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Bone marrow involvement by an isolated interstitial lymphoid aggregate exhibiting the pattern and phenotype described for follicular lymphoma in situ (FLIS) has not been reported before. The detection of clinically silent FL in this case highlights the necessity of complete staging workup when such lesions are encountered in biopsies.

Background: Celiac disease (CD) is underdiagnosed, and iron-deficiency anemia (IDA) is a common presentation of CD. No guidelines exist in the literature for screening for CD among those with IDA in the United States. We surveyed hematologists to deter- mine rates of CD screening in patients with IDA., Methods: A survey was e-mailed to members of the American Society of Hematology., Results: There were 385 complete responses from 4551 e-mails. Most respondents were practicing clinicians (74%), clinical researchers (10%), or laboratory researchers (6%). Specialists in benign hematology accounted for 45% of respondents, oncologists accounted for 33%, and specialists in malignant hematology accounted for 22%. The most common practice types were university-affiliated hospital (43%), private clinic (29%), community hospital (12%), and Veterans Affairs or military hospital (9%). Only 8.6% believed all patients with IDA should be screened for CD. Respondents who had completed their fellowship within 5 years were more likely than more experienced clinicians to believe that all patients with IDA should receive CD screening (OR, 2.8; CI; 1.1-7.5; P=.04). Having a higher volume of IDA patients per month also increased the likelihood of testing (P=.01). In multivariate analysis, specialists in malignant hematology (OR, 3.2; CI, 1.1-9.5; P=.04) and oncologists (OR, 3.5; CI, 1.3-9.5; P=.02) were more likely than specialists in benign hematology to screen all patients for CD, as were those who saw predominately pediatric patients with IDA vs adult patients (OR, 16.9; CI, 3.0-97.0; P=.002)., Conclusions: Practicing hematologists infrequently screen for CD in IDA. Physicians who have recently finished their fellowship and those who see a high volume of patients with IDA are more likely to screen for CD.

After liver transplantation, reinfection of the newly engrafted liver with hepatitis C virus is essentially universal in patients who are viremic at the time of transplantation. Treatment with interferon preparations with or without ribavirin is recommended in patients with marked histologic injury; however, hematologic toxicity associated with therapy has been reported, which is usually treated with growth factor support, including erythropoietin analogues. We present the first reported case of anti-erythropoietin antibody-mediated pure red cell aplasia arising in the setting of hepatitis C virus therapy in a patient who underwent living donor liver transplantation., ((c) 2007 AASLD.)

An increased incidence of acute myeloid leukemia (AML) has recently been documented in patients post-solid organ transplantation but the incidence and types of myelodysplastic syndromes (MDS) occurring in this patient population are not known. We identified 5 patients (3M, 2F, age 48-64 years) who developed MDS ranging from 1.8 to 25 years (median 4.2 years) post-solid organ transplantation, only 2 patients had received azathioprine. The cumulative incidence of MDS in heart and lung transplant recipients at 15 years was 0.5% and 1.8%, respectively, which is markedly higher compared to the general population. Low-risk types of MDS predominated, 3 of 5 patients are alive (median 3.9 years) since diagnosis. Deletions of chromosome 20q, which have not been previously reported in post-transplant MDS/AML, were identified in 3 cases. Our findings expand the morphologic and cytogenetic spectrum of MDS occurring post-solid organ transplantation and suggest that mechanisms beside azathioprine toxicity might be important in disease pathogenesis.

As both fludarabine and rituximab are active against indolent lymphoproliferative disorders, we have studied the combination of fludarabine and rituximab in patients with low-grade lymphoma and chronic lymphocytic leukemia (CLL) in phase I/II fashion. Of 33 patients enrolled, 21(63.6%) had low-grade lymphoma and 12 (36.4%) had CLL. They received fludarabine 30 mg/m2 on days 1-4 and rituximab 125, 250 or 375 mg/m2 on day 5 at intervals of 28 days to a maximum of 8 cycles. Three patients were removed from the study because of rituximab-associated anaphylaxis and four because of prolonged hematopoietic toxicity. Toxicity and responsiveness did not differ at the different dose levels of rituximab. For 29 evaluable patients, responses were seen in 82.8% and complete responses in 34.5%. Of 7 responding patients not referred for stem cell transplantation, 6 remain in complete remission at a median follow-up of 16 months (range 4-30 months). Of 13 previously untreated patients, all responded and 46.2% had a complete response. Of 16 previously treated patients, 68.5% responded and 25% had a complete response. The combination of fludarabine and rituximab has major activity and acceptable toxicity in patients with low-grade lymphoma and CLL.

Progressive multiple myeloma may manifest features of 'de-differentiation', including a plasmablastic appearance, failure to secrete paraprotein, extramedullary involvement, and resistance to treatment. A 44-year-old woman with kappa-light chain myeloma underwent allogeneic stem cell transplantation (SCT). Twenty months later she developed paraspinal plasmablastic myeloma in the absence of paraprotein in urine or myeloma in the marrow. The paraspinal masses responded to chemotherapy. At 30 months she developed myelomatous meningitis, which proved resistant to intrathecal chemotherapy, irradiation, and donor lymphocyte infusion (DLI). The leptomeningeal disease led to death at 38 months. This is the first report of leptomeningeal relapse of myeloma after allografting.

We report a case of high grade lymphoma which appeared at the site of prior injections of medications into the shaft of the penis for erectile dysfunction. We discuss the possible mechanisms of causation for this unusual form of lymphoma.

Background: Posttransplantation lymphoproliferative disorders (PT-LPDs) are a well-known complication of immunosuppression associated with solid organ transplantation. The clinical course of PT-LPDs is unpredictable; some patients experience regression of all lesions with a reduction in immunosuppression, whereas other patients, despite chemotherapy, radiation therapy, or surgery, rapidly die of their disease. In this study, the authors attempted to establish whether the previously described morphologic and molecular genetic categories of PT-LPD--plasmacytic hyperplasia (PH), polymorphic PT-LPD (polymorphic), and malignant lymphoma/multiple myeloma (ML/MM)--are clinically relevant and helpful in predicting the clinical outcome of patients who develop these lesions., Methods: To determine the clinical significance of the morphologic and molecular genetic categories of PT-LPDs, the clinical characteristics of 32 solid organ transplant recipients (26 heart, 5 kidney, and 1 lung), including age, time from transplantation to development of PT-LPD, stage of disease, and clinical outcome, were compared with the morphologic and molecular genetic features of the 41 PT-LPDs that they developed (15 PH in 12 patients, 19 polymorphic in 16 patients, and 7 ML/MM in 6 patients). Clinical outcome was defined by the following categories: 1) regression (after a reduction in immunosuppression) and surgical resolution (by surgical excision, with or without a reduction in immunosuppression); 2) medical resolution (by chemotherapy and/or radiation therapy); and 3) no response., Results: Although there was no difference in the time from transplantation to PT-LPD development among patients belonging to the three morphologic and molecular genetic categories, there was a significant difference in patient age at the time of PT-LPD development (P < 0.0098). Younger patients developed PH (mean age of 19 years), whereas older patients developed polymorphic PT-LPD (mean age of 35 years) and ML/MM (mean age of 56 years). Patients with PH presented with lower stages of disease (Stages I-II) than patients with ML/MM (P < 0.0004). Furthermore, there was a statistically significant trend between morphologic and molecular genetic category and clinical outcome, with decreased likelihood that lesions categorized as PH, polymorphic, or ML/MM would regress with a reduction in immunosuppression or be resolved by surgery, whereas those classified as ML/MM were more likely to exhibit no response to aggressive clinical intervention (P < 0.00006). Furthermore, no patients with PH died, whereas 20% with polymorphic PT-LPD and 67% with ML/MM died as a direct result of their PT-LPDs., Conclusions: This study strongly suggests that classification of PT-LPDs into the morphologic and molecular genetic categories PH, polymorphic, PT-LPD and ML/MM is clinically relevant.

Background: Primary pulmonary hypertension (PPH) is characterized by vascular injury of pulmonary arterioles, in which endothelial dysfunction may play a major role. Although continuous infusion of prostacyclin (prostaglandin I2, a potent vasodilator released by vascular endothelial cells) improves the clinical status and survival in PPH, its mechanism or mechanisms of action remain unclear., Methods and Results: We measured endothelium-derived clotting factors and assayed platelet aggregation in 64 patients (26 adults and 38 children) with PPH before long-term PGI2 therapy. Repeat studies were performed in 42 patients (18 adults, 24 children) after one year of PGI2 therapy. At baseline, 87% of adults and 79% of children had abnormal platelet aggregation. In addition, factor VIII, von Willebrand (vW) antigen, and ristocetin cofactor levels were abnormally high in 92%, 72%, and 52%, respectively, of the adults versus 29%, 16%, and 16%, respectively, of the children (P<.005 adults versus children). With long-term PGI2, platelet aggregation normalized in 83% of the adults and 80% of the children who had platelet aggregation abnormalities at baseline (P<.01). Factor VIII, vW antigen, and ristocetin cofactor also decreased with long-term PGI2 in both groups (P<.02). The ratio of ristocetin cofactor to vW antigen, which may reflect biological activity of vW factor, increased with long-term PGI2 in adults from an abnormally low level (0.6+/-0.2) to normal level (1.10+/-0.4), and in children the ratio increased from 0.8+/-0.3 to 1.3+/-0.4 (normal, 0.8 to 1.4)., Conclusions: Alterations in the coagulation system may contribute to the pathogenesis of PPH; the normalization of these endothelial markers concomitant with improvement in hemodynamic parameters with long-term PGI2 suggests that long-term PGI2 remodels the pulmonary vascular bed with subsequent decreases in endothelial cell injury and hypercoagulability.

The translocation between chromosome 9 and chromosome 22 which creates the Philadelphia chromosome moves the ABL oncogene from its normal location on chromosome 9 and fuses it with a portion of the BCR gene on chromosome 22. This new BCR/ABL fusion gene generates a unique 8.7 kilobase (kb) RNA which codes for a new 210 kilodalton (kd, p210) protein which has a protein tyrosine kinase activity that is greatly increased in comparison to the normal ABL protein. The human K562 cell line was derived from a patient with CML, and serves as one model for the regulation of expression of the ABL and BCR/ABL genes. This study examines the expression of the BCR/ABL fusion gene and the normal ABL gene in relation to differentiation and changes in proliferative state. The expression of both the normal ABL transcripts and the BCR/ABL fusion transcript decrease approximately ten-fold when the cells are induced to differentiate with hemin. In contrast, expression of the MYC oncogene is unaffected by hemin-induced differentiation. The results suggest that both ABL and BCR/ABL expression vary in proportion to the differentiation of the cells, but minimally if at all as a function of the cells' proliferative state.

Chronic myelogenous leukemia (CML) is characterized by the presence of a novel fusion gene comprised of portions of the BCR gene from chromosome (ch) 22 and the ABL gene from ch 9. The present study was designed to identify regulatory DNA regions as determined by DNAase I hypersensitivity to address the question of whether altered chromatin contributes to changes in ABL expression. We identify five hypersensitive (HS) sites within the abnormal BCR/ABL allele in K562 cells in a pattern different from the normal BCR. The pattern of hypersensitivity is modified when the cells undergo hemin induced differentiation. These results indicate that the normal BCR has a chromatin configuration consistent with active transcription and that the BCR/ABL fusion gene chromatin is different. This may be important in the pathogenesis of CML.

Previous studies of the Hpa I cleavage site-sickle cell hemoglobin gene linkage in various African populations suggested that the sickle gene arose independently more than once. In the present study we have performed restriction endonuclease haplotype analysis for the beta-globin-like gene cluster from four separate geographic areas in Africa, all of which possess the sickle gene. In Benin (Central West Africa) and Algeria (Arab North Africa) all chromosomes carrying the sickle gene possess an identical haplotype as defined by 11 different polymorphic restriction endonuclease sites within the 60-kilobase region of the beta-globin-like gene cluster. In the Central African Republic (Bantu-speaking Africa) and in Senegal (Atlantic West Africa) a very large proportion of the sickle gene chromosomes were associated with a haplotype specific for each country. Thus, three different haplotypes are shown to be associated with the sickle gene in Africa, and each is present at a very high frequency in geographically separate regions. Since the three haplotypes differ from each other by at least three sites residing both 5' and 3' to a putative hot spot for recombination, it is most likely that the sickle gene arose at least three times on separate preexisting chromosomal haplotypes. This may have implications for a better understanding of the variable nature of the expression of sickle cell anemia, because clinically relevant sequences (for example, gamma-globin gene regulatory sequences responsive to anemia) might be linked polymorphically to these haplotypes.

Linked DNA polymorphisms can be used to study the evolution of structural gene mutations. Both the beta S-(beta 6Glu leads to Val) and beta C-(beta 6Glu leads to Lys) genes are common in West Africa. We have analyzed their linkage to a polymorphic Hpa 1 site appearing 3' to the beta-globin gene locus in selected populations from Wes Africa. A large reservoir of beta A-genes linked to 13-kilobase Hpa 1 fragments with a frequency of 17-18% has been identified. In addition, the beta S- and beta C-genes in Togo are found to be tightly linked to the 13-kilobase Hpa 1 fragment, whereas 72% of the beta S-genes in the Ivory Coast reside on the 7.6-kilobase Hpa 1 fragment. These studies are consistent with the selection and expansion of two different chromosomes bearing beta S-genes in at least two physically close, but ethnically separate regions of West Africa, with subsequent diffusion to North, Equatorial, and East Africa.

Studies of the human hemoglobin system have provided new insights into the regulation of expression of a group of linked human genes, the gamma-delta-beta-globin gene complex in man. In particular, the thalassemia syndromes and related disorders of man are inherited anemias that provide mutations for the study of the regulation of globin gene expression. New methods, including restriction enzyme analysis and cloning of cellular DNA, have made it feasible to define more precisely the structure and organization of the globin genes in cellular DNA. Deletions of specific globin gene fragments have already been found in certain of these disorders and have been applied in prenatal diagnosis.

Globin messenger RNA (mRNA) isolated from three patients homozygous for hemoglobin Lepore is shown to have a marked reduction of the amount of beta-like globin mRNA (Lepore-globin mRNA sequences) compared with alpha-globin mRNA by molecular hybridization. The relative amounts of alpha- and Lepore mRNA are similar to the amounts of alpha- and Lepore globin synthesized in intact cells and by isolated mRNA in a cell-free system. It is also demonstrated that Lepore-globin mRNA can completely hybridize to full-length or nearly full-length beta-globin specific complementary DNA and protect it from nuclease digestion, indicating close homology between the delta-mRNA sequences present in Lepore mRNA and the beta-complementary-DNA probe. We have also quantitated the numbers of beta-like globin gene sequences in genomic Lepore DNA by molecular hybridization and demonstrated a reduction in their number consistent with the Lepore gene being a delta beta-gene fusion product.

We have probed the chromatin conformation of the G gamma-A gamma-delta-beta globin gene locus of K562 cells, a human hematopoietic cell line, with the enzyme pancreatic DNAse I. This enzyme preferentially digests genes in an active configuration. We have found that in K562 cells, which produce embryonic and fetal but not adult hemoglobins, both the active gamma and inactive beta genes are DNAse I sensitive. However, only the active gamma genes have DNAse I hypersensitive regions. The hypersensitive regions have been mapped to an area approximately 100 base pairs 5' to the G gamma and A gamma genes.

We have analyzed human cellular DNA for its delta--and beta-globin gene sequence content by separation of restriction enzyme fragments by agarose gel electrophoresis; transfer of the DNA fragments to nitrocellulose filters; hybridization of filters with 32P--beta-globin cDNA; and analysis by autoradiography. A short cDNA has been used to identify specifically the 3' end of the genes and to orient the fragments. A comparison of the globin gene fragments generated by normal and Lepore DNA has been used to distinguish fragments representing DNA sequences between the delta and beta genes and those containing sequences flanking either 5' to the delta gene or 3' to the beta gene. The results indicate that unique restriction fragments are presented in normal DNA and absent in Lepore DNA, and allow preliminary ordering of these fragments on a restriction enzyme map. In addition, the Lepore, delta--and beta-globin genes have been found to contain at least one inserted nucleotide sequence of about 1000 bases which is not represented in mature globin mRNA.

The structure and organization of the human globin genes at the nucleotide level has been established by restriction endonuclease digestion of cellular DNA, and by the isolation and purification of these genes in phage vectors. With this approach it has been possible to define alterations at the DNA level resulting in a group of inherited diseases of man known as the thalassemia syndromes, and related disorders. Combined with other known genetic and biochemical data, these studies provide a framework for understanding the pathogenesis of these disorders at the molecular level.

Three clones containing both the human G gamma and A gamma globlin genes have been isolated and characterized from a library of DNA fragments generated by partial Eco RI digestion of cellular DNA using charon 4A phage as vector. Two of the clones (NY 2 and 3) are identical and have an insert of 14.0 kb. The third clone (NY 1) has a 15.4 kb insert by virtue of an extra 1.4 kb Eco RI fragment at its 5' most end. This clone also has a Kpn I site not present in the other two suggesting it is the product of the gamma gene on the opposite chromosome. Restriction analysis of the three clones indicates that the G gamma and A gamma genes are linked on a single continuous piece of DNA and are separated by 3.5 kb and each contains at least one large intervening sequence of 0.85 kg between the Bam HI and Eco RI sites. These findings in cloned DNA provide direct evidence for linkage and organization of the gamma genes in man.

The (9;22) translocation which produces the Philadelphia (Ph1) chromosome activates the abl oncogene from chromosome 9 by recombination with the bcr gene from chromosome 22. This fusion gene is transcribed into a new 8.5-kilobase chimeric mRNA which is translated into a novel Mr 210,000 fusion protein which has a protein tyrosine kinase activity that is greatly increased in comparison to the activity of the normal abl protein. Studies from this laboratory and others have shown that virtually all patients with chronic myelogenous leukemia have this new bcr/abl fusion gene. In contrast to these findings in chronic myelogenous leukemia, a small number of patients with Ph1(+) acute lymphoblastic leukemia (ALL) have been studied and were found to lack the bcr/abl fusion gene [bcr(-)], but to have a new activation of abl, by recombination with an as yet undetermined region on chromosome 22. In this study, nine adults with Ph1(+)-ALL have been examined for evidence of a bcr/abl fusion gene. Of the nine patients, five have a bcr/abl recombination, whereas the remaining four patients do not. In contrast, the children studied to date have all been bcr(-). These data suggest that adults with Ph1(+)-ALL are a more heterogeneous group on a molecular level than are children, and that further studies will be required to determine the spectrum of molecular defects in patients with Ph1(+)-ALL, and the relationship of these various molecular defects to the clinical disease state of the individuals.

Restriction enzyme digestion analysis and direct human globin gene cloning have permitted analysis of the physical arrangement of nucleotide sequences within and surrounding the human globin genes. With these methods it has been shown that the linear arrangement 5' to 3' of the globin genes is G gamma-A gamma-delta-beta. The G gamma and A gamma genes are separated by about 3.5 kilobases (kb), while the A gamma and delta genes are 15 kb apart, and the delta and beta 6.5 kb apart. Each of these genes contains a large intervening sequence (IVS) of approximately 1 kb in precisely the same position between condons 104 and 105. In addition, each of these genes has a small IVS between codons 30 and 31. In homozygous delta beta thalassemia DNA, there is deletion of all of the normal delta and beta gene fragments. However, a new fragment 4.2 kb in size containing the 5' end of the delta globin gene is retained. Retention of this fragment in delta beta thalassemia, but not in HPFH is consistent with a role for sequences in this region for limiting gamma globin gene expression. Studies to date suggest that the beta + and beta 0 thalassemias will be due to a heterogeneous group of DNA defects affecting either beta globin gene transcription or beta mRNA processing. In most cases of beta + and beta 0 thalassemia DNA analyzed, there is no detectable deletion of beta or delta genes. In three India beta 0 patients, deletion of the 3' end of the beta gene has been found. Analysis of cloned beta globin genes from a patient with beta + thalasseia shows differences from normal in the fragments generated by restriction enzymes which cut frequently. Whether these differences are responsible for the defect in thalassemia or are polymorphisms unrelated to thalassemia remains to be determined.

Human cellular DNA fragments from cells of normal subjects and patients with thalassemia obtained by restriction enzyme digestion were analyzed for their globin gene content. The fragments were separated on agarose gels, transferred to nitrocellulose filters, hybridized to globin [(32)P]cDNA, and radioautographed. One to ten picograms of globin gene sequences were detectable. With EcoRI digestion, eight to nine cellular DNA fragments were found to contain globin genes. Three of these contained beta-like gene sequences assayed with beta globin cDNA probe. One beta-like fragment was absent in DNA from a homozygous subject for hemoglobin Lepore. Two of the three beta gene-containing fragments present in normal DNA were absent in DNA from a patient with hereditary persistence of fetal hemoglobin. The same two fragments containing beta-like genes were absent from deltabeta thalassemic DNA and one new fragment containing beta-like genes was found. Together with results obtained by hybridization of these DNAs in solution, the data are consistent with deletion of specific restriction human DNA fragments in subjects with these disorders and a greater deletion of beta-like gene sequences in subjects with hereditary persistence of fetal hemoglobin than in those with deltabeta thalassemia.

We have examined the genetic polymorphism previously reported to be associated with the sickle-cell (beta s) gene. The polymorphism involves an alteration of the DNA sequence 3' to the beta-globin gene as detected with the restriction endonuclease, Hpa I. In normal individuals, the beta-globin gene is contained within a DNA fragment of 7.6 kilobases (kb), whereas 87% of individuals with sickle-cell anemia have been reported to have the beta s-gene associated with a 13.0-kb Hpa I fragment. We have studied this polymorphism in 31 New York Black individuals homozygous for sickle-cell anemia to ascertain its genetic and biochemical significance and to evaluate its potential use in the prenatal diagnosis of sickle-cell disease. Our results show only a 58% association of the beta s-gene and the 13.0-kb Hpa I fragment, as well as the presence of additional variants involving the Hpa I site. In addition, the 13.0-kb fragment is also found associated with the beta c- and beta A-genes. Thus, the Hpa I polymorphism probably represents a change in DNA not specifically associated with the beta s-gene, and appears to antedate the beta s-and beta c-mutations.