Scand I Haematol (1975) 14,201-209
Cytogenetic Studies in Myelomatosis MOGENSKRQGHJENSEN, M.D., JANERIKSEN, M.D. & BIRGITTE WILTON DJERNES, M.D. Division of Haematology, Department of Medicine B (Chief, M . Krogh Jensen), Aalborg Sygehus Syd, Aalborg, Denmark
Cytogenetic studies have been carried out on bone marrow aspirates from 25 patients with myelomatosis. Abnormal stem lines were present in 7 of the patients; the remainder had a diploid chromosome complement. In most patients - also in those without an abnormal clone - some metaphases had a blurred appearance similar to that seen in bone marrow aspirates from patients with acute leukaemia. In many of the chromosome preparations obtained before cytostatic therapy some metaphases with structural aberrations of the chromosomes were seen. Evidence is presented that in the patients with abnormal stem lines in the bone marrow, the chromosome abnormalities are confined to the myeloma cells and are not found in the erythrocytic or granulocytic precursors, which thus do not seem to be involved by the neoplastic process. Based on the present results and on a review of the relevant literature some general cytogenetic features are emphasized which may contribute to a better understanding of the disorder. Especially, it is demonstrated that in myelomatosis the cytogenetic changes are much more uniform than in other malignant disorders with the exception of chronic myeloid leukaemia. K e y words: cytogenetics
- myelomatosis
Accepted for publication January 31, 1975 Correspondence to: Dr. M. Krogh Jensen, Aalborg Sygehus Syd, DK-9100 Aalborg, Denmark
Abnormalities of the chromosome complement frequently occur in neoplastic cells of patients with haematologic malignancies. A specific chromosome abnormality has only been demonstrated in chronic myeloid leukaemia, whereas in acute leukaemia the chromosome abnormalities are unspecific and vary from patient to patient. In some
patients with myelomatosis extra marker chromosomes, the size of chromosomes nos. 1-3, have been described. However, whereas the cytogenetic findings in acute leukaemia and chronic myeloid leukaemia have been extensively studied, the chromosome constitution of patients with myelomatosis remains rather unclarified, probably due to
This work has been supported by a grant from ‘The Scientific Fund of North Jutland’ Scand J Haematol (1975) 14
14
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M. KROGH JENSEN, JAN ERIKSEN & BIRGITTE W. DJERNES
technical difficulties in obtaining satisfactory chromosome preparations from bone marrow aspirates in myelomatosis. This report presents the cytogenetic findifigs in 25 patients with myelomatosis and reviews the literature on cytogenetics in myelomatosis. The purpose has been primarily to describe the frequency and types of chromosome abnormalities present in myelomatosis and secondarily to clarify whether the chromosome abnormalities are confined to the plasma cells or whether they are also present in other cell lines of the bone marrow (i.e. the granulocytic and erythrocytic precursors) as is the case in acute and chronic myeloid leukaemia.
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MATERIAL AND METHODS From 1969 t o 1974 cytogenetic studies were performed on bone marrow aspirates from 25 unselected patients with myelomatosis. Their ages ranged from 29 to 77 years. 17 were males and 8 females. 16 of the patients were studied before treatment, the remainder had received therapy with either melphalan or cyclophosphamide. In the patients with abnormal stem lines in the bone marrow and in some of the patients with normal diploid modes differential counts of the mitotic figures were performed in smears from the same bone marrow aspirates which were used for chromosome study. Mitoses were classified as plasmocytic only if the mitotic cells corresponded in size and tinctorial characteristics to plasma cells or myeloma cells. All other mitoses were considered 'non-plasmocytic'. Between 50 and 100 mitoses were counted. The chromosome studies were performed according to the method described by Lam-PooTang (1968).
RESULTS
Three types of abnormalities of the chromosome complement were seen, viz. abnormal
0000000
mmmmmlrrm
CYTOGENETIC STUDIES I N MYELOMATOSIS
stem lines, structural aberrations and abnormal appearance of the chromatin. Abnormal stem lines. 7 of the 25 patients had an abnormal stem line in the bone marrow. The remainder had a diploid chromosome complement. The abnormal clones were hyperdiploid in all cases (Table I). Patient no. 1. All the metaphases with 47 chromosomes contained a submetacentric marker chromosome but 1Y2 x the size of a chromosome 1 (Figure 1). Patient no. 2. The metaphases with 54 chromosomes could not be completely analyzed. However, an abnormal acrocentric marker chromosome the size of a chromosome 2 and two extra chromosomes in group 2122 were seen. Patient no. 3. The metaphases with 53 chro-
Figure 1. Metaphase from patient no. 1 with 47 chromosomes and a large submetacentric chromosome.
203
mosomes could not be completely analyzed. However, there were two supernumerary large submetacentric chromosomes similar in size and proportion to a normal chromosome 2. There were three supernumerary chromosomes in group 6-12 and two additional acrocentric chromosomes half the size of the chromosomes belonging to group 21-22. 8 of the 14 metaphases with 44 chromosomes could be analyzed. There was a random loss of chromosomes and thus n o evidence of the presence of a hypodiploid stem line. Patient no. 4. The metaphases with 48 chromosomes contained an abnormal acrocentric marker chromosome approximately the size of a chromosome 3. There were two supernumerary chromosomes in both group 6-12 and group 18-20, whereas two chromosomes were missing in group 13-15 and one chromosome in group 21-22.
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TABLE I1 Comparison between the proportion of ‘plasmocytic’ and ‘non-plasmocytic’ mitoses in the direct marrow smears and the proportion of abnormal and normal metaphases in the chromosome preparations
1
Mitosses
19 60 16 16 88 92
Patient
% plasma cell
mitosels
% ‘non-plasnio-
cytic’ mitoses
Metaphases
Patient no. 81 40 84 84 12 8
TABLE 111 Percentage of plasma cell mitoses in bone marrow smears of 8 patients without abnormal stem lines in the bone marrow
80 40 52 66 8 4
20 60 48 38 92 96
Patient no. 5 . The metaphases with 47 chromosomes had an extra acrocentric chromosome half the size of the chromosomes belonging to group 21-22. Patient no. 6. None of the four metaphases with 47 chromosomes could be analyzed. Patient no. 7 . In both metaphases with 47 chromosomes the hyperdiploidy was due to an extra acrocentric chromosome slightly larger than the chromosomes in group 2122. About 50 % of the hypodiploid and diploid metaphases present in the marrow aspirates of the 25 patients could be analyzed. No evidence of a hypodiploid or pseudodiploid clone of cells was found.
In Table I1 the percentage of plasma cell mitoses in the direct bone marrow smears from the patients with abnormal stem lines is compared to the percentage of aneuploid metaphases in the chromosome preparations from the same marrow aspirates. From 8 to 84 % of the mitotic figures in the direct marrow smears belonged to the plasma cell series. The frequency of aneuploid metaphases in the chromosome preparations ranged from 4 to 80 %. Con-
A B C D E F G H
8 36 8 15 64 18 74 3
92 64 92 85 36 82 26 97
sidering the data on the individual patients it is evident that in most of the patients the proportion of plasma cell mitoses corresponded fairly well to the proportion of aneuploid metaphases. It may be observed that in none of the patients more aneuploid metaphases than plasma cell mitoses were found. Table I11 shows the results of the differential counts of bone marrow cells in mitoses in 8 of the patients in whom no abnormal stem line could be demonstrated. The percentage of plasma cell mitoses varied from 3 to 74. Abnormal appearance of the chromatin. It was a characteristic finding that in many of the patients the chromosomes of some of the metaphases studied had a blurred appearance with an ill-defined boundary of the chromatids. In those patients which had abnormal stem lines it was characteristic that it was the aneuploid metaphases which had blurred chromosomes, whereas the diploid metaphases had a more natural looking appearance. Likewise, in patients without aneuploid stem lines in the bone marrow a varied number of the diploid metaphases had blurred chromosomes, whereas the remainder of the metaphases had a normal
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CYTOGENETIC STUDIES IN MYELOMATOSIS
appearance. However, in many of the metaphases the appearance of the chromatin was difficult to evaluate as many of the marrow aspirates yielded technically unsatisfactory metaphases with ill-defined contracted chromosomes.
Chromatid and chromosome breaks. Structural chromosome aberrations were present in 9 of the 16 patients who were studied before cytostatic therapy. They were almost exclusively breaks of the chromatid type, but a few chromosome breaks were also present. The proportion of bone marrow metaphases with breaks varied from 2 to 22 % (mean 4.4 %). In 10 control patients without a haematologic disorder structural aberrations were found in 1.7 % of 350 metaphases studied. In none of these controls aberrations were present in more than 4 % of the metaphases. It was a characteristic feature that breaks were mainly seen in the aneuploid metaphases and in the diploid metaphases which had a blurred appearance. The cytogenetic findings could not be correlated to the type of abnormal immunoglobulin present in the serum of the patients. Likewise, it was not possible to find any correlation between the cytogenetic findings and the course of the disease. DISCUSSION
Cytogenetic studies have not been performed nearly as extensively in myelomatosis as in chronic myeloid and acute leukaemia. This may partly be due to the fact that the quality of chromosome preparations from bone marrow aspirates in myelomatosis is inferior to that obtainable in other haematologic disorders (Lewis et a1 1963, Dartnall et a1 1973).To our opinion
TABLE IV The frequency of abnormal stem lines in bone marrow aspirates from 127 patients with myelomatosis
Source of data
Baikie et a1 Bottura Lewis et a1 Hayhoe & Hammouda 1965 Ponti et a1 Siebner et a1 Dubrova et a1 1966 Das & Aikat 1967 Kanzow et a1 1967 Tassoni et a1 1967 Itani et a1 1970 Rochon et a1 1971 Dartnall et a1 1973 Anday et a1 1974 Present series Total
No. of No#.of patients with patients examined abnormal stem lines 2 5 3 4 4 2
0 1 1 0
5 2 14 7 8 4 38 25
0 2 2 4 0 8 2 1 0 7 7
127
35
4
it is difficult to accomplish a perfect spreading of the bone marrow metaphases in myelomatosis; however, we have been able to study 50 metaphases from most of the patients of the present series. Due to the difficult techniques the types of chromosome abnormalities, the frequency with which they appear, and their relation to the cytologic findings are not very much elucidated. Nevertheless, a review of the cytogenetic literature together with some of the findings in the present study make several general aspects appear which may lead to a better understanding of myelomatosis and its chromosome constitution. Some of these are summarized below. 1. Abnormal stem lines were present in 7 of the 25 patient; of the present series. Table IV shows the results of previous cytogenetic studies on bone marrow aspirates from patients with myelomatosis. It
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M. KROGH JENSEN, J A N ERIKSEN & BIRGITTE W. DJERNES
TABLE V The ploidy of abnormal stern lines in 22 patients with mvelomatosis Hypodiploid Pseudodiploid Hyperdiploid
5 patients 4 13 -
is seen that in about 25 % of the patients abnormal stem lines could be detected in the bone marrow. Table V shows that both hypodiploid, pseudodiploid and hyperdiploid clones are seen, the hyperdiploid being the most common. The chromosome counts of the hypodiploid stem lines varied from 42 to 45, the hyperdiploid stem lines contained between 47 and 86 chromosomes. Abnormal stem lines seem to occur less frequently than in acute leukaemia, where about 50 % of the patients have abnormal clones in the bone marrow (Krogh Jensen 1969). Also in acute leukaemia both hypodiploid, pseudodiploid and hyperdiploid stem lines are present. Thus, cytogenetic findings seem to set apart the haematologic neoplasias (acute leukaemia and myelomatosis) from the solid neoplasms in which nearly 100 % have an abnormal chromosome constitution and where hyperdiploid clones are almost exclusively seen (Yamada et a1 1966). In order to elucidate why abnormal stem lines are found in so relatively few patients with myelomatosis we performed differential counts of mitotic cells from some of the patients in whom no abnormal stem line could be seen. Table I11 shows that from 3 t o 74 % of the mitoses belonged to the plasma cell series, in 5 patients less than 20 % of the cells in mitosis being plasma cells. Probably, the rather low frequency with which abnormal stem lines are found in patients with myelomatosis is due to the fact that in many cases the number of plas-
ma cells in mitosis is so low that they are not represented when only 50 metaphases can be counted in the chromosome preparations. On the other hand, in some cases of myelomatosis the myeloma cells must have a normal diploid chromosome constitution, as in some of our patients without abnormal stem lines more than 50 % of the mitotic figures in the direct bone marrow smears belonged to the plasma cell series. However, it may be noted that all metaphases with 46 chromosomes could not be analyzed. 2. So far the only specific chromosome abnormality in malignant diseases has been the Phl chromosome in chronic myeloid leukaemia. However, in 1961 in a patient with Waldenstrom’s macroglobulinaemia Bottura et a1 described a large supernumerary submetacentric chromosome the size of the chromosomes no. 1-2. In the following years similar marker chromosomes have been described in several other patients with this disorder (German et a1 1961, Houston et a1 1967) and also in patients with myelomatosis (Siebner et a1 1965, Houston et a1 1967, Tassoni et a1 1967, Rochon et al 1971, Anday et a1 1974). In 4 of the 7 cytogenetically abnormal patients of the present series supernumerary chromosomes the size of which ranged between a chromosome no. 3 to larger than no. 1were seen. In 2 cases the chromosomes were acrocentric and in 2 cases the chromosomes were submetacentric. 28 of the abnormal stem lines mentioned in Table IV could be analyzed. 21 contained an abnormal marker chromosome of similar size as described in the present 4 patients. In 11 cases the chromosome was acrocentric, in 9 cases submetacentric and in 1 case metacentric. It has been proposed - and some evidence has been presented - that the large
CYTOGENET’IC STUDIES IN MYELOMATOSIS
acrocentric marker chromosome originates from a translocation from some other chromosome to a normal group D (Tassoni et a1 1967, Mancinelli et a1 1969). Similarly, evidence has been presented that the large submetacentric chromosome originates from a chromosome no. 1 or 2 possibly due to pericentric inversion and mitotic non-disjunction (Patau 1961). Marker chromosomes of a similar appearance have been described in some cases of acute leukaemia (Krogh Jensen 1969) and solid tumors (Yamada et al 1966) and are thus not unique for myelomatosis. However, they appear so often (in 21 out of 28 patients cytogenetically studied (Table 111) that the cytogenetic changes in myelomatosis (and macroglobulinaemia) seem to be much more uniform than in any other malignant disorder - with the exception of chronic myeloid leukaemia. 3. Evidence has been presented that in acute leukaemia (Krogh Jensen & Killmann 1967, 1971) and chronic myeloid leukaemia (Clein & Flemans 1966, Rastric et a1 1968) the chromosome abnormalities are not confined to the ‘leukaemic’ blast cells alone but are also present in the erythroblasts and possibly in the megakarycxytes. In the patients of the present series with abnormal stem lines we therefore made differential counts of the mitotic figures in the direct bone marrow smears and compared the results to the findings in the chromosome preparations. It was found that the percentage of abnormal metaphases in the chromosome preparations corresponded well to the percentage of plasma cell mitoses. These findings indicate that in myelomatosis the chromosome abnormalities are only present in the cells belonging to the plasma cell series. Thus, in myelomatosis the neoplastic pro-
207
cess seems to be confined to the plasma cells, whereas in acute and chronic myeloid leukaemia both the erythrocytic and granulocytic precursors and possibly also the megakaryocytes are involved by the neoplastic process. This fundamental difference between the degree of ‘neoplastic involvement’ of the bone marrow cells in leukaemia and myelomatosis may explain why severe anaemia, granulocytopenia and thrombocytopenia are nearly invariably seen at the debut of leukaemia, whereas these findings are usually only seen in patients with an advanced myelomatosis. Furthermore, the cytogenetic differences mentioned above may offer an explanation of the different therapeutic response to cytostatic therapy seen in these two disorders. 4. It is a characteristic feature of the chromosome findings in acute leukaemia that the chromatin of most of the bone marrow metaphases has a blurred and ill-defined appearance (Sandberg et a1 1964, Krogh Jensen 1969, 1971). In patients with abnormal stem lines this finding is confined to the aneuploid metaphases, whereas in patients with diploid modes both diploid blurred metaphases and diploid metaphases with normal chromatin are seen. Usually, the abnormally looking metaphases make up the majority of the metaphases in chromosome preparations in acute leukaemia. To our opinion the metaphases - whether aneuploid or diploid - with abnormal chromatin constitute the neoplastic (leukaemic) cell population. Similarly, the present report shows that in many bone marrows from patients with myelomatosis some metaphases with abnormally looking chromatin are present. At present we have no methods which allow us to estimate for certain
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whether the chromatin of different metaphases have an abnormal appearance, wherefore it is very difficult to make comparisons between the appearance of metaphases in different chromosome preparations. However, it is evident that a much smaller proportion of the metaphases has a blurred appearance in patients with myelomatosis than in patients with acute leukaemia. Thus, these findings also indicate that the neoplastic cell population is considerably smaller in myelomatosis than in leukaemia. The demonstration of an increased number of metaphases with chromatid and chromosome breaks in the untreated patients of the present series shows that structural chromosome abnormalities are characteristic of some metaphases from neoplastic bone marrow aspirates. Similar findings have been described in acute leukaemia (Krogh Jensen 1969). The present material is too small to evaluate whether the cytostatic therapy employed in some of the patients produced further structural aberrations. Melphalan (Wantzin & Krogh Jensen 1973) as well as cyclophosphamide (Arrighi et a1 1962) have been shown to be chromosome-breaking agents in vivo.
In conclusion cytogenetic studies in myelomatosis have shown that similar abnormalities of the chromosome complement are present as has been described in acute leukaemia and other types of neoplasia, viz. abnormal stem lines and metaphases -whether aneuploid or diploid - with blurred chromatin. In addition the presence of supernumerary marker chromosomes the size of chromosomes 1-3 is a characteristic finding in many cases of myelomatosis. The cytogenetic studies indicate that the neoplastic process is confined to the myeloma cells
and do not comprise the other cell types of the bone marrow. Additional cytogenetic studies ought to be performed in myelomatosis with the modern banding techniques in order to elucidate the origin of the characteristic abnormal marker chromosomes and to see whether some of the diploid cases will turn out to be pseudodiploid. However, it is to be expected that the banding techniques will be employed with little success in myelomatosis as even the preparations obtained with conventional techniques are of unsatisfactory quality. Finally, the chromosomal findings in the peripheral blood cells of patients with myelomatosis are at present rather confusing. Studies are clearly needed which compare the cytogenetic findings in bone marrow cells and both stimulated and unstimulated peripheral blood cells from the same patients. Preferably mitogens which stimulate either T cells or B cells should be used, such as phytohaemagglutinin and lipopolysaccharides. REFERENCES Anday G J, Fishkin B & Gabor F P (1974) Cytogenetic studies in multiple myeloma. J Nut1 Cancer Inst 52, 1069-79. Arrihgi F E, Hsu T C & Bergsagel D E (1962) Chromosome damage in murine and human cells following cytoxan therapy. Tex Rep Biol Med 20, 545-49. Baikie A G, Court Brown W M, Jacobs P A & Milne J S (1959) Chromosome studies in human leukaemia. Lancet ii, 425-28. Bottura C (1963) Chromosome abnormalities in multiple myeloma. Acta Haematol (Basel) 30, 27479. Bottura C, Ferrari I & Veiga A A (1961) Chromosome abnormalities in Waldenstrom’s macroglobulinaemia. Lancet i, 1070. Clein G P & Flemans R J (1966) Involvement of the erythroid series in blastic crisis of chronic myeloid leukaemia. Further evidence for the
CYTOGENETIC STUDIES IN MYELOMATOSIS presence of Philadelphia chromosome in erythroblasts. Brit J Haematol 12, 754-58. Dartnall J A, Mundy G R & Baikie A G (1973) Cytogenetic studies in myeloma. Blood 42, 22939. Das K C & Aikat B K (1967) Chromosomal abnormalities in multiple myeloma. Blood 30, 738-48. Dubrova S E, Dygin V P & Ushakova E A (1966) The cytogenetical and clinico-hematological changes in myeloma disease. Tsitologiia 8, 24149. German J L, Biro C E & Bearn A G (1961) Chromosomal abnormalities in Waldenstrom’s macroglobulinaemia. Lancet ii, 48. Hayhoe F G J & Hammouda F (1965) Cytogenetic and metabolic observations in leukaemias and allied states. In F G J Hayhoe (ed) Current Research in Leukaemia. University Press, Cambridge, England. Houston EW, HoshinoT, Kawasaki S & Nahayama S (1970) Chromosome abnormality and its significance in human multiple myeloma. Acta Haematol Jap 33, 54-66. Kanzow V, Lange B, Niederalt G & Gropp A (1967) Chromosomenuntersuchungen bei Paraproteinamien. Klin Wochenschr 45, 1076-84. Krogh Jensex M (1969) Chromosome studies in acute leukaemia. Munksgaard, Copenhagen. Krogh Jensen M (1971) Cytogenetic studies in acute myeloid leukaemia. Acta Med Scand 190, 429-34. Krogh Jensen M & Killmann S-Aa (1967) Chromosome studies in acute leukaemia. Evidence for chromosomal abnormalities common to erythroblasts and leukaemic white cells. Acta M e d Scand 181, 47-53. Krogh Jensen M & Killmann S-Aa (1971) Additional evidence for chromosome abnormalities in the erythroid precursors in acute leukaemia. Acta M e d Scand 189, 97-100. Lam-Poo-Tang P R L C (1968) An improved method of processing bone marrow for chromosomes. Scand J Haematol 5, 158-60.
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Lewis F J W, Fraser I L & MacTaggart M (1963) An abnormal chromosomal pattern in myelomatosis. Lancet ii, 1013-15. Lewis F J W, MacTaggart M, Crow R S & Wills M R (1963) Chromosomal abnormalities in multiple myeloma. Lancet i, 1183-84. Mancinelli S, Durant J R & Hammack W J (1969) Cytogenetic abnormalities in a plasmocytoma. Blood 33, 225-33. Patau K (1961) Chromosomal abnormalities in Waldenstrom’s macroglobulinaemia. Lancet ii, 600-01. Ponti G B, Valentini R, Carrara P M & Eridani S (1965) Investigations on the chromosome complement in some myeloproliferative disorders. Acta Haematol (Basel) 34, 36-43. Rastrick J M, Fitzgerald P H & Gunz F W (1968) Direct evidence for presence of Phl chromosome in erythroid cells. Brit Med J 1, 96-98. Rochon M, Cadotte M, Pretty H M & Long L A (1971) Les anomalies chromosomiques du my& lome multiple. Experience personelle et revue de la littkrature. Union med Can 100, 1750-54. Sandberg A A, Ishihara T, Kikuchi Y & Crosswhite L H (1964) Chromosomal differences among the acute leukemias. A n n N Y Acad Sci 113, 663-716. Siebner H, Spengler G A, Butler R, Heni F & Riva G (1965) Chromosomenanomalien bei Paraproteinamie. Schweiz Med Wochenschr 95, 1767-77. Tassoni E M, Durant J R, Becker S & Kravitz B (1967) Cytogenetic studies in multiple myeloma: A study of fourteen cases. Cancer Res 27, 80610. Wantzin G L & Krogh Jensen M (1973) The induction of chromosome abnormalities by melphalan in rat bone marrow cells. Scand J Haematol 11, 135-39. Y a r a d a K, Takagi N & Sandberg A A (1966) Chromosomes and causation of human cancer and leukaemia. 11. Karyotypes of human solid tumors. Cancer 19, 1879-90.
Cytogenetic Studies in Myelomatosis MOGENSKRQGHJENSEN, M.D., JANERIKSEN, M.D. & BIRGITTE WILTON DJERNES, M.D. Division of Haematology, Department of Medicine B (Chief, M . Krogh Jensen), Aalborg Sygehus Syd, Aalborg, Denmark
Cytogenetic studies have been carried out on bone marrow aspirates from 25 patients with myelomatosis. Abnormal stem lines were present in 7 of the patients; the remainder had a diploid chromosome complement. In most patients - also in those without an abnormal clone - some metaphases had a blurred appearance similar to that seen in bone marrow aspirates from patients with acute leukaemia. In many of the chromosome preparations obtained before cytostatic therapy some metaphases with structural aberrations of the chromosomes were seen. Evidence is presented that in the patients with abnormal stem lines in the bone marrow, the chromosome abnormalities are confined to the myeloma cells and are not found in the erythrocytic or granulocytic precursors, which thus do not seem to be involved by the neoplastic process. Based on the present results and on a review of the relevant literature some general cytogenetic features are emphasized which may contribute to a better understanding of the disorder. Especially, it is demonstrated that in myelomatosis the cytogenetic changes are much more uniform than in other malignant disorders with the exception of chronic myeloid leukaemia. K e y words: cytogenetics
- myelomatosis
Accepted for publication January 31, 1975 Correspondence to: Dr. M. Krogh Jensen, Aalborg Sygehus Syd, DK-9100 Aalborg, Denmark
Abnormalities of the chromosome complement frequently occur in neoplastic cells of patients with haematologic malignancies. A specific chromosome abnormality has only been demonstrated in chronic myeloid leukaemia, whereas in acute leukaemia the chromosome abnormalities are unspecific and vary from patient to patient. In some
patients with myelomatosis extra marker chromosomes, the size of chromosomes nos. 1-3, have been described. However, whereas the cytogenetic findings in acute leukaemia and chronic myeloid leukaemia have been extensively studied, the chromosome constitution of patients with myelomatosis remains rather unclarified, probably due to
This work has been supported by a grant from ‘The Scientific Fund of North Jutland’ Scand J Haematol (1975) 14
14
202
M. KROGH JENSEN, JAN ERIKSEN & BIRGITTE W. DJERNES
technical difficulties in obtaining satisfactory chromosome preparations from bone marrow aspirates in myelomatosis. This report presents the cytogenetic findifigs in 25 patients with myelomatosis and reviews the literature on cytogenetics in myelomatosis. The purpose has been primarily to describe the frequency and types of chromosome abnormalities present in myelomatosis and secondarily to clarify whether the chromosome abnormalities are confined to the plasma cells or whether they are also present in other cell lines of the bone marrow (i.e. the granulocytic and erythrocytic precursors) as is the case in acute and chronic myeloid leukaemia.
t-N
N
d
MATERIAL AND METHODS From 1969 t o 1974 cytogenetic studies were performed on bone marrow aspirates from 25 unselected patients with myelomatosis. Their ages ranged from 29 to 77 years. 17 were males and 8 females. 16 of the patients were studied before treatment, the remainder had received therapy with either melphalan or cyclophosphamide. In the patients with abnormal stem lines in the bone marrow and in some of the patients with normal diploid modes differential counts of the mitotic figures were performed in smears from the same bone marrow aspirates which were used for chromosome study. Mitoses were classified as plasmocytic only if the mitotic cells corresponded in size and tinctorial characteristics to plasma cells or myeloma cells. All other mitoses were considered 'non-plasmocytic'. Between 50 and 100 mitoses were counted. The chromosome studies were performed according to the method described by Lam-PooTang (1968).
RESULTS
Three types of abnormalities of the chromosome complement were seen, viz. abnormal
0000000
mmmmmlrrm
CYTOGENETIC STUDIES I N MYELOMATOSIS
stem lines, structural aberrations and abnormal appearance of the chromatin. Abnormal stem lines. 7 of the 25 patients had an abnormal stem line in the bone marrow. The remainder had a diploid chromosome complement. The abnormal clones were hyperdiploid in all cases (Table I). Patient no. 1. All the metaphases with 47 chromosomes contained a submetacentric marker chromosome but 1Y2 x the size of a chromosome 1 (Figure 1). Patient no. 2. The metaphases with 54 chromosomes could not be completely analyzed. However, an abnormal acrocentric marker chromosome the size of a chromosome 2 and two extra chromosomes in group 2122 were seen. Patient no. 3. The metaphases with 53 chro-
Figure 1. Metaphase from patient no. 1 with 47 chromosomes and a large submetacentric chromosome.
203
mosomes could not be completely analyzed. However, there were two supernumerary large submetacentric chromosomes similar in size and proportion to a normal chromosome 2. There were three supernumerary chromosomes in group 6-12 and two additional acrocentric chromosomes half the size of the chromosomes belonging to group 21-22. 8 of the 14 metaphases with 44 chromosomes could be analyzed. There was a random loss of chromosomes and thus n o evidence of the presence of a hypodiploid stem line. Patient no. 4. The metaphases with 48 chromosomes contained an abnormal acrocentric marker chromosome approximately the size of a chromosome 3. There were two supernumerary chromosomes in both group 6-12 and group 18-20, whereas two chromosomes were missing in group 13-15 and one chromosome in group 21-22.
M. KROGH JENSEN, JAN ERIKSEN & BIRGITTE W. DJERNES
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TABLE I1 Comparison between the proportion of ‘plasmocytic’ and ‘non-plasmocytic’ mitoses in the direct marrow smears and the proportion of abnormal and normal metaphases in the chromosome preparations
1
Mitosses
19 60 16 16 88 92
Patient
% plasma cell
mitosels
% ‘non-plasnio-
cytic’ mitoses
Metaphases
Patient no. 81 40 84 84 12 8
TABLE 111 Percentage of plasma cell mitoses in bone marrow smears of 8 patients without abnormal stem lines in the bone marrow
80 40 52 66 8 4
20 60 48 38 92 96
Patient no. 5 . The metaphases with 47 chromosomes had an extra acrocentric chromosome half the size of the chromosomes belonging to group 21-22. Patient no. 6. None of the four metaphases with 47 chromosomes could be analyzed. Patient no. 7 . In both metaphases with 47 chromosomes the hyperdiploidy was due to an extra acrocentric chromosome slightly larger than the chromosomes in group 2122. About 50 % of the hypodiploid and diploid metaphases present in the marrow aspirates of the 25 patients could be analyzed. No evidence of a hypodiploid or pseudodiploid clone of cells was found.
In Table I1 the percentage of plasma cell mitoses in the direct bone marrow smears from the patients with abnormal stem lines is compared to the percentage of aneuploid metaphases in the chromosome preparations from the same marrow aspirates. From 8 to 84 % of the mitotic figures in the direct marrow smears belonged to the plasma cell series. The frequency of aneuploid metaphases in the chromosome preparations ranged from 4 to 80 %. Con-
A B C D E F G H
8 36 8 15 64 18 74 3
92 64 92 85 36 82 26 97
sidering the data on the individual patients it is evident that in most of the patients the proportion of plasma cell mitoses corresponded fairly well to the proportion of aneuploid metaphases. It may be observed that in none of the patients more aneuploid metaphases than plasma cell mitoses were found. Table I11 shows the results of the differential counts of bone marrow cells in mitoses in 8 of the patients in whom no abnormal stem line could be demonstrated. The percentage of plasma cell mitoses varied from 3 to 74. Abnormal appearance of the chromatin. It was a characteristic finding that in many of the patients the chromosomes of some of the metaphases studied had a blurred appearance with an ill-defined boundary of the chromatids. In those patients which had abnormal stem lines it was characteristic that it was the aneuploid metaphases which had blurred chromosomes, whereas the diploid metaphases had a more natural looking appearance. Likewise, in patients without aneuploid stem lines in the bone marrow a varied number of the diploid metaphases had blurred chromosomes, whereas the remainder of the metaphases had a normal
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appearance. However, in many of the metaphases the appearance of the chromatin was difficult to evaluate as many of the marrow aspirates yielded technically unsatisfactory metaphases with ill-defined contracted chromosomes.
Chromatid and chromosome breaks. Structural chromosome aberrations were present in 9 of the 16 patients who were studied before cytostatic therapy. They were almost exclusively breaks of the chromatid type, but a few chromosome breaks were also present. The proportion of bone marrow metaphases with breaks varied from 2 to 22 % (mean 4.4 %). In 10 control patients without a haematologic disorder structural aberrations were found in 1.7 % of 350 metaphases studied. In none of these controls aberrations were present in more than 4 % of the metaphases. It was a characteristic feature that breaks were mainly seen in the aneuploid metaphases and in the diploid metaphases which had a blurred appearance. The cytogenetic findings could not be correlated to the type of abnormal immunoglobulin present in the serum of the patients. Likewise, it was not possible to find any correlation between the cytogenetic findings and the course of the disease. DISCUSSION
Cytogenetic studies have not been performed nearly as extensively in myelomatosis as in chronic myeloid and acute leukaemia. This may partly be due to the fact that the quality of chromosome preparations from bone marrow aspirates in myelomatosis is inferior to that obtainable in other haematologic disorders (Lewis et a1 1963, Dartnall et a1 1973).To our opinion
TABLE IV The frequency of abnormal stem lines in bone marrow aspirates from 127 patients with myelomatosis
Source of data
Baikie et a1 Bottura Lewis et a1 Hayhoe & Hammouda 1965 Ponti et a1 Siebner et a1 Dubrova et a1 1966 Das & Aikat 1967 Kanzow et a1 1967 Tassoni et a1 1967 Itani et a1 1970 Rochon et a1 1971 Dartnall et a1 1973 Anday et a1 1974 Present series Total
No. of No#.of patients with patients examined abnormal stem lines 2 5 3 4 4 2
0 1 1 0
5 2 14 7 8 4 38 25
0 2 2 4 0 8 2 1 0 7 7
127
35
4
it is difficult to accomplish a perfect spreading of the bone marrow metaphases in myelomatosis; however, we have been able to study 50 metaphases from most of the patients of the present series. Due to the difficult techniques the types of chromosome abnormalities, the frequency with which they appear, and their relation to the cytologic findings are not very much elucidated. Nevertheless, a review of the cytogenetic literature together with some of the findings in the present study make several general aspects appear which may lead to a better understanding of myelomatosis and its chromosome constitution. Some of these are summarized below. 1. Abnormal stem lines were present in 7 of the 25 patient; of the present series. Table IV shows the results of previous cytogenetic studies on bone marrow aspirates from patients with myelomatosis. It
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M. KROGH JENSEN, J A N ERIKSEN & BIRGITTE W. DJERNES
TABLE V The ploidy of abnormal stern lines in 22 patients with mvelomatosis Hypodiploid Pseudodiploid Hyperdiploid
5 patients 4 13 -
is seen that in about 25 % of the patients abnormal stem lines could be detected in the bone marrow. Table V shows that both hypodiploid, pseudodiploid and hyperdiploid clones are seen, the hyperdiploid being the most common. The chromosome counts of the hypodiploid stem lines varied from 42 to 45, the hyperdiploid stem lines contained between 47 and 86 chromosomes. Abnormal stem lines seem to occur less frequently than in acute leukaemia, where about 50 % of the patients have abnormal clones in the bone marrow (Krogh Jensen 1969). Also in acute leukaemia both hypodiploid, pseudodiploid and hyperdiploid stem lines are present. Thus, cytogenetic findings seem to set apart the haematologic neoplasias (acute leukaemia and myelomatosis) from the solid neoplasms in which nearly 100 % have an abnormal chromosome constitution and where hyperdiploid clones are almost exclusively seen (Yamada et a1 1966). In order to elucidate why abnormal stem lines are found in so relatively few patients with myelomatosis we performed differential counts of mitotic cells from some of the patients in whom no abnormal stem line could be seen. Table I11 shows that from 3 t o 74 % of the mitoses belonged to the plasma cell series, in 5 patients less than 20 % of the cells in mitosis being plasma cells. Probably, the rather low frequency with which abnormal stem lines are found in patients with myelomatosis is due to the fact that in many cases the number of plas-
ma cells in mitosis is so low that they are not represented when only 50 metaphases can be counted in the chromosome preparations. On the other hand, in some cases of myelomatosis the myeloma cells must have a normal diploid chromosome constitution, as in some of our patients without abnormal stem lines more than 50 % of the mitotic figures in the direct bone marrow smears belonged to the plasma cell series. However, it may be noted that all metaphases with 46 chromosomes could not be analyzed. 2. So far the only specific chromosome abnormality in malignant diseases has been the Phl chromosome in chronic myeloid leukaemia. However, in 1961 in a patient with Waldenstrom’s macroglobulinaemia Bottura et a1 described a large supernumerary submetacentric chromosome the size of the chromosomes no. 1-2. In the following years similar marker chromosomes have been described in several other patients with this disorder (German et a1 1961, Houston et a1 1967) and also in patients with myelomatosis (Siebner et a1 1965, Houston et a1 1967, Tassoni et a1 1967, Rochon et al 1971, Anday et a1 1974). In 4 of the 7 cytogenetically abnormal patients of the present series supernumerary chromosomes the size of which ranged between a chromosome no. 3 to larger than no. 1were seen. In 2 cases the chromosomes were acrocentric and in 2 cases the chromosomes were submetacentric. 28 of the abnormal stem lines mentioned in Table IV could be analyzed. 21 contained an abnormal marker chromosome of similar size as described in the present 4 patients. In 11 cases the chromosome was acrocentric, in 9 cases submetacentric and in 1 case metacentric. It has been proposed - and some evidence has been presented - that the large
CYTOGENET’IC STUDIES IN MYELOMATOSIS
acrocentric marker chromosome originates from a translocation from some other chromosome to a normal group D (Tassoni et a1 1967, Mancinelli et a1 1969). Similarly, evidence has been presented that the large submetacentric chromosome originates from a chromosome no. 1 or 2 possibly due to pericentric inversion and mitotic non-disjunction (Patau 1961). Marker chromosomes of a similar appearance have been described in some cases of acute leukaemia (Krogh Jensen 1969) and solid tumors (Yamada et al 1966) and are thus not unique for myelomatosis. However, they appear so often (in 21 out of 28 patients cytogenetically studied (Table 111) that the cytogenetic changes in myelomatosis (and macroglobulinaemia) seem to be much more uniform than in any other malignant disorder - with the exception of chronic myeloid leukaemia. 3. Evidence has been presented that in acute leukaemia (Krogh Jensen & Killmann 1967, 1971) and chronic myeloid leukaemia (Clein & Flemans 1966, Rastric et a1 1968) the chromosome abnormalities are not confined to the ‘leukaemic’ blast cells alone but are also present in the erythroblasts and possibly in the megakarycxytes. In the patients of the present series with abnormal stem lines we therefore made differential counts of the mitotic figures in the direct bone marrow smears and compared the results to the findings in the chromosome preparations. It was found that the percentage of abnormal metaphases in the chromosome preparations corresponded well to the percentage of plasma cell mitoses. These findings indicate that in myelomatosis the chromosome abnormalities are only present in the cells belonging to the plasma cell series. Thus, in myelomatosis the neoplastic pro-
207
cess seems to be confined to the plasma cells, whereas in acute and chronic myeloid leukaemia both the erythrocytic and granulocytic precursors and possibly also the megakaryocytes are involved by the neoplastic process. This fundamental difference between the degree of ‘neoplastic involvement’ of the bone marrow cells in leukaemia and myelomatosis may explain why severe anaemia, granulocytopenia and thrombocytopenia are nearly invariably seen at the debut of leukaemia, whereas these findings are usually only seen in patients with an advanced myelomatosis. Furthermore, the cytogenetic differences mentioned above may offer an explanation of the different therapeutic response to cytostatic therapy seen in these two disorders. 4. It is a characteristic feature of the chromosome findings in acute leukaemia that the chromatin of most of the bone marrow metaphases has a blurred and ill-defined appearance (Sandberg et a1 1964, Krogh Jensen 1969, 1971). In patients with abnormal stem lines this finding is confined to the aneuploid metaphases, whereas in patients with diploid modes both diploid blurred metaphases and diploid metaphases with normal chromatin are seen. Usually, the abnormally looking metaphases make up the majority of the metaphases in chromosome preparations in acute leukaemia. To our opinion the metaphases - whether aneuploid or diploid - with abnormal chromatin constitute the neoplastic (leukaemic) cell population. Similarly, the present report shows that in many bone marrows from patients with myelomatosis some metaphases with abnormally looking chromatin are present. At present we have no methods which allow us to estimate for certain
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whether the chromatin of different metaphases have an abnormal appearance, wherefore it is very difficult to make comparisons between the appearance of metaphases in different chromosome preparations. However, it is evident that a much smaller proportion of the metaphases has a blurred appearance in patients with myelomatosis than in patients with acute leukaemia. Thus, these findings also indicate that the neoplastic cell population is considerably smaller in myelomatosis than in leukaemia. The demonstration of an increased number of metaphases with chromatid and chromosome breaks in the untreated patients of the present series shows that structural chromosome abnormalities are characteristic of some metaphases from neoplastic bone marrow aspirates. Similar findings have been described in acute leukaemia (Krogh Jensen 1969). The present material is too small to evaluate whether the cytostatic therapy employed in some of the patients produced further structural aberrations. Melphalan (Wantzin & Krogh Jensen 1973) as well as cyclophosphamide (Arrighi et a1 1962) have been shown to be chromosome-breaking agents in vivo.
In conclusion cytogenetic studies in myelomatosis have shown that similar abnormalities of the chromosome complement are present as has been described in acute leukaemia and other types of neoplasia, viz. abnormal stem lines and metaphases -whether aneuploid or diploid - with blurred chromatin. In addition the presence of supernumerary marker chromosomes the size of chromosomes 1-3 is a characteristic finding in many cases of myelomatosis. The cytogenetic studies indicate that the neoplastic process is confined to the myeloma cells
and do not comprise the other cell types of the bone marrow. Additional cytogenetic studies ought to be performed in myelomatosis with the modern banding techniques in order to elucidate the origin of the characteristic abnormal marker chromosomes and to see whether some of the diploid cases will turn out to be pseudodiploid. However, it is to be expected that the banding techniques will be employed with little success in myelomatosis as even the preparations obtained with conventional techniques are of unsatisfactory quality. Finally, the chromosomal findings in the peripheral blood cells of patients with myelomatosis are at present rather confusing. Studies are clearly needed which compare the cytogenetic findings in bone marrow cells and both stimulated and unstimulated peripheral blood cells from the same patients. Preferably mitogens which stimulate either T cells or B cells should be used, such as phytohaemagglutinin and lipopolysaccharides. REFERENCES Anday G J, Fishkin B & Gabor F P (1974) Cytogenetic studies in multiple myeloma. J Nut1 Cancer Inst 52, 1069-79. Arrihgi F E, Hsu T C & Bergsagel D E (1962) Chromosome damage in murine and human cells following cytoxan therapy. Tex Rep Biol Med 20, 545-49. Baikie A G, Court Brown W M, Jacobs P A & Milne J S (1959) Chromosome studies in human leukaemia. Lancet ii, 425-28. Bottura C (1963) Chromosome abnormalities in multiple myeloma. Acta Haematol (Basel) 30, 27479. Bottura C, Ferrari I & Veiga A A (1961) Chromosome abnormalities in Waldenstrom’s macroglobulinaemia. Lancet i, 1070. Clein G P & Flemans R J (1966) Involvement of the erythroid series in blastic crisis of chronic myeloid leukaemia. Further evidence for the
CYTOGENETIC STUDIES IN MYELOMATOSIS presence of Philadelphia chromosome in erythroblasts. Brit J Haematol 12, 754-58. Dartnall J A, Mundy G R & Baikie A G (1973) Cytogenetic studies in myeloma. Blood 42, 22939. Das K C & Aikat B K (1967) Chromosomal abnormalities in multiple myeloma. Blood 30, 738-48. Dubrova S E, Dygin V P & Ushakova E A (1966) The cytogenetical and clinico-hematological changes in myeloma disease. Tsitologiia 8, 24149. German J L, Biro C E & Bearn A G (1961) Chromosomal abnormalities in Waldenstrom’s macroglobulinaemia. Lancet ii, 48. Hayhoe F G J & Hammouda F (1965) Cytogenetic and metabolic observations in leukaemias and allied states. In F G J Hayhoe (ed) Current Research in Leukaemia. University Press, Cambridge, England. Houston EW, HoshinoT, Kawasaki S & Nahayama S (1970) Chromosome abnormality and its significance in human multiple myeloma. Acta Haematol Jap 33, 54-66. Kanzow V, Lange B, Niederalt G & Gropp A (1967) Chromosomenuntersuchungen bei Paraproteinamien. Klin Wochenschr 45, 1076-84. Krogh Jensex M (1969) Chromosome studies in acute leukaemia. Munksgaard, Copenhagen. Krogh Jensen M (1971) Cytogenetic studies in acute myeloid leukaemia. Acta Med Scand 190, 429-34. Krogh Jensen M & Killmann S-Aa (1967) Chromosome studies in acute leukaemia. Evidence for chromosomal abnormalities common to erythroblasts and leukaemic white cells. Acta M e d Scand 181, 47-53. Krogh Jensen M & Killmann S-Aa (1971) Additional evidence for chromosome abnormalities in the erythroid precursors in acute leukaemia. Acta M e d Scand 189, 97-100. Lam-Poo-Tang P R L C (1968) An improved method of processing bone marrow for chromosomes. Scand J Haematol 5, 158-60.
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Lewis F J W, Fraser I L & MacTaggart M (1963) An abnormal chromosomal pattern in myelomatosis. Lancet ii, 1013-15. Lewis F J W, MacTaggart M, Crow R S & Wills M R (1963) Chromosomal abnormalities in multiple myeloma. Lancet i, 1183-84. Mancinelli S, Durant J R & Hammack W J (1969) Cytogenetic abnormalities in a plasmocytoma. Blood 33, 225-33. Patau K (1961) Chromosomal abnormalities in Waldenstrom’s macroglobulinaemia. Lancet ii, 600-01. Ponti G B, Valentini R, Carrara P M & Eridani S (1965) Investigations on the chromosome complement in some myeloproliferative disorders. Acta Haematol (Basel) 34, 36-43. Rastrick J M, Fitzgerald P H & Gunz F W (1968) Direct evidence for presence of Phl chromosome in erythroid cells. Brit Med J 1, 96-98. Rochon M, Cadotte M, Pretty H M & Long L A (1971) Les anomalies chromosomiques du my& lome multiple. Experience personelle et revue de la littkrature. Union med Can 100, 1750-54. Sandberg A A, Ishihara T, Kikuchi Y & Crosswhite L H (1964) Chromosomal differences among the acute leukemias. A n n N Y Acad Sci 113, 663-716. Siebner H, Spengler G A, Butler R, Heni F & Riva G (1965) Chromosomenanomalien bei Paraproteinamie. Schweiz Med Wochenschr 95, 1767-77. Tassoni E M, Durant J R, Becker S & Kravitz B (1967) Cytogenetic studies in multiple myeloma: A study of fourteen cases. Cancer Res 27, 80610. Wantzin G L & Krogh Jensen M (1973) The induction of chromosome abnormalities by melphalan in rat bone marrow cells. Scand J Haematol 11, 135-39. Y a r a d a K, Takagi N & Sandberg A A (1966) Chromosomes and causation of human cancer and leukaemia. 11. Karyotypes of human solid tumors. Cancer 19, 1879-90.
