- Email: [email protected]
A photometric study of the standard Philadelphia (Ph1) translocation of chronic myeloid leukemia (CML)
A Photometric Study of the Standard Philadelphia (Ph1) Translocation of Chronic Myeloid Leukemia (CML) Andrew W. Wayne and James C. Sharp
ABSTRACT:
A newly developed form of high resolution microscope photometry has been employed in a study of the relationships between the chromosomes involved in the standard Philadelphia (Ph 1) translocation of chronic myeloid leukemia (CML). The intention of the investigation was to provide quantitative data on the relative amount of material exchanged between the No. 9 and No. 22 chromosames. A marked consistency in the amount of material lost from the Ph I was observed, and the balanced nature of the standard Ph 1 translocation was confirmed. The data are also in accordance with the concept of a reciprocal exchange between the No. 9 and No. 22 chromosomes.
INTRODUCTION The P h i l a d e l p h i a chromosome (Phi), first described by Nowell and Hungerford in 1960 [1], is a universally recognized chromosome abnormality in leukemia. The Ph 1 was positively identified as a No. 22 derivative in 1970 [2], and Rowley in 1973 [3] noted that the Ph 1 was usually formed as a result of a translocation of material from the No. 22 (q) long arm onto the end of the No. 9 long arm. This standard form of the translocation is seen in over 90% of patients with chronic m y e l o i d leukemia (CML). The overall balance of the Ph ~ translocation has been s h o w n by DNA cytofluorometry [4]; the reciprocal nature of the exchange has also been inferred. The occurrence of a reciprocal exchange of material between two chromosomes is probable in m a n y balanced translocations. This concept is s u p p o r t e d by Muller [5], who postulated that the integrity of the telomere must be m a i n t a i n e d in the exchange, and by the apparent absence of a repair m e c h a n i s m for "sticky" chromosome ends p r o d u c e d during mitosis. The p u r p o s e of this study was to analyze the amount of material exchanged in the standard Ph ~ translocation, using a new form of high resolution microscope p h o t o m e t r y [6]. The area of the long arm material of the chromosomes involved in the translocation were m e a s u r e d and c o m p a r e d to their normal homologs in the same cell.
From
the Department of Haematology,King's CollegeHospital, London.
Address requests for reprints to Dr. A. W. Wayne, Department of Haematology, King's College Hospital, Denmark Hill, London SE5 8RX, U.K. Received June 8, 1981; accepted July 20, 1981.
253 © Elsevier Science Publishing Co., Inc. 52 Vanderbilt Ave., New York, NY 1 0 0 1 7
Cancer Genetics and Cytogenetics5, 253-256 (1982] 0165-4608/82/03025304502.75
A. W. W a y n e and J. C. Sharp
254 MATERIALS AND METHODS Photometry
The e q u i p m e n t e m p l o y e d was based on the Reichert "Univar" microscope with photometer system. Measurements were performed using a p l a n a p o c h r o m a t i c objective of numerical aperture 1.32 at a wavelength of 550 nm. Photometric scanning was performed using a specially developed "fine scanning lens" that operates in the plane of the magnified image. A 0.08-~,m step interval was e m p l o y e d between each reading, and a 0.16-p,m circular diaphragm was m o u n t e d in the photometer head to give a 39% overlap between measurements to m i n i m i z e peripheral field aberration. A full description of the technical procedures e m p l o y e d and the theoretical basis of high resolution microscope photometry has recently been p u b l i s h e d [6].
Cytogenetic Techniques All studies were performed on GTG-banded material from appropriate bone marrow specimens. Each chromosome was analyzed i n d i v i d u a l l y and the measured area c o m p a r e d with that obtained for its homolog. Only chromosomes that o c c u p i e d equivalent central or peripheral locations within the cell were analyzed to avoid discrepancies due to differential chromosome elongation w h e n comparing homologs. Chromosome n o m e n c l a t u r e is in accordance with the International System for H u m a n Cytogenetic Nomenclature (ISCN, 1978) [7]. RESULTS The ratios for the amount of material exchanged between the chromosomes involved in the standard Ph 1 translocation are given in Table 1; their derivation is e x p l a i n e d in Figure 1. The readings are based on the pooled results obtained from 10 patients to give a total of 20 to 30 comparisons for each of the five sets of data presented. This material was selected from an original investigation of over 50 patients with chronic m y e l o i d leukemia, studied using GTG banding, in w h i c h the standard Ph ~ translocation was demonstrated to be the sole abnormality present
Table 1 Area c o m p a r i s o n s of the chromosomes and their homologs involved in the standard Ph 1 translocation ° Ratio (1:)
No. of readings
Range
e:d f:d g:d c:c c:a
30 30 20 30 20
1.71-2.14 0.61-0.88 1.57-1.63 1.01-1.15 1.25-1.42
Mean - S.D. 1.88 0.77 1.60 1.08 1.35
-*- 0.14 ~ 0.07 - 0.02 - 0.05 ± 0.06
The readings are based on the pooled data derived from 10 patients to give 20 or 30 separate comparisons for each set of data "See Figure 1.
Note:
255
Photometric Study of Ph 1 Translocation of CML
22
iI:.iii" i.i'
- ~ : ~-:-
i e'~-eeoe
ph 8
• • • • eeooo
C
•
o e e e e
ooo
•
Figure 1 Representation of the parameters employed in the area comparisons between homologs given in Table 1. For example, g:d provides the ratio of the area of q arm material from the centromere to the q arm terminus of the Ph ' compared to the whole of the q arm material of its normal No. 22 homolog.
during the chronic phase. The overall percentage loss and gain of material from the No. 22 and No. 9 chromosomes, respectively, is also tabulated (Table 2), using the measurements obtained from five paired comparisons on two patients.
DISCUSSION The overall balance of the standard Ph I translocation was confirmed by the parity between the percentage loss of material from the Ph 1 and the percentage gain of material by the 9q + chromosome. This study is in agreement with the data presented by Mayall et al. [4], which showed that there was no net gain or loss of DNA as a result of the Ph 1 translocation; however, the actual measurements derived from the two studies cannot be directly compared due to the differences in their methods of determination. The present study employed data based on comparative ratios only and thus avoided m a n y of the problems associated with the measurement of absolute values. It has been suggested [8,9] that the Ph 1 is heteromorphic. The results of this study contradict this concept insofar as a marked consistency in the a m o u n t of material lost from the Ph 1, relative to its normal No. 22 homolog, is always readily apparent in the standard form of the translocation. However, the situation i n cases involving an u n u s u a l or complex translocation has not been specifically studied, and a difference in the relative amount of material lost from the Ph 1 may result in at least some of these. It is widely assumed that the presumptive break point in the No. 22 chromosome is at band q11; normal visual analysis of G-banded material seems to indicate that none of the dark band material of q12 remains on the Ph' chromosome, at least in
Table 2
Patient A Patient B
Measurements for the overall percentage loss (from 22q) and gain (onto 9q) of chromosome material (based on five paired comparisons from two patients) Mean % loss _+ SD
Mean % gain ± SD
37.11 _+ 0.02 37.12 ± 0.01
37.09 _ 0.02 37.10 ± 0.02
256
A . W . Wayne and J. C. Sharp the standard form. Thus a determination of the amount of material lost from the Ph' can be simulated by measuring the area of long arm material in the No. 22 from the centromere down to band q12, relative to its normal homolog, and this value can then be compared to that obtained for the actual area of the Ph 1 long arm material, relative to its homolog. The values for the ratios g:d (Phlq:22q) and e : d (22q11:22q) should be equivalent if the break-point assignment has been correct and all the material below q l l is lost from the Ph 1. The value for g:d was consistently lower than that for e:d, indicating that more material is present in the Ph 1 than w o u l d be expected from the above assumptions. Since no net gain of material occurs in the exchange between the No. 9 and No. 22 chromosomes, a likely explanation for this discrepancy is the reciprocal acquistion of material from the terminal end of the No. 9 long arm onto the terminus of the 2 2 q - . This data would then be in accord with the concept of the Ph 1 being formed as a result of a balanced and reciprocal translocation. However, the possibility of a difference in the relative density, or related property, of chromosomal material according to its location, leading to differential elongation or contraction of a particular region or band, cannot be eliminated at this stage. We thank the Lions International Blood Research Appeal and the Szeben Peto Foundation for support of this project.
REFERENCES 1. Nowell PC, Hungerford DA (1960): A minute chromosome in human chronic granulocytic leukemia. Science 312, 1497. 2. Caspersson T, Gahrton G, Lindsten J, Zech L (1970): Identification of the Philadelphia chromosome as a number 22 by quinacrine mustard fluorescence analysis. Exp Cell Res 63, 238-240. 3. Rowley JD (1973): A new consistent chromosomal abnormality in chronic myelogenous leukemia identified by quinacrine fluorescence and Giemsa banding. Nature 243, 290-293. 4. Mayall BH, Carrano AV, Moore DH, Rowley JD (1977): Quantification by DNA-based cytophotometry of the 9q+/22q- chromosomal translocation associated with chronic myelogenous leukemia. Cancer Res 37, 3590-3597. 5. Muller HJ (1940): An Analysis of the process of structural change in chromosomes of Drosophila. J Genet 40, 1-66. 6. Wayne AW, Sharp JC (1981): The use of high resolution microscope photometry in the discrimination of chromosome bands. J Microsc 124, 163-167. 7. An International System for Human Cytogenetic Nomenclature, ISCN (1978): Birth Defects: Original Article Series, vol. 14, no. 8. The National Foundation, New York. 8. Watt JL, Hamilton PJ, Page BM (1977): Variation in the Philadelphia chromosome. Hum Genet 37, 141-148. 9. Verma RS, Dosik H (1980): Heteromorphisms of the Philadelphia (Ph1) chromosome in patients with chronic myelogenous leukaemia (CML). Br J Haematol 45, 215-222.
ABSTRACT:
A newly developed form of high resolution microscope photometry has been employed in a study of the relationships between the chromosomes involved in the standard Philadelphia (Ph 1) translocation of chronic myeloid leukemia (CML). The intention of the investigation was to provide quantitative data on the relative amount of material exchanged between the No. 9 and No. 22 chromosames. A marked consistency in the amount of material lost from the Ph I was observed, and the balanced nature of the standard Ph 1 translocation was confirmed. The data are also in accordance with the concept of a reciprocal exchange between the No. 9 and No. 22 chromosomes.
INTRODUCTION The P h i l a d e l p h i a chromosome (Phi), first described by Nowell and Hungerford in 1960 [1], is a universally recognized chromosome abnormality in leukemia. The Ph 1 was positively identified as a No. 22 derivative in 1970 [2], and Rowley in 1973 [3] noted that the Ph 1 was usually formed as a result of a translocation of material from the No. 22 (q) long arm onto the end of the No. 9 long arm. This standard form of the translocation is seen in over 90% of patients with chronic m y e l o i d leukemia (CML). The overall balance of the Ph ~ translocation has been s h o w n by DNA cytofluorometry [4]; the reciprocal nature of the exchange has also been inferred. The occurrence of a reciprocal exchange of material between two chromosomes is probable in m a n y balanced translocations. This concept is s u p p o r t e d by Muller [5], who postulated that the integrity of the telomere must be m a i n t a i n e d in the exchange, and by the apparent absence of a repair m e c h a n i s m for "sticky" chromosome ends p r o d u c e d during mitosis. The p u r p o s e of this study was to analyze the amount of material exchanged in the standard Ph ~ translocation, using a new form of high resolution microscope p h o t o m e t r y [6]. The area of the long arm material of the chromosomes involved in the translocation were m e a s u r e d and c o m p a r e d to their normal homologs in the same cell.
From
the Department of Haematology,King's CollegeHospital, London.
Address requests for reprints to Dr. A. W. Wayne, Department of Haematology, King's College Hospital, Denmark Hill, London SE5 8RX, U.K. Received June 8, 1981; accepted July 20, 1981.
253 © Elsevier Science Publishing Co., Inc. 52 Vanderbilt Ave., New York, NY 1 0 0 1 7
Cancer Genetics and Cytogenetics5, 253-256 (1982] 0165-4608/82/03025304502.75
A. W. W a y n e and J. C. Sharp
254 MATERIALS AND METHODS Photometry
The e q u i p m e n t e m p l o y e d was based on the Reichert "Univar" microscope with photometer system. Measurements were performed using a p l a n a p o c h r o m a t i c objective of numerical aperture 1.32 at a wavelength of 550 nm. Photometric scanning was performed using a specially developed "fine scanning lens" that operates in the plane of the magnified image. A 0.08-~,m step interval was e m p l o y e d between each reading, and a 0.16-p,m circular diaphragm was m o u n t e d in the photometer head to give a 39% overlap between measurements to m i n i m i z e peripheral field aberration. A full description of the technical procedures e m p l o y e d and the theoretical basis of high resolution microscope photometry has recently been p u b l i s h e d [6].
Cytogenetic Techniques All studies were performed on GTG-banded material from appropriate bone marrow specimens. Each chromosome was analyzed i n d i v i d u a l l y and the measured area c o m p a r e d with that obtained for its homolog. Only chromosomes that o c c u p i e d equivalent central or peripheral locations within the cell were analyzed to avoid discrepancies due to differential chromosome elongation w h e n comparing homologs. Chromosome n o m e n c l a t u r e is in accordance with the International System for H u m a n Cytogenetic Nomenclature (ISCN, 1978) [7]. RESULTS The ratios for the amount of material exchanged between the chromosomes involved in the standard Ph 1 translocation are given in Table 1; their derivation is e x p l a i n e d in Figure 1. The readings are based on the pooled results obtained from 10 patients to give a total of 20 to 30 comparisons for each of the five sets of data presented. This material was selected from an original investigation of over 50 patients with chronic m y e l o i d leukemia, studied using GTG banding, in w h i c h the standard Ph ~ translocation was demonstrated to be the sole abnormality present
Table 1 Area c o m p a r i s o n s of the chromosomes and their homologs involved in the standard Ph 1 translocation ° Ratio (1:)
No. of readings
Range
e:d f:d g:d c:c c:a
30 30 20 30 20
1.71-2.14 0.61-0.88 1.57-1.63 1.01-1.15 1.25-1.42
Mean - S.D. 1.88 0.77 1.60 1.08 1.35
-*- 0.14 ~ 0.07 - 0.02 - 0.05 ± 0.06
The readings are based on the pooled data derived from 10 patients to give 20 or 30 separate comparisons for each set of data "See Figure 1.
Note:
255
Photometric Study of Ph 1 Translocation of CML
22
iI:.iii" i.i'
- ~ : ~-:-
i e'~-eeoe
ph 8
• • • • eeooo
C
•
o e e e e
ooo
•
Figure 1 Representation of the parameters employed in the area comparisons between homologs given in Table 1. For example, g:d provides the ratio of the area of q arm material from the centromere to the q arm terminus of the Ph ' compared to the whole of the q arm material of its normal No. 22 homolog.
during the chronic phase. The overall percentage loss and gain of material from the No. 22 and No. 9 chromosomes, respectively, is also tabulated (Table 2), using the measurements obtained from five paired comparisons on two patients.
DISCUSSION The overall balance of the standard Ph I translocation was confirmed by the parity between the percentage loss of material from the Ph 1 and the percentage gain of material by the 9q + chromosome. This study is in agreement with the data presented by Mayall et al. [4], which showed that there was no net gain or loss of DNA as a result of the Ph 1 translocation; however, the actual measurements derived from the two studies cannot be directly compared due to the differences in their methods of determination. The present study employed data based on comparative ratios only and thus avoided m a n y of the problems associated with the measurement of absolute values. It has been suggested [8,9] that the Ph 1 is heteromorphic. The results of this study contradict this concept insofar as a marked consistency in the a m o u n t of material lost from the Ph 1, relative to its normal No. 22 homolog, is always readily apparent in the standard form of the translocation. However, the situation i n cases involving an u n u s u a l or complex translocation has not been specifically studied, and a difference in the relative amount of material lost from the Ph 1 may result in at least some of these. It is widely assumed that the presumptive break point in the No. 22 chromosome is at band q11; normal visual analysis of G-banded material seems to indicate that none of the dark band material of q12 remains on the Ph' chromosome, at least in
Table 2
Patient A Patient B
Measurements for the overall percentage loss (from 22q) and gain (onto 9q) of chromosome material (based on five paired comparisons from two patients) Mean % loss _+ SD
Mean % gain ± SD
37.11 _+ 0.02 37.12 ± 0.01
37.09 _ 0.02 37.10 ± 0.02
256
A . W . Wayne and J. C. Sharp the standard form. Thus a determination of the amount of material lost from the Ph' can be simulated by measuring the area of long arm material in the No. 22 from the centromere down to band q12, relative to its normal homolog, and this value can then be compared to that obtained for the actual area of the Ph 1 long arm material, relative to its homolog. The values for the ratios g:d (Phlq:22q) and e : d (22q11:22q) should be equivalent if the break-point assignment has been correct and all the material below q l l is lost from the Ph 1. The value for g:d was consistently lower than that for e:d, indicating that more material is present in the Ph 1 than w o u l d be expected from the above assumptions. Since no net gain of material occurs in the exchange between the No. 9 and No. 22 chromosomes, a likely explanation for this discrepancy is the reciprocal acquistion of material from the terminal end of the No. 9 long arm onto the terminus of the 2 2 q - . This data would then be in accord with the concept of the Ph 1 being formed as a result of a balanced and reciprocal translocation. However, the possibility of a difference in the relative density, or related property, of chromosomal material according to its location, leading to differential elongation or contraction of a particular region or band, cannot be eliminated at this stage. We thank the Lions International Blood Research Appeal and the Szeben Peto Foundation for support of this project.
REFERENCES 1. Nowell PC, Hungerford DA (1960): A minute chromosome in human chronic granulocytic leukemia. Science 312, 1497. 2. Caspersson T, Gahrton G, Lindsten J, Zech L (1970): Identification of the Philadelphia chromosome as a number 22 by quinacrine mustard fluorescence analysis. Exp Cell Res 63, 238-240. 3. Rowley JD (1973): A new consistent chromosomal abnormality in chronic myelogenous leukemia identified by quinacrine fluorescence and Giemsa banding. Nature 243, 290-293. 4. Mayall BH, Carrano AV, Moore DH, Rowley JD (1977): Quantification by DNA-based cytophotometry of the 9q+/22q- chromosomal translocation associated with chronic myelogenous leukemia. Cancer Res 37, 3590-3597. 5. Muller HJ (1940): An Analysis of the process of structural change in chromosomes of Drosophila. J Genet 40, 1-66. 6. Wayne AW, Sharp JC (1981): The use of high resolution microscope photometry in the discrimination of chromosome bands. J Microsc 124, 163-167. 7. An International System for Human Cytogenetic Nomenclature, ISCN (1978): Birth Defects: Original Article Series, vol. 14, no. 8. The National Foundation, New York. 8. Watt JL, Hamilton PJ, Page BM (1977): Variation in the Philadelphia chromosome. Hum Genet 37, 141-148. 9. Verma RS, Dosik H (1980): Heteromorphisms of the Philadelphia (Ph1) chromosome in patients with chronic myelogenous leukaemia (CML). Br J Haematol 45, 215-222.