-induced apoptosis. In contrast, expression of wild-type HRX gave rise to an increase in apoptosis. The difference observed here between wild-type HRX and the leukemic HRX fusion proteins suggests that inhibition of GADD34-mediated apoptosis may be important to leukemogenesis. We also show here that GADD34 binds the human SNF5/INI1 protein, a member of the SNF/SWI complex that can remodel chromatin and activate transcription. These studies demonstrate, for the first time, a gain of function for leukemic HRX fusion proteins compared to wild-type protein. We propose that the role of HRX fusion proteins as negative regulators of post-DNA-damage-induced apoptosis is important to leukemia progression.

Signaling by ectopically expressed Drosophila Src64 requires the protein-tyrosine phosphatase corkscrew and the adapter downstream of receptor kinases.

Vertebrate Src can be activated by specific mutations to become oncogenic. Analogous mutations in Drosophila Src64 (DSrc) induce abnormal differentiation of photoreceptor cells when expressed ectopically in the developing Drosophila adult eye. We have investigated the roles that the adapter protein, Downstream of receptor kinases (Drk), and the SH2 domain-containing tyrosine phosphatase, Corkscrew (Csw), play in this process. We find that dominant-negative mutations in either the drk or csw genes ameliorate the developmental abnormalities induced by activated DSrc. This suggests that Drk and Csw are required downstream of, or parallel to, DSrc. Csw does not act solely as an upstream activator of DSrc. The results are discussed in relation to potential roles for the vertebrate homologues of Drk and Csw (Grb2 and SHP2, respectively) in the transformation of fibroblasts by vertebrate Src.

Ras1-dependent signaling by ectopically-expressed Drosophila src gene product in the embryo and developing eye.

The cellular functions of the Drosophila src 64B (Dsrc) gene product, Dsrc, and of most vertebrate Src-family kinases, are unknown. We have examined the effects of over-expression of wild type and mutated forms of Dsrc in transgenic Drosophila. Expression of both wild type Dsrc and a C-terminally truncated mutant at high levels during embryonic development induced extensive tyrosine phosphorylation of cellular proteins and caused considerable lethality, correlating with a block to germ-band retraction. Over-expression in the eye imaginal disc led to excess production of photoreceptor cells in the adult ommatidia. In contrast, expression of a kinase-inactive form of Dsrc caused distinct nervous system abnormalities in embryos and decreased the numbers of photoreceptor cells in the adult eye ommatidia. This suggests that active forms of Dsrc alter development by phosphorylation. Both the lethality and the eye roughening caused by activated Dsrc were partially suppressed by mutations in the Drosophila Ras1 gene. These results suggest that over-expressed Dsrc may function through Ras1 to stimulate differentiation in the embryonic nervous system and eye imaginal disc, and that kinase-active Dsrc interferes with these processes.

Overexpressed Drosophila src 64B is phosphorylated at its carboxy-terminal tyrosine, but is not catalytically repressed, in cultured Drosophila cells.

Little is known about the regulation of non-receptor tyrosine kinases in invertebrates. We have studied the relationship between the phosphorylation state of the Drosophila src 64B (Dsrc) gene product, p62D, and its tyrosine kinase activity in Drosophila Schneider 2 cells, using wild-type and mutated Dsrc constructs that were overexpressed by transient transfection. Phosphopeptide mapping showed that the putative regulatory C-terminal tyrosine (Tyr-547) of p62D was phosphorylated in vivo. In contrast to vertebrate src family kinases overexpressed in fibroblasts, wild-type p62D overexpressed in Schneider 2 cells was phosphorylated at additional tyrosines outside of the C-terminus. These tyrosines corresponded to the major in vitro autophosphorylation sites. Overexpression of wild-type p62D or several catalytically active p62D mutants significantly increased the phosphorylation of numerous Schneider cell proteins on tyrosine, while expression of catalytically inactive mutants of p62D had no such effect. Thus, in contrast to the repression of src family kinase activity in fibroblasts, p62D is catalytically active when overexpressed in Drosophila cells, perhaps because of substoichiometric C-terminal tyrosine phosphorylation. These results raise the possibility that fly development will be sensitive to ectopic expression of p62D.

Phosphorylation and regulatory effects of the carboxy terminus of a Drosophila src homolog.

The kinase activities of the vertebrate src family members are repressed by phosphorylation of a tyrosine residue in the carboxy-terminal 'tail' of these molecules. To explore whether the tail of an invertebrate src homolog might also serve a regulatory function, we examined the ability of the carboxy terminus of a Drosophila src homolog (p62D), which contains a tyrosine homologous to those in the vertebrate src family members, to regulate the following molecules in mammalian fibroblasts: (1) a chimeric protein, p60CD, containing the amino terminus and catalytic domains of chicken p60c-src joined to the C-terminus of p62D; and (2) full-length p62D itself. By a variety of criteria p60CD appears to be a partially, rather than fully, repressed form of p60c-src. Phosphopeptide mapping indicates that partial repression correlates with partial phosphorylation of the tyrosine in the p62D tail of the chimera. Phosphorylation of the tail may also regulate full-length p62D. Expression of p62D in fibroblasts does not affect cell morphology or the overall abundance of tyrosine-phosphorylated proteins. The molecule is phosphorylated at its C-terminal tyrosine (Tyr-547), but not at its in vitro autophosphorylation sites, suggesting that it is catalytically repressed in fibroblasts. Expression of a truncated p62D mutant lacking Tyr-547 is associated with a clear alteration in cellular morphology and a two- to threefold increase in cellular phosphotyrosine levels. These results suggest that phosphorylation of the C-terminal tyrosine of the tail of an invertebrate src-like kinase can repress the activity of adjacent catalytic domains.

Giving up smoking and the risk of heart attacks. A report from The British Regional Heart Study.

In a prospective study of 7735 middle-aged men, both current and ex-cigarette-smokers had a risk of a major IHD event, within an average 6.2 years of screening, more than twice that in men who had never smoked cigarettes; men who had given up smoking more than 20 years ago still had an increased risk. This excess risk among ex-smokers is only to a small extent explained by their higher blood pressure, serum total cholesterol, and body-mass index. An increased prevalence of IHD in men who had recently given up smoking also made a small contribution to excess risk. In both current and former cigarette smokers, the number of years a man had smoked cigarettes ("smoking-years") was the clearest indicator of IHD risk due to cigarettes. The major benefit of giving up smoking may lie in halting the accumulation of smoking years.