Hypocupremia and bone marrow failure.

Copper deficiency associated with neurological disorders is a well-documented condition. However, hypocupremia is less often recognized as a cause of cytopenias or bone marrow failure. We report an illustrative series of three new cases of bi-lineage cytopenia associated with copper deficiency. We have analyzed clinical features of current and historical cases to identify clues that could facilitate application of appropriate laboratory testing and heighten the level of clinical suspicion. By maintaining an appropriately high level of suspicion for potential copper deficiency and obtaining a serum copper level, bone marrow failure due to this condition can be correctly diagnosed and treated. We suggest that copper deficiency be included in the differential diagnosis of reversible causes of bone marrow failure syndromes including myelodysplastic syndrome.

Influence of killer immunoglobulin-like receptor/HLA ligand matching on achievement of T-cell complete donor chimerism in related donor nonmyeloablative allogeneic hematopoietic stem cell transplantation.

Achievement of complete donor chimerism (CDC) after allogeneic nonmyeloablative hematopoietic stem cell transplantation (NMHSCT) is important for preventing graft rejection and for generating a graft-vs-malignancy effect. The alloreactivity of NK cells and some T-cell subsets is mediated through the interaction of their killer immunoglobulin-like receptors (KIRs) with target cell HLA/KIR ligands. The influence of KIR matching on the achievement of T-cell CDC after NMHSCT has not been previously described. We analyzed 31 patients undergoing T-cell replete related donor NMHSCT following fludarabine and 200 cGy TBI. Recipient inhibitory KIR genotype and donor HLA/KIR ligand matches were used to generate an inhibitory KIR score from 1 to 4 based upon the potential number of recipient inhibitory KIRs that could be engaged with donor HLA/KIR ligands. Patients with a score of 1 were less likely to achieve T-cell CDC (P=0.016) and more likely to develop graft rejection (P=0.011) than those with scores greater than 1. Thus, patients with lower inhibitory KIR scores may have more active anti-donor immune effector cells that may reduce donor chimerism. Conversely, patients with greater inhibitory KIR scores may have less active NK cell and T-cell populations, which may make them more likely to achieve CDC.

Survival of AML patients receiving HLA-matched sibling donor allogeneic bone marrow transplantation correlates with HLA-Cw ligand groups for killer immunoglobulin-like receptors.

The reactivity of natural killer cells and some T-cell populations is regulated by killer immunoglobulin-like receptors (KIR) interactions with target cell HLA class I molecules. Such interactions have been suggested to influence outcomes after allogeneic hematopoietic stem cell transplantation, particularly for myeloid malignancies and with T-cell depletion. Donor KIR genotypes and recipient HLA KIR ligands were analyzed in 60 AML patients receiving T-cell replete, HLA-matched-related donor allogeneic bone marrow transplants. Patients were categorized according to their HLA inhibitory KIR ligand groups by determining whether or not they expressed: HLA-A3 or -A11; HLA-Bw4 and HLA-Cw groups (homozygous C1, homozygous C2 or heterozygous C1/C2). Heterozygous C1/C2 patients had significantly worse survival than those homozygous for C1 or C2 (5.8 vs 43.5 months, respectively, P=0.018) and the C1/C2 group had a higher relapse rate (47 vs 31%, respectively, P=0.048). Multivariate analysis found C1/C2 status to be an independent predictor for mortality (P=0.007, HR 2.54, confidence interval 1.29-5.00). C1/C2 heterozygosity was also associated with a delayed time to platelet engraftment, particularly for those with concurrent HLA-Bw4 expression (P=0.003). Since C1/C2 heterozygotes have a greater opportunity to engage inhibitory KIRs than do C1 or C2 homozygotes, they may more effectively inhibit KIR-positive NK- and T-cell populations involved in graft vs leukemia responses.

The partial nontandem duplication of the MLL (ALL1) gene is a novel rearrangement that generates three distinct fusion transcripts in B-cell acute lymphoblastic leukemia.

A partial nontandem duplication (PNTD) of mixed lineage leukemia (MLL) gene is described in B-cell acute lymphoid leukemia without structural cytogenetic abnormalities at 11q23 and 9p22. A duplicated portion of MLL is interrupted by the insertion of a region of 9p22 that includes the 3'-end of the AF9 gene. The PNTD encodes: (a) a PNTD transcript; (b) a partial tandem duplication of MLL; and (c) a chimeric transcript fusing MLL to the 3'-end of AF9, mimicking the t(9;11)(p22;q23) and expressed 1024-fold higher than the other two. The MLL PNTD, therefore, contributes toward leukemogenesis through simultaneous production of fusion transcripts that are otherwise encoded by three distinct genetic defects.

Comparison of cytogenetic and molecular genetic detection of t(8;21) and inv(16) in a prospective series of adults with de novo acute myeloid leukemia: a Cancer and Leukemia Group B Study.

PURPOSE: To prospectively compare cytogenetics and reverse transcriptase-polymerase chain reaction (RT-PCR) for detection of t(8;21)(q22;q22) and inv(16)(p13q22)/t(16;16)(p13;q22), aberrations characteristic of core-binding factor (CBF) acute myeloid leukemia (AML), in 284 adults newly diagnosed with primary AML. PATIENTS AND METHODS: Cytogenetic analyses were performed at local laboratories, with results reviewed centrally. RT-PCR for AML1/ETO and CBFbeta/MYH11 was performed centrally. RESULTS: CBF AML was ultimately identified in 48 patients: 21 had t(8;21) or its variant and AML1/ETO, and 27 had inv(16)/t(16;16), CBFbeta/MYH11, or both. Initial cytogenetic and RT-PCR analyses correctly classified 95.7% and 96.1% of patients, respectively (P =.83). Initial cytogenetic results were considered to be false-negative in three AML1/ETO-positive patients with unique variants of t(8;21), and in three CBFbeta/MYH11-positive patients with, respectively, an isolated +22; del(16)(q22),+22; and a normal karyotype. The latter three patients were later confirmed to have inv(16)/t(16;16) cytogenetically. Only one of 124 patients reported initially as cytogenetically normal was ultimately RT-PCR-positive. There was no false-positive cytogenetic result. Initial RT-PCR was falsely negative in two patients with inv(16) and falsely positive for AML1/ETO in two and for CBFbeta/MYH11 in another two patients. Two patients with del(16)(q22) were found to be CBFbeta/MYH11-negative. M4Eo marrow morphology was a good predictor of the presence of inv(16)/t(16;16). CONCLUSION: Patients with t(8;21) or inv(16) can be successfully identified in prospective multi-institutional clinical trials. Both cytogenetics and RT-PCR detect most such patients, although each method has limitations. RT-PCR is required when the cytogenetic study fails; it is also required to determine whether patients with suspected variants of t(8;21), del(16)(q22), or +22 represent CBF AML. RT-PCR should not replace cytogenetics and should not be used as the only diagnostic test for detection of CBF AML because of the possibility of obtaining false-positive or false-negative results.

Expression, exon-intron organization, and chromosome mapping of the human sodium iodide symporter.

The active iodide uptake of the thyroid gland in humans is mediated by the human sodium iodide symporter (hNIS). In this report, we show that hNIS expression was detected primarily in thyroid tissue, but also in breast, colon, and ovary tissues. Expression of hNIS is greatly reduced in thyroid tumors compared to normal thyroid tissue. Among tumor tissues, hNIS expression appears to be variable, consistent with the variable response to radioiodide treatment observed for thyroid carcinomas. The coding region of hNIS is interrupted by 14 introns, and the nucleotide sequence of each exon-intron junction is reported. Using this information, an alternatively spliced form of hNIS was identified. Finally, the chromosome location of the hNIS gene was mapped to chromosome 19p.

Familial eosinophilia: clinical and laboratory results on a U.S. kindred.

We describe a five-generation kindred with familial eosinophilia (FE; MIM131400), characterized by the occurrence of sustained eosinophilia of unidentifiable cause in multiple relatives. The inheritance pattern is consistent with an autosomal dominant pattern. Among 52 related subjects studied, 19 were affected and 33 were unaffected. Ten unaffected spouses were also evaluated. Four subjects with sustained eosinophilia were diagnosed with cardiac abnormalities and two of them also had neurologic symptoms. In comparison with the unaffected or spouses, evaluation of complete blood counts showed that the affected relatives had, as expected, significantly higher white cell (P < 0.005) and absolute eosinophil counts (P < 0.001) and lower red cell counts (P < 0.05). Evaluation of serum cytokine levels (IL-5, IL-3, and granulocyte-macrophage colony-stimulating factor (GMCSF) and serology for parasitic helminth infection demonstrated no differences between the affected and unaffected individuals; no individuals studied had serologic evidence for parasitic infection. There were also no differences in anti-nuclear antibody, serum cobalamin (vitamin B12) level, immunoglobulin level, leukocyte alkaline phosphatase, rheumatoid factor, HLA analysis, and stool findings for ova and parasites. Among eight affected persons who had peripheral blood or bone marrow karyotype analysis, two carried the same chromosome abnormality, a pericentric inversion of chromosome 10, inv (10) (p11.2q21.2). A gene mapping study is currently underway to study the underlying genetic mechanism(s) of this syndrome.

Testicular stromal tumor with myofilaments: ultrastructural comparison with normal gonadal stroma.

The ultrastructural features of two testicular stromal tumors were compared with those of normal gonadal stroma. The two patients with tumor were 28 and 48 years old and had no endocrine abnormalities. No metastases or recurrences occurred after 32 and 12 months of follow-up, respectively. The tumors were composed of bundles of oval to spindle-shaped cells. Ultrastructurally, intracytoplasmic myofilaments were characteristic of the tumor cells, which resembled the contractile peritubular and interfollicular cells of normal testis and ovary. In normal testicular tissue, an intertubular mesenchymal cell may differentiate into a peritubular contractile cell or into an interstitial (Leydig) cell. Therefore, testicular stromal tumors with myofilaments may originate from an intertubular mesenchymal cell that is capable of differentiating into a cell with contractile elements.

The influence of early transplantation, age, GVHD prevention regimen, and other factors on outcome of allogeneic transplantation for CML following BuCy.

Results in 164 patients who underwent allogeneic marrow transplantation following busulfan and cyclophosphamide over a 15 year period were analyzed. Age (median 37, range 14-66 years) did not significantly affect the incidence of graft-versus-host disease (GVHD), but patients who received methotrexate with cyclosporine had a significantly lower incidence (P = 0.002) of chronic GVHD compared to those who received methylprednisolone with cyclosporine. Hepatic veno-occlusive disease (VOD) occurred less frequently in chronic phase patients (P = 0.002) and in those transplanted shortly after diagnosis (P = 0.001). Five year leukemia-free survival (LFS) for the entire group was 49% (95% CI 41-57%). For 102 patients who underwent transplantation in chronic phase, results were significantly improved by transplantation at a short interval following diagnosis, particularly within 3 months (P = 0.01), by the use of methotrexate and not corticosteroids for GVHD prevention (P = 0.03), and by use of HLA-identical sibling donors (P = 0.01). Age was not a significant adverse prognostic factor and transplantation was successfully performed in individuals up to age 66. Allogeneic transplantation in CML, including older patients and those with unrelated donors, can be most safely and effectively performed shortly after diagnosis.

Abnormalities of leukocyte histograms resulting from microorganisms.

The authors report a series of 13 patients seen in their laboratory during October 1985 to August 1988 in which the presence of bacterial, fungal, or malarial parasites visible on peripheral smear was correlated with an abnormal leukocyte histogram. Samples submitted for complete blood count and differential counts were analyzed with Coulter S-Plus VI (seven specimens) or S-Plus STKR (six specimens) instrumentation. Organisms visualized on the Wright-stained peripheral smears included Histoplasma capsulatum (two), Candida sp. (four), Plasmodium sp. (three), and Staphylococcus sp. (four). Two patients had a diagnosis of acquired immune deficiency syndrome (AIDS); intravascular catheters were present in five other patients. In all cases the leukocyte histograms were abnormal. The instrument flagged abnormalities of the R1 region in four patients and multiple regions in nine patients. Similar flags were produced by the in vitro addition of bacteria or fungi to whole blood. These studies document that the presence of microorganisms in the peripheral blood can result in spurious white blood cell (WBC) counts or electronic differentials. The authors' findings indicate that the possibility of circulating organisms should be considered when abnormal WBC flags are detected with Coulter instrumentation.

Aberrant expression of CD19 as a marker of monocytic lineage in acute myelogenous leukemia.

Immunophenotypic analysis plays a critical role in the diagnosis and classification of acute leukemia. Certain characteristic immunophenotypic patterns have emerged that aid in the classification of acute myelogenous leukemia (AML). We describe a unique pattern of expression of CD19, a B cell-associated cell surface antigen, in cases of AML. We reviewed 59 cases of de novo AML to determine the pattern of CD19 expression in cases of AML using three different CD19 monoclonal antibodies, including B4 (Lytic), B4 89B (Coulter, Miami, Fla) and SJ25-C1 (GenTrak, Plymouth Meeting, Pa). We confirmed the known relationship between CD19 expression and t(8;21)-positive AML M2; in these cases, CD19 was detected with all three antibodies. We also found a unique pattern of CD19 expression in cases of AML with a substantial monocytic-monoblastic component. In 6 of 12 cases of AML M4 or M5, CD19 expression was evident only with the B4 (Lytic) antibody; CD19 expression was not observed using B4 89B or SJ25-C1. We did not observe any recurring chromosomal abnormalities in these cases of CD19-positive AML M4/M5; furthermore, none of these cases demonstrated a t(8;21). Using CD11b, CD14, and other myeloid markers, we found that AML M4 and AML M5 were characterized by dual populations of blasts. With the exception of a case of AML M4 eo, cases of AML M4 were associated with one population of blasts lacking both CD11b and CD14 and a second population with one or both of these antigens. Cases of AML M5 also had dual populations of blasts, but in contrast with AML M4, each population expressed CD11b, CD14, or both. Our findings suggest that specific immunophenotypic patterns, including the unique pattern of CD19 expression with the B4 (Lytic) monoclonal antibody, may prove useful in classifying cases of AML M4 and M5.

TEL/AML-1 fusion gene. its frequency and prognostic significance in childhood acute lymphoblastic leukemia.

TEL gene rearrangement due to the 12;21 chromosome translocation is believed to be the most common molecular genetic abnormality in childhood acute lymphoblastic leukemia (ALL). A study was conducted to investigate the frequency and prognostic significance of TEL/AML-1 fusion gene resulting from a cryptic t(12;21)(p13;q22). Bone marrow samples from 86 patients diagnosed over the past 5 years at Columbus Children's Hospital were analyzed by fluorescence in situ hybridization (FISH) technique for TEL/AML-1 fusion gene, using LSI((R)) DNA probes. The positive cases were analyzed for clinical outcome. Patients in this study received treatment according to Children's Cancer Group (CCG) protocols. Fifteen of the 86 cases (17%) were positive for the fusion gene. All were B-cell lineage and except for one, all were CD10 positive. TEL/AML-1 was not found in any T-cell ALL. The mean overall survival (OS) following diagnosis for the TEL/AML-1-positive group was significantly longer than for the TEL/AML-1-negative group by log-rank = 7.84, P = 0.005. Similarly, the event-free survival (EFS) after remission for the positive group (median 94.5 months) was longer than the negative group (median 57 months) by log-rank = 7.19, P = 0.007. This study confirms that the TEL/AML-1 fusion gene may be the most common genetic event in childhood ALL, occurring in 17% of the patients. It appears restricted to the B-cell lineage. In this study, the presence of a TEL/AML-1 fusion gene was statistically significant in predicting both OS and EFS, indicating a favorable clinical outcome for these patients. Screening for TEL/AML-1 should become routine at diagnosis and a useful biological variable for risk stratification in future clinical trials.

Rapid diagnosis of cytomegalovirus in the cerebrospinal fluid of a patient with AIDS-associated polyradiculopathy.

Polyradiculopathy is an uncommon but serious neurologic disorder that can complicate the course of the acquired immunodeficiency syndrome. We report a case in which the presence of cytomegalovirus was detected in cerebrospinal fluid by immunoperoxidase staining. In addition, large atypical cells with flocculogranular cytoplasmic inclusions were present. These cells were found to be positive for cytomegalovirus by immunoperoxidase staining. Rapid diagnosis by this method permitted early intervention with ganciclovir.

Comparison of Papanicolaou's and Wright-Giemsa stains in the examination of body fluids for Hodgkin's disease.

We reviewed 36 body fluid specimens from 18 patients with Hodgkin's disease (HD) to characterize the cytologic features of HD as seen in Wright-Giemsa (WG)-stained cytocentrifuge preparations, and to compare diagnostic agreement between WG- and Papanicolaou-stained samples. Slides were examined independently by two pathologists without knowledge of the original diagnosis, and were classified as either positive, inconclusive, or negative for malignant cells. There was diagnostic agreement between both methods in 35 (97%) of 36 samples. Features in cytocentrifuged WG-stained specimens that were most helpful in recognizing HD included mirror image nuclei in typical Reed-Sternberg cells and an axis of symmetry in polylobate Reed-Sternberg variants, with even distribution of the nuclear material within the cytoplasm.

400 cGy TBI with fludarabine for reduced-intensity conditioning allogeneic hematopoietic stem cell transplantation.

Fludarabine and 200 cGy TBI are commonly used for reduced-intensity conditioning preceding allogeneic hematopoietic SCT (HSCT). However, graft rejection and disease relapse are significant causes of treatment failure with this regimen. We modified this regimen by escalating the TBI dose to 400 cGy in 40 patients with hematologic malignancies. Thirty-four patients achieved complete donor T-cell chimerism at a median of 40 days following HSCT. The incidences of grades II-IV and III-IV acute GVHD were 40 and 15%, respectively, whereas that of limited and extensive chronic GVHD were 12 and 20%, respectively. Two patients rejected their grafts and 12 relapsed. The 100-day mortality was 18%, 2-year transplant-related mortality 20% and overall survival was 58% at a median follow-up of 16 months. There were no significant survival differences between patients with lymphoid compared to myeloid malignancies. A dose of 400 cGy TBI administered with fludarabine is well tolerated and further study is needed to determine whether outcomes are superior to those with 200 cGy TBI.

The three genes of the human FCGR1 gene family encoding Fc gamma RI flank the centromere of chromosome 1 at 1p12 and 1q21.

The high-affinity receptor for immunoglobulin G, Fc gamma RI (FCGR1), is encoded by a family of three genes within humans that share over 98% of DNA sequence homology. Efforts to define the location of the FCGR1 genes within chromosome 1 have been made to determine if they are tightly linked to the five other FCGR genes present at 1q23. Our results, obtained through both fluorescence in situ hybridization analysis of human cells and Southern analysis of cell lines containing 1p and 1q, show instead that the three genes flank the centromere of chromosome 1 at bands 1p12 and 1q21. FCGR1B was found at 1p12, whereas both FCGR1A and FCGR1C were localized to 1q21. This places the FCGR1 gene family within a large pericentric linkage group which is conserved between humans and mice. We hypothesize that the three FCGR1 genes were separated by a pericentric inversion known to have occurred on human chromosome 1, which relocated FCGR1A and FCGR1C to the long arm and left FCGR1B positioned on the short arm. We have also performed FCGR1 gene copy number experiments which indicate the existence of three FCGR1 genes within the human genome.

Spontaneous development of a chromosomal translocation 5;14 in an Epstein-Barr-virus-associated B-cell lymphoma in a SCID mouse.

C.B-17 SCID mice were inoculated with human peripheral blood leukocytes (PBLs) from normal Epstein-Barr virus (EBV)-seropositive and -seronegative donors. Confirmation of a functioning human immune response was demonstrated by the detection of human antibody after inoculation with rotavirus, tetanus toxoid, or EBV. One group of animals inoculated with PBLs from an EBV-seropositive donor developed immunoblastic lymphomas approximately 9 weeks after transplantation. Confirmation of the species and sex of origin of the tumor cells was established using a spontaneous cell line prepared from the tumor. At passage I, the tumor-cell line (AGTI) showed 15% of the metaphases with a translocation involving chromosomes 5 and 14. A lymphoblastoid cell line (AGLCL) established from the same PBLs from the same donor at the time of inoculation of the mice had a normal female karyotype. The AGLCL and a clone of AGTI cells were analyzed for rearrangement of immunoglobulin heavy chain (IgH) genes; both cell lines showed rearrangement of both IgH alleles. The results outlined in this report suggest that a spontaneous chromosomal translocation involving chromosome 14 occurred in normal PBLs in the SCID mouse.

HER-2/neu amplification and overexpression in endometrial carcinoma.

HER-2/neu is a proto-oncogene associated with poor prognosis in women with breast and ovarian carcinoma. The significance of HER-2/neu in endometrial carcinoma is less clearly established. The authors compared HER-2/neu gene amplification using fluorescence in situ hybridization and protein overexpression using immunohistochemistry with survival in patients with endometrial carcinoma. Fluorescence in situ hybridization and immunohistochemical staining were performed on 72 formalin-fixed, paraffin-embedded endometrial carcinoma specimens. Vysis combination HER-2/neu and centromere 17 probe mixture was applied to isolated tumor cell nuclei. A minimum of 200 nuclei were scored for each specimen using standard signal enumeration criteria. A specimen was considered amplified with 5% or greater amplified nuclei. Tissue sections were immunostained with polyclonal antibody against p185erb-2 transmembrane glycoprotein. Immunohistochemical reactivity was scored on a three-tiered scale. HER-2/neu gene amplification and protein overexpression were detected in 15 of 72 (21%) and 12 of 72 (17%) of the specimens, respectively, with 2 cases of normal copy overexpression and 5 cases of amplification without overexpression. Both amplification and overexpression were associated with higher grade tumors. Amplification was associated with clear cell and serous subtypes (p = 0.002), and overexpression with only clear cell type (p = 0.006). Using the proportional hazards model of survival, amplification was found to have significant negative predictive value beyond stage, grade, and cell type (p = 0.002). HER-2/neu gene amplification as detected by fluorescence in situ hybridization in archival material has significant prognostic value.