Chromosome studies in a case of monoblastic leukemia

Chromosome analysis of a direct bone marrow preparation from a patient with acute, undifferentiated monoblastic leukemia showed a predominant clone of 51,XX,+6,+8,+14,+19,+21,11q+, along with a chromosomally normal clone. Studies with cytofluorometry confirmed the two populations of cells with different DNA content.     

Patterns of late replication in X chromosomes of human lymphoid cells

Two predominant patterns of late X replication were observed in both short-term and established human lymphoid cultures. One pattern was found in a minority of short-term cultured T-cell metaphases, in most lectin-stimulated B cells, and, with minor variations, in established B-cell lines. In these cells, DNA replication terminated in the distal part of the long arm of the late X. A different pattern was found in the majority of lectin-simulated T cells and in the T-cell line CCRF-CEM. These cells exhibited terminal replication in a region of the long arm of the late X that was nearer to the centromere. It is speculated that the variations in replication patterns correlate with phenotypic and functional characteristics of human lymphoid subsets.     

Absence of a clastogenic factor in ataxia telangiectasia lymphoblastoid cells

Based on the observation of a diffusable “clastogenic factor” in the plasma of ataxia telangiectasia (AT) patients and the medium used to culture their skin fibroblasts, studies were performed to discern its possible presence in AT long-term lymphoblastoid cell lines. Cocultivation of normal and AT lymphoblasts showed no increase in chromosome damage in the normal cells. Additionally, tissue culture medium, used for the propogation of the AT lymphoblasts, demonstrated no damaging effect on the chromosomes of normal phytohemagglutinin-stimulated lymphocytes. Therefore, contrary to the findings in other cell types (T lymphocytes and skin fibroblasts), the AT B cell apparently does not produce this clastogenic factor.     

A direct bone marrow chromosome technique for acute lymphoblastic leukemia

We describe a direct bone marrow chromosome technique that was developed especially for use in studies of acute lymphoblastic leukemia (ALL). The features responsible for technical improvements include: the use of RPMI 1640 medium, supplemented with 30% fetal calf serum, to support cellular activity during both specimen transport and Colcemid treatment; the processing of only 0.1 ml of sedimented cells or less per centrifuge tube; the exposure of cells to Colcemid for a maximum of 25 min; control of the total time of exposure to hypotonic solution; the use of a steel wire as a stirring rod (fashioned to fit the centrifuge tube) for mixing cells; slide preparation by a specific edging-flaming technique; the natural aging of the slides to achieve optimal drying; and the use of a modified G-banding procedure that employs Wright's stain. This technique has been used in more than 350 cases of ALL and has consistently provided analyzable banded chromosomes, even in hyperdiploid cases with up to 91 chromosomes. It makes the previously recognized morphological difference between metaphases of residual normal cells and those from the leukemic clone less apparent. The edging-flaming technique of slide preparation is the most important component and is especially appropriate for spreading large numbers of chromosomes in ALL.     

EBV transformed B cell lines present to antigen specific T cell clones determinants different from those recognized by antibody in an HLA-DR linked restricted fashion

Human B cells nonspecifically activated by Epstein Barr virus (EBV) can present tetanus toxoid (TT) antigen to TT specific helper T cell lines and T cell clones. Presentation of TT antigen by EBV-B cells did not require the presence of TT specific B cells and did not involve B cell surface immunoglobulins. Immunosorbent purified antibody to TT added in great excess to EBV-B cells pulsed with TT for 18 hr did not inhibit the capacity of the EBV-B cells to present TT antigen. Furthermore, EBV-B cells induced proliferation in T cells in the presence of urea denatured TT which contained less than 1% TT reactive with serum anti TT antibody. Studies of TT presentation by a panel of EBV-B cells obtained from HLA typed donors indicated that T cell recognition of TT presented by EBV-B cells was MHC restricted by products of the HLA-D region and some of which differed from serologically defined HLA-DR. These results indicate that the immunogenic moiety presented by EBV activated human B cells consists of antigenic determinants, which differ from those recognized by serum antibody, and of HLA-DR linked MHC determinants. This moiety is similar to that presented by monocytes. The implications of these findings for the amplification of the immune response by activated B cells are discussed.     

Chromosomal anomaly of 6q in chronic myelogenous leukemia (CML)

Anomalies of chromosome 6q, along with other chromosomal anomalies, are described in the bone marrow cells of two patients with chronic myelogenous leukemia (CML). One patient, a 14-year-old male, developed the karyotype 46,XY,t(1;6)(p36;q15),del(3)(q25),del(17)(p11),? inv(17)(q12q24) during blastic crisis of his disease. The other patient, a 24-year-old male, had the karyotype 46,XY,del(6)(q13),t(9;22)(q34;q11) during the early phase of his disease and evolution of i(17q) in the karyotype late in the disease.     

Energy transfer-enhanced chromosome banding. An overview

The conditions necessary for energy transfer-enhanced chromosome banding are described and the results of chromosome staining produced by different energy donor-acceptor dye pairs are presented. Energy donors with A-T binding or fluorescence quantum yield specificity, such as 33258 Hoechst or quinacrine, in combination with energy acceptors with G-C specific binding (e.g., actinomycins) produce enhanced Q banding, with especially prominent Q-bright polymorphisms. Conversely, G-C specific energy donors, such as chromomycin A₃ or 7-aminoactinomycin D, when used with methyl green, as an A-T specific energy acceptor, produce a reverse type banding in which different, putative polymorphic regions are highlighted. Donor fluorescence should be insensitive to quenching by the energy acceptor in chromosomal regions compatible with donor binding and fluorescence but not acceptor binding (e.g., clusters of 10–30 A-T or G-C base pairs). In addition to providing information about the molecular basis of chromosome banding and facilitating the detection of certain chromosome polymorphisms, the methodology described may prove useful in characterizing chromosome rearrangements, such as the Philadelphia chromosome (t(9q;22q)) translocation found in chronic myelogenous leukemia.     

Chromosome pattern,occupation, and clinical features in patients with acute nonlymphocytic leukemia

Chromosome banding pattern of bone marrow cells, cell morphology according to the FAB classification, and clinical findings were compared in two groups of adult patients with acute nonlymphocytic leukemia (ANLL): 52 patients occupationally exposed to chemical solvents, insecticides, or petrol products, and 110 patients with no history of occupational exposure to potential mutagenic/carcinogenic agents. Striking differences were found between the two groups: (1) Clonal chromosomal aberrations were present in 75% of exposed patients compared with only 32% in the nonexposed group. (2) Of the patients exposed to solvents and insecticides 92% had abnormal chromosomes, whereas only 29% of patients exposed to petrol products showed abnormalities; in the total material $\text{10}\text{13}$ exposed patients with normal chromosomes were exposed to petrol products. (3) The relationship between chromosomal abnormality and exposure was evident in both females and males. However, only 29% of women with an abnormal karyotype were exposed, whereas 70% of males with an abnormal karyotype were exposed. (4) The incidence of certain characteristic karyotypic abnormalities, i.e., $\text{?5}\text{5}\text{q}\text{?}$, $\text{?7}\text{7}\text{q}\text{?}$, +8, +21, t(8;21), and t(9;22), were decidedly more common in exposed than in nonexposed patients. At least one of these changes were present in 92% of exposed patients with aberrations, whereas in the nonexposed group the incidence was only 60%. (5) The monocytic varieties of ANLL (M4 + M5) were more common in the nonexposed patients, whereas erythroleukemia (M6) was more common in the exposed group. The predominance of abnormal karyotypes in the exposed compared to the nonexposed patients was similar in leukemia types M1 + M2 and in M4 + M5. (6) There was no difference in survival time between the two groups and the same correlation was obvious in both exposed and nonexposed patients: patients who had only abnormal metaphases had poorer prognosis than those with normal bone marrow metaphases only (6 vs 1.5 months). This correlation was obvious in patients classified as acute myeloid leukemia (AML) as well as in the monocytic varieties of ANLL.     

Prognostic subgroups for undifferentiated neuroblastoma: immunohistochemical study with anti-S-100 protein antibody

Pathologic prognostic factors in 36 undifferentiated neuroblastomas were studied by an immunohistochemical staining technique with anti-S-100 protein antibody. Nuclear abnormalities of neuroblastic cells (mitosis and karyorrhexis/5,000 cells [MKI]), the incidence of S-100 protein-positive cells in the supportive stroma, and the age of the patients at diagnosis were analyzed both individually and in combination. Based on the evaluation of these factors, two prognostic subgroups were identified for these tumors. Tumors for which the prognosis was good (all nine patients alive) occurred in younger patients (younger than 1.5 years), showed low (less than 100) or intermediate (100 to 200) MKI, and had positive to intensely positive staining for S-100 protein. Cases in which the prognosis was poor (7 per cent survival) had single or multiple factors of high (greater than 200) MKI and/or absent or weak staining for S-100 protein and/or older age (older than 1.5 years) at diagnosis. These results were compared with other clinical information.     

Reduction in Nephrotoxic Antimicrobial Exposure Decreases Associated Acute Kidney Injury in Pediatric Hematopoietic Stem Cell Transplant Patients

Exposure to nephrotoxic medications is a common risk factor for acute kidney injury (AKI) in pediatric stem cell transplantation (SCT). We hypothesized that reducing nephrotoxic antimicrobial exposure for SCT patients would be associated with lower nephrotoxin-associated AKI (NTMx-AKI) rates and no increase in infection treatment failures. We conducted a prospective cohort analysis of all inpatient SCT patients at Cincinnati Children's Hospital Medical Center between January 2014 and December 2017. In January 2016, first line fever coverage was changed from piperacillin-tazobactam to cefepime, acknowledging that the change resulted in a loss of enterococcal coverage, and the duration of antimicrobial exposures was limited, specifically including vancomycin. We collected data using prospective NTMx-AKI and antimicrobial utilization monitoring platforms within the electronic health record. AKI days and severity were extracted for patients exposed to 3+ nephrotoxins, 3+ days of IV aminoglycosides, or 3+ days of IV vancomycin. AKI was identified using KDIGO serum creatinine criteria. We assessed rates of nephrotoxin exposure and NTMx-AKI in all SCT inpatients for 2 years pre- and post-intervention. Data were grouped and analyzed by calendar month, normalized to a denominator of 1000 patient-days. Statistical process control methods were used to monitor adherence to the intervention and identify changes in mean rate of nephrotoxin exposure and NTMx-AKI. Infection rates, alternate antimicrobial usage rates, and the fraction of repeat positive cultures were used to identify treatment failures. PTZ usage decreased from 196 to 33 days/1000 patient days, cefepime usage increased from 62 to 290 days/1000 patient days, and vancomycin usage decreased from 62 to 41 days/1000 patient days. High nephrotoxin exposure decreased by 33% (143 to 96 days/1000 patient days), and NTMx-AKI decreased by 74% (24 to 6 days/1000 patient days). Rates of all KDIGO stages of NTMx-AKI decreased ≥50% after the intervention. Stage 3, the most severe, decreased by >80%. The fraction of repeat positive cultures remained stable between the two eras at .1 (standard deviation 0.21) and .07 (standard deviation 0.17), respectively. There were no increases in infection rates, alternate antimicrobial usage rates, or treatment failures. Reduction of nephrotoxic antimicrobial exposure can decrease the amount and severity of NTMx-AKI in SCT patients without an increase in treatment failures.     

Noninvolvement of chromosome 16 in karyotype evolution of acute myeloid leukemia in a patient with a heritable fragile site on 16q22

A fragile site on the long arm of chromosome #16 (q22) was detected in a 24-year-old man with pancytopenia. During the course of the disease he developed an inverted duplication of region q11-12 of chromosome #1 and a translocation between chromosomes #9 and #13: t(9;13)(p22;q32). These abnormalities, as well as an additional iso-like marker chromosome that consisted of one normal 9p and the abnormal 9p arm, were detected in Epstein-Barr nuclear antigen-positive B-cell cultures. Two years later, evolution of the abnormal clone with loss of chromosome #7 and, subsequently, chromosome #22 occurred in connection with development of acute myeloid leukemia. Although the heritable fragile site on chromosome #16 was present in all cell populations investigated, it was not involved in the evolution of the abnormal karyotype. This fragile chromosome #16 also was found in 4 of 11 family members in whom chromosome analysis was performed, thus suggesting this aberration was inherited in a dominant autosomal pattern. The incidence of the heritable fragile site in normal and leukemic cells of the patient, as well as stimulated blood cultures of his relatives, are reported. In addition, the possible relationship between this constitutional chromosome breakage syndrome and the occurrence of leukemia is analyzed.     

Nonrandom chromosomal changes in untreated retinoblastomas

The karotypic patterns of 15 retinoblastomas were examined. Five tumors were found to have two distinct stem lines and, therefore, the chromosomal patterns of 20 tumor cell lines are reported. Three nonrandom chromosomal changes, namely, a loss of a chromosome #13, the presence of an i(6p), or a trisomy of 1q were observed. The potential importance of these chromosomal changes in tumor development is discussed, particularly the loss of a chromosome #13 or the gain of an i(6p). At least one of the three chromosomal changes was found in 75% of the tumor lines analyzed.     

Chemical clastogenicity in lymphoid cell lines of chromosomal instability syndromes

Long-term lymphoid cell lines (LCL) derived from normal individuals, patients with ataxia telangiectasia (A-T), xeroderma pigmentosum (XP), and Fanconi anemia (FA) were exposed to various concentrations of 11 chemical clastogens. The agents were chosen to represent a variety of suggested modes of action. In contrast to all other genotypes, the FA lines demonstrated significant rates of spontaneous chromosomal breakage and showed hypersensitivity to all of the clastogens employed. Variability among lines within a genotype suggested individual responses to specific agents. Computation of “corrected values” to address the problem of baseline disparity removed some of the significant differences between the FA and other lines. Nonetheless, following correction, the FA genotype was still delineated by clastogens which are not DNA cross-linkers. The A-T lines were specifically identified by the induction of chromosome damage by bleomycin and neocarzinostatin.     

Hypotetraploidy in erythroleukemia

Cytogenetic study of a case of erythroleukemia in a 9-year-old white male showed a modal number of 72 with tetraploidy of several chromosomes and duplication of markers. The disease had a very rapid initial response to therapy. The uniqueness of this karyotype and its relationship to previously reported cytogenetic studies on erythroleukemia are discussed.     

Chromosome analysis of three seminomas

Each of three seminomas revealed chromosome #1 and #12 structural changes in direct preparations and short-term cultures. The #1 changes involved duplication of 1q and loss of 1p; in two, the breakpoint was in the heterochromatic region. The anomaly in #12 was a short arm isochromosome, usually present in duplicate. In one tumor, these were the only structural changes; in the other two, there was also involvement of #7, with extra copies of 7p. In one of these two tumors, a heterochromatic minute was identified after C-banding, and in the other, aside from two different markers containing part of #7, there was a dicentric derived from two chromosomes #15; this tumor proved to be prognostically unfavorable. Three normal chromosomes #1 and XXY sex chromosomes were present in each tumor. Chromosomes #11 and #13 were generally underrepresented, and #12 and #19-22 were over-represented.     

Fragile sites and cancer breakpoints

To determine whether there might be a statistically significant association between fragile sites and cancer breakpoints, we examined the locations of the 21 fragile sites and the 50 cancer breakpoints recently accepted by the Seventh Human Gene Mapping Workshop. Nine of the 21 fragile sites appeared to be located at or near a cancer breakpoint. The chi-square test for association gives a value of 15.8 (p < 0.001) indicating that there is a very highly significant statistical association between human fragile sites and cancer breakpoints. This association is not narrowly limited to one class of fragile site, such as those sensitive to folate or to one type of cancer, but appears to extend to leukemia, lymphoma, and solid cancer. To more fully understand the meaning of this intriguing association between fragile sites and cancer breakpoints, future research will need to locate additional fragile sites and cancer breakpoints with precision, record their concurrence in individuals and families, determine if fragile site families are predisposed to cancer, and prove that a fragile site and a cancer breakpoint that appear to be coincident are at the same point on the DNA level.     

Chromosome replication in normal and transformed human lymphocytes

We analyzed the late-replication patterns of human B-lymphocyte chromosomes before and after transformation by Epstein-Barr virus. There were no statistically significant differences between normal cells and transformed cells derived from the same male individual; therefore, the order of termination of chromosome replication was unchanged by transformation. We also examined the replication patterns of T lymphocytes from the same donor and found no differences between normal B and T cells.     

Abnormalities of chromosome #13 in retinoblastomas from individuals with normal constitutional karyotypes

Constitutional chromsome abnormalities have been associated with retinoblastoma, Wilms' tumor, and a familial form of renal cell carcinoma. For each tumor type, the particular chromosome segment involved in the observed rearrangements is different: in retinoblastoma, that segment is band q14 on chromosome #13. We now present evidence that in retinoblastoma, structural abnormalities involving the particular chromosome segment identified in the constitutional cases can also occur in the tumors of individuals with normal constitutional karyotypes. Six cases with retinoblastomas in one or both eyes were analyzed; deletions/rearrangements involving 13q14 were found in the tumor cell karyotypes of five of the six. These observations suggest that changes in a gene or genes at a common site (13q14) play a role in tumorigenesis in all forms of retinoblastoma, sporadic as well as heritable.