Biomedical subjects
Publications and source records attributed to J D Rowley.
Cultures of bone core specimens have proved satisfactory for cytogenetic analysis in patients from whom it was impossible to obtain a bone marrow aspirate, or in whose peripheral blood dividing myeloid cells were absent or insufficient in number. The quality of the metaphase chromosome is adequate for banding studies.
Metaphase chromosomes from the Chinese hamster cell line M3-1 were separated by means of a flow sorter. Two chromosome fractions were used for this study: A, which consisted of 95% pure chromosome no. 1, and B, which was 90% pure chromosome no. 2. The DNA of 10(6) chromosomes of each type was purified, and a 125I-cRNA transcript was synthesized in a reaction containing E. coli RNA polymerase and carrier-free 125I-CTP (1.7 Ci/mumole). The cRNA product synthesized with template DNA from 10(5) sorted chromosomes contained more than 10(6) dpm. The electrophoretic mobility profiles of the cRNAs on 7.5% SDS acrylamide gels demonstrated that more than 50% of the ribo-polymers were equal to or longer than marker E. coli met-tRNAf. In hybridization reactions 21% and 17% of the transcripts from Chinese hamster whole cell and sorted chromosome DNA hybridized to Chinese hamster DNA and did not hybridize significantly over background in reactions containing calf DNA at Crt values of 1.3 and 1.9 x 10(2) mole sec/l. Labelled cRNAs transcribed from the DNA of sorted chromosomes hybridized with the DNA of each sorted chromosome fractions at a Crt of 0.6 mole sec/l. This study demonstrated that the DNA can be (1) recovered from small numbers of highly purified flow sorted chromosomes, (2) used as template by E. coli RNA polymerase and (3) used to prepare a cRNA in reactions containing polymerase and carrier-free 125I-CTP to yield a product which can be employed for hybridization analysis.
Ten of 55 patients with chronic myelogenous leukemia (CML) diagnosed between 1972 and 1977 were found to lack the Philadelphia (Ph1) chromosome. Serial clinical, morphologic, and cytogenetic studies of patients with Ph1-negative CML showed that 30% of them had chromosomal abnormalities. Two had an extra chromosome No. 8 at the time of blast crisis, with a morphological picture of myeloblasts in the bone marrow. A third patient had a 6:14 translocation initially Abnormalities of chromosome No. 14 are frequently seen in lymphoproliferative disorders, and the bone marrow and peripheral blood contained a significant population of lymphoblasts as well as myeloblasts. The median survival for the 10 patients was 19 months. The exact nature of Ph1-negative CML is not yet clear; disease appears to be a distinct entity among the myeloproliferative disorders.
Karyotypic abnormalities were detected in the malignant cells of 6 of 18 patients with multiple myeloma (MM). Six patients with benign monoclonal gammopathy, one with amyloidosis of immunoglobulin origin, and two with Waldenström's macroglobulinemia had normal karyotypes. All six MM patients with aneuploidy were in a group of 10 patients in an accelerated or relapse phase of their disease and four had high serum paraprotein levels (7.92, 6.24, 6.80, and 4.24 g/dl, respectively) when their abnormal karyotypes were detected. Five of the 6 MM patients with aneuploidy had received prior chemotherapy. Aneuploidy was not observed in 8 stable MM patients. Abnormalities of chromosome 14 were present in all 6 patients, with a 14q+ marker in 5 and loss of No. 14 in 1. A translocation between Nos. 11 and 14 was found in aneuploid cells of 2 patients who had plasma cell leukemia (PCL). However, the break point in the long arm of No. 11 differed in the 2 patients. A gain of Nos. 5, 9, and 11 was seen in 3 patients, a gain of No 1 in 2, and rearrangements of No. 1 in 5 MM patients, including all 4 who had a 14q+ marker chromosome initially. A deletion of chromosome 6 at band q25 was detected in 2 MM patients and a pericentric inversion of No. 6 (6p21 to 6q13) was seen in the patient with PCL. Three of 4 MM patients had a nonrandom loss of one chromosome 8. Two other MM patients, who were treated with melphalan and prednisone, developed acute nonlymphocytic leukemia (ANLL) 2+ and 4+ years after the diagnosis of MM. Marrow cells of one patient showed a 5q- chromosome and a constitutional translocation involving Nos. 13 and 14 during the preleukemic stage; during the leukemic phase, the karyotype evolved to 50 chromosomes including extra chromosomes 1, 6, 8, 10, and 21 and a missing 7, in addition to the originally detected 5q- and the 13/14 translocation. The peripheral blood from the other patient was hypodiploid, with a missing chromosome 7 and a translocation between 3q and 9p. These patterns of chromosome change resemble those of ANLL rather than MM and are similar to the changes seen in ANLL after treated malignant lymphoma.
The consistent occurrence of nonrandom chromosome changes in human malignancies suggests that they are not trivial epiphonomena. Whereas we do not understand their significance at present, one possible role which they may fulfill is to provide the chromosomally aberrant cells with a proliferative advantage as the result of alteration in the number or location of genes related to nucleic acid biosynthesis. The proliferative advantage provided by various chromosome aberrations is likely to differ in patients with different genetic constitutions.
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An adult patient with acute lymphoblastic leukemia associated with a 14q+ marker chromosome is presented. The abnormality resulted from a translocation of material from the long arm of chromosome 11. The leukemic cells were found to be B cells on the basis of surface immunoglobulins, lack of receptors for sheep erythrocytes, and a characteristically low level of adenosine deaminase activity. In other patients with ALL studied by us or reported by others in whom chromosome banding was done, a 14q+ chromosome was present in only one instance, also a case of B cell ALL. These two cases are the only examples of B cell ALL studied with chromosome banding reported to date. The frequent occurrence of a 14q+ chromosome in other malignant lymphoproliferative diseases of B cell origin suggests that a general association may exist between the 14q+ abnormality and B cell neoplasms. Cytogenetic analysis may therefore be useful in defining subtypes of ALL and in relating specific chromosomal abnormalities to lymphoproliferative disorders.
Karyotypes were analyzed by routine Giemsa and quinacrine fluorescence for 16 patients with acute lymphocytic leukemia [ten adults (18 to 51 years) and six children (3 to 15 years)]. Four patients had received previous therapy, but all 16 had active disease when they were first studied. Eight patients (five untreated) had a normal karyotype initially; however, three of these developed a chromosomal abnormality during relapse. Eight patients had a chromosomal abnormality in their initial samples. Each of the 11 patients had different abnormalities. All chromosomes except Nos. 3, 5, 15, 16, and Y were involved in the various aneuploidies. One patient had a Ph1 chromosome due to a translocation with No. 21: t(21;22)(q22;q11). A patient with B-cell acute lymphocytic leukemia had a 14q+ marker in addition to other abnormalities. The median survival of patients with initially normal karyotypes may be longer than that of patients whose karyotypes are abnormal initially.
DNA cytophotometry was used to quantify the chromosomal alterations in the bone marrow and blood of three patients with nonspecific myeloid disorders. All patients possessed a population of cells with a morphologically abnormal chromosome 20, del(20)(qll). In two of the patients, the abnormal chromosome 20 showed nearly identical DNA measurements with a net loss of 0.37% of the total autosomal DNA in one patient and 0.38% in the second. The third patient had a net loss of only 0.25% of the autosomal DNA. Analysis of the DNA content of the long arm and short arm of the abnormal No. 20 indicated that all three cases had chromosomal material added to the short arm (0.10 to 0.14% of the autosomal DNA). About 0.50% of the autosomal DNA was deleted from the long arm in two of the patients; only 0.35% of the autosomal DNA was deleted from the long arm in the third case. Within the limit of resolution, there is no evidence that the material lost has been translocated intact to another chromosome. The origin of the 20q- chromosome as the result of an incomplete pericentric inversion is suggested.
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Analysis of chromosomal banding patterns in acute nonlymphocytic leukemia (ANLL) reveals that approximately 50% of patients have an abnormal karyotype. Although there is substantial variability, certain nonrandom abnormalities occur, e.g., +8, -7, and the 8;21 translocation (often accompanied by loss of an X or Y chromosome). The 15;17 translocation appears to be highly specific for acute promyelocytic leukemia. These abnormalities usually are not seen in remission, but reappear in relapse, sometimes exhibiting further clonal evolution; a +8 is the most frequently observed evolutionary change. Patients with ANLL following treatment of a malignant lymphoma tend to have hypodiploid modal numbers and frequently show loss of a chromosome No. 5 or No. 7.
We studied the chromosomes from 26 patients with hairy cell leukemia (HCL) to ascertain the frequency and types of consistent chromosomal abnormalities. Samples from 21 patients were obtained from peripheral blood cultures grown 24 and 48 h without phytohemagglutinin, or from bone marrow samples. Two male patients had similar, consistent abnormalities; one patient's karyotype was 46, X, +12; that of the second was 46, X, +C marker. In the latter case, the distal long arm of the C marker most closely resembled chromosome No. 12 from band q14 to q terminal, but the short arm and proximal long arm were of undetermined origin. Both karyotypes lacked the Y chromosome. Nine of the 21 patients had abnormalities in single cells. One patient had, in one sample, a single abnormal cell with an extra No. 3 and an extra No. 12 (48, XY, +3, +12), and in a later sample, a second cell of poor morphology which also could have been trisomic for No. 12. Another patient had one cell with an unusually bright short arm, as well as two cells, with different abnormalities, both involving the short arm of chromosome No. 1. The two patients with consistent chromosome abnormalities had rapidly progressive disease in spite of splenectomy, and their clinical course from the time of diagnosis was relatively short (5 and 7 months, respectively).
Studies of human hematologic malignancies have provided sufficient data not only for the identification of nonrandom abnormalities of whole chromosomes, but also for determination of the specific chromosome regions involved. In clonal aberrations leading to an excess of chromosome No. 1, or a partial excess of No. 1, trisomy for bands 1q25 to 1q32 was noted in the myeloid cells obtained from every one of 35 patients who had various disorders, such as acute leukemia, polycythemia vera, or myelofibrosis. Similar chromosome changes were a consistent finding in various solid tumors as well. This rearrangement was not the result of a particularly fragile site in that region of the chromosome, since the break points in reciprocal translocations that involve No. 1 occurred almost exclusively in the short arm. The nonrandom chromosome changes found in neoplastic cells can now be correlated with the gene loci on these chromosomes or chromosome segments as an attempt is made to identify specific genes that might be related to malignancy.
We observed chromosome-banding abnormalities in leukemic cells of 46 of 90 (51 per cent) adults with acute nonlymphocytic leukemia at initial hospital admission. The difference in survival between 37 treated patients with an initially normal karyotype (10 months) and 43 with an initially abnormal karyotype (four months) was significant (P less than 0.01). When patients were classified as having acute myelogenous leukemia or acute myelomonocytic leukemia, this difference in survival was even more pronounced. Of 16 treated patients with acute myelogenous leukemia and a normal karyotype, 11 (69 per cent) had a complete remission and a median survival of 13 months. Of eight patients with acute myelogenous leukemia in whom only abnormal metaphases were observed, none had a complete remission, and the median survival was only two months (P approximately 0.50). Remission rate and median survival were not significantly different in patients with acute myelomonocytic leukemia grouped according to initial karyotypes.
Chromosome studies were performed on malignant cells obtained from 27 patients with non-Burkitt lymphomas. A marker chromosome affecting the long arm of No. 14 (14q+) was the single most frequent abnormality and was noted in 17 of these patients. The frequency of the 14q+ marker varied with the type of lymphoma. For patients with malignant lymphoma, histiocytic, the frequency was 5 of 8; for mixed-cell type, 1 of 3; for poorly differentiated lymphocytic, 8 of 8; for well-differentiated lymphocytic, 0 of 3; for lymphoblastic, 0 of 1; for Hodgkin's disease, 2 of 3; and for mycosis fungoides, 1 of 1. The donor chromosome involved in the 14q translocation was identified in 12 cases; certain chromosomes appeared to be affected more frequently than others. Although the break point was band 14q32 in most cases, the exact location of the receptor site on 14q was not always consistent. The distal part of 14q24 was also involved as a receptor site in at least one translocation. These findings suggest that, in some types of lymphoid malignancy, cells with a 14q translocation have a proliferative advantage over cells with other chromosome rearrangments. The presence of the 14q translocation may be important in the future for the distinction among morphologically different, but functionally comparable, subgroups of lymphoid malignancies.
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