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Fine needle aspiration biopsy: A useful tool in tumor cytogenetics with special reference to malignant lymphomas
Fine Needle Aspiration Biopsy: A Useful Tool in Tumor Cytogenetics with Special Reference to Malignant Lymphomas Ulf Kristoffersson, H~kan Olsson, Eva Mark-Vendel, and Felix Mitelman
ABSTRACT: A method for chromosome analysis of malignant cells by means of fine needle aspiration biopsy has been developed. The method has some obvious advantages compared to conventional surgical biopsy. During a 1-year period 25 consecutive non-Hodgkin, non-Burkitt lymphoma biopsies from 19 patients have been studied. In 14 out of 21 lymph node biopsies chromosome analysis was successful; whereas, four biopsies of subcutaneous lymphomas were unsuccessful. INTRODUCTION In spite of the great e x p a n s i o n of our knowledge in tumor cytogenetics brought about by the a p p l i c a t i o n of the various banding techniques to malignant cells, very little information is still available as regards the detailed karyotypic changes in solid tumors of m a n [1,2]. This is m a i n l y due to technical problems; the single most important obstacle in solid tumor cytogenetics being the great difficulty in obtaining a sufficient n u m b e r of free t u m o r cells, especially repeated samples since this requires surgical intervention. Fine needle aspiration b i o p s y has been used for more than 20 years as a valuable tool to obtain cells from solid tumors for cytological diagnosis [3]. Theoretically, this offers an excellent o p p o r t u n i t y to obtain tumor cells for c h r o m o s o m e analysis, but this technique has not been systematically evaluated in solid t u m o r cytogenetics, although previous work on spleen cells has d e m o n s t r a t e d the feasibility of this approach, at least in the myeloproliferative disorders [4]. Fine n e e d l e aspiration b i o p s y of tumor cells for cytogenetic analysis has been a d o p t e d in our laboratory during the last year in patients presenting with the diagnosis of malignant l y m p h o m a . Some p r e l i m i n a r y results, focused on the reliability of the cytogenetic m e t h o d used, are described below.
From the Departments of Clinical Genetics,Oncology,and Cytology,University Hospital, Lund, Sweden. Address requests for reprints to Dr. Ulf Kristoffersson, Department of Clinical Genetics, University Hospital, S-221 85 Lund. Sweden. Received November 24, 1980; accepted February 1O, 1981.
53 ~) Elsevier North Holland, Inc., 1981 52 Vanderbilt Ave., New York, NY 1 0 0 1 7
Cancer Genetics and Cytogenetics4, 53-60 (1981) 0165-4608/81/050530852.50
54
u . Kristoffersson et al.
Figure 1
The instrument used for fine needle aspiration biopsy (Cameco, Sweden).
MATERIALS AND METHODS Biopsies were obtained by aspirations from the tumors using a special holder for 10 ml disposable syringes (Fig. 1). This h o l d e r permits a one h a n d grip during the aspiration; thus enabling fixation of the enlarged l y m p h node with the other hand. The needles used had an outer diameter of 0.7 m m (gauge 22) and were 25 or 50 m m in length. The p u n c t u r e s were performed after disinfection of the skin and u n d e r aseptic conditions. Local anesthesia was not used. A s p i r a t i o n was started w h e n the needle had reached the target and was terminated w h e n the needle tip left the object. The cells were i m m e d i a t e l y s u s p e n d e d in 5 - 1 0 ml of McCoy's 5A m e d i u m supplem e n t e d with 20% n e w b o r n calf serum, 2% glntamin, and 2 IU/ml heparin. The cell n u m b e r was counted, and since the best results were obtained if the cell n u m b e r was adjusted to a p p r o x i m a t e l y 1 m i l l i o n cells/ml, the cells, if necessary, were centrifuged at 1100 r p m and r e s u s p e n d e d in the adjusted a m o u n t of the same culture m e d i u m and incubated at 37°C. Normally a culture contained 5 - 1 0 m i l l i o n cells s u s p e n d e d in 5 - 1 0 ml culture m e d i u m . Two to three aspirations were u s u a l l y necessary to obtain this n u m b e r of cells (Table 1). Cultures were incubated for 1 8 - 48 hr w i t h colchicine treatment in most cases carried out overnight at a final concentration of 0.0025 /~g/ml. Preparations were m a d e according to the standard air-drying technique of our laboratory i n c l u d i n g h y p o t o n i c treatment for 20 min in 0.075 M KC1 and fixation with 4 : 1 methanol:acetic acid followed by three changes in 3 : 1 methanol:acetic acid. The c h r o m o s o m e s were stained with the trypsin-Giemsa b a n d i n g technique: slides were treated for 2 - 30 sec in trypsin, d i l u t e d in 0.02% versene in a calcium-magnesium free buffer to a final concentration varying between 0 . 0 4 - 0 . 2 5 % ; different tumors s h o w e d a great variation in sensitivity to trypsin treatment. After rinsing in calc i u m - m a g n e s i u m free buffer, slides were i m m e d i a t e l y stained for 5 - 7 rain in 2% Giemsa in SSrensen p h o s p h a t e buffer at pH 7.0, and after rinsing twice in distilled water, the slides were blotted dry and mounted. Twenty-five consecutive biopsies from non-Hodgkin, non-Burkitt l y m p h o m a s from the files at the Department of Oncology in Lurid have been investigated. Four biopsies from three patients were from subcutaneous l y m p h o m a s and 21 biopsies from 16 patients were from l y m p h node manifestations of l y m p h o m a s .
Fine Needle Aspiration Biopsy
Table 1
55
Results obtained by fine needle aspiration biopsy from 25 consecutive l y m p h o m a biopsies Estimated amount of cells at aspiration
Case 1 2 3 4 5 6a 6b
Tissue"
Adequate (>5 × 106)
In In In In in In In
x x x x × x x
Doubtful (<5 x 106)
Number of analyzable metaphases 0
<15
15-20
>20
X ×
7
sc
X
X
8 9a 9b 10 11a llb 12a 12b 13a 13b 14 15 16 17 18 19a 19b
In In In In In In sc sc In In sc In In In In In In
X
X
x
X
x
X X
X
x x X
X
X
X
X
X
X
X
X
X
x x x x X
X
X
X
"ln = lymph node;sc = subcutaneouslymphoma.
The chromosomes were classified according to the system r e c o m m e n d e d by ISCN (1978) [5]. A clone was defined as two cells with the same extra chromosome or structural rearrangement, or three cells with the same missing chromosame. A stem line was defined as the most frequent chromosome constitution of the cell population. RESULTS Out of the 21 l y m p h node biopsies, analyzable chromosomes were found in 14 biopsies representing 11 cases. The remaining seven l y m p h node biopsies and the four biopsies from subcutaneous ]ymphomas failed owing to an insufficient a m o u n t of cells obtained at aspiration. The results from each biopsy are presented in Table 1. It was possible to obtain complete banded karyotype analyses in 12 out of the 14 biopsies where mitoses were found. Representative karyotypes from three different l y m p h o m a s are shown in Figs. 2 - 4 . As the m a i n purpose of this paper is to evaluate the feasibility of fine needle aspiration biopsy in cytogenetic studies of malignant disorders, neither detailed chromosome analysis nor cytologic classification are presented; these matters will be discussed elsewhere. Also, the present material is too small to permit any conclusions as regards specific or n o n r a n d o m chromosome i n v o l v e m e n t or attempts at clinical/
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cytogenetical correlations. At this stage, only a few points may be worth mentioning. Of the 12 biopsies representing nine patients where complete banding analysis was possible, chromosome #7 was involved in clonal aberrations in five cases, and chromosomes #1, #3, #6, #12, and #14 in three cases each. A 14q÷ marker was found as a clonal aberration in two cases. In addition, both cases where complete banding analysis was not possible presented a Dq÷ marker, which might have originated from a chromosome #14, but this could not be decided unequivocally. In one case (No. 6) the two biopsies from different lymph node regions of the same patient revealed different karyotypes: 46,XY,14q÷ and 46,XY,12q÷, respectively. The origin of the 14q÷ marker was interpreted as a t(14;18) (q32;q22); the extra segment on chromosome #12 could not be identified unequivocally. Metaphases with a completely normal diploid karyotype were found in only one case (No. 4). In all remaining 13 biopsies from ten patients all mitoses analyzed had abnormal karyotypes; clonal changes were identified in all of these biopsies. DISCUSSION
In this series of 25 biopsies from 19 patients we failed to obtain adequate samples for chromosome analysis in 11 biopsies from eight patients. Among these were the three patients with subcutaneous manifestations of the lymphomas; none of these were available for repeated biopsy. Seven lymph node biopsies failed. Two of these were unsuccessful in spite of repeated attempts; the other five could not be repeated because the patients were not available for a new biopsy. In the unsuccessful cases the tumor was either very small or at a site difficult to reach, so no or very few cells could be obtained by aspiration. In three of the successful biopsies the number of countable metaphases was less than 15, but in the remaining 11 biopsies 17 - 50 metaphases were counted and 4 - 1 7 cells could be analyzed in detail by means of G banding. Thus, so far, the success rate qf this technique as regards lymph node biopsies is about 70%; whereas, the results h o , n the few subcutaneous lymphomas studied suggest that the technique may not be suitable for tumors with an infiltrative growth. Only when more tumors have been studied, it will become apparent whether this difficulty can be overcome. The present technique has many advantages in cancer cytogenetic work. The cells are in suspension immediately after biopsy and, therefore, no mechanical mincing is necessary to obtain free cells. The method is very simple to handle, requiring no surgical intervention, and it causes little distress to the patient. Furthermore, it is possible to obtain repeated biopsies from the same lymphoma, biopsies from different sites, and additional biopsies if relapse occurs. Thus, cytogenetic analysis of malignant lymphomas by means of fine needle aspiration biopsy may be useful to 1. study the cytogenetic evolution in various lymphoma types at different sites in relation to clinical parameters, 2. investigate whether the cytogenetic picture will correlate with response to therapy or survival or both, 3. correlate the cytogenetic findings with other biological parameters during the course of the disease. A broad collaborative study covering these aspects is in progress. It may be mentioned that this method h~s been tried also in other types of solid tumors, such as bronchial and breast carcinomas. The results are very promising, but further experience is needed before any firm conclusion can be drawn.
60
U. K r i s t o f f e r s s o n et al.
This study was supported by grants from the Swedish Cancer Society, and the John and Augusta Persson Foundation for Medical Research.
REFERENCES
1. Sandberg AA (1980): The Chromosomes in Human Cancer and Leukemia. Elsevier North Holland, New York. 2. Mitelman F (1980): Cytogenetics of experimental neoplasms and non-random chromosome correlations in man. Clin Haematol 9,195 - 219.
3. Zajicek J (1974): Aspiration Biopsy Cytology. Part I. In: Monographs in Clinical Cytology, vol. 4, GL Wied, ed. S. Karger, Basel, pp. 1 - 29. 4. Mitelman F (1975): Comparative cytogenetic studies of bone marrow and extramedullary tissues in chronic myeloid leukemia. Ser Haemat 8, 113-117. 5. ISCN (1978): An international system for h u m a n cytogenetic nomenclature. Cytogenet Cell Genet 21:309-404.
ABSTRACT: A method for chromosome analysis of malignant cells by means of fine needle aspiration biopsy has been developed. The method has some obvious advantages compared to conventional surgical biopsy. During a 1-year period 25 consecutive non-Hodgkin, non-Burkitt lymphoma biopsies from 19 patients have been studied. In 14 out of 21 lymph node biopsies chromosome analysis was successful; whereas, four biopsies of subcutaneous lymphomas were unsuccessful. INTRODUCTION In spite of the great e x p a n s i o n of our knowledge in tumor cytogenetics brought about by the a p p l i c a t i o n of the various banding techniques to malignant cells, very little information is still available as regards the detailed karyotypic changes in solid tumors of m a n [1,2]. This is m a i n l y due to technical problems; the single most important obstacle in solid tumor cytogenetics being the great difficulty in obtaining a sufficient n u m b e r of free t u m o r cells, especially repeated samples since this requires surgical intervention. Fine needle aspiration b i o p s y has been used for more than 20 years as a valuable tool to obtain cells from solid tumors for cytological diagnosis [3]. Theoretically, this offers an excellent o p p o r t u n i t y to obtain tumor cells for c h r o m o s o m e analysis, but this technique has not been systematically evaluated in solid t u m o r cytogenetics, although previous work on spleen cells has d e m o n s t r a t e d the feasibility of this approach, at least in the myeloproliferative disorders [4]. Fine n e e d l e aspiration b i o p s y of tumor cells for cytogenetic analysis has been a d o p t e d in our laboratory during the last year in patients presenting with the diagnosis of malignant l y m p h o m a . Some p r e l i m i n a r y results, focused on the reliability of the cytogenetic m e t h o d used, are described below.
From the Departments of Clinical Genetics,Oncology,and Cytology,University Hospital, Lund, Sweden. Address requests for reprints to Dr. Ulf Kristoffersson, Department of Clinical Genetics, University Hospital, S-221 85 Lund. Sweden. Received November 24, 1980; accepted February 1O, 1981.
53 ~) Elsevier North Holland, Inc., 1981 52 Vanderbilt Ave., New York, NY 1 0 0 1 7
Cancer Genetics and Cytogenetics4, 53-60 (1981) 0165-4608/81/050530852.50
54
u . Kristoffersson et al.
Figure 1
The instrument used for fine needle aspiration biopsy (Cameco, Sweden).
MATERIALS AND METHODS Biopsies were obtained by aspirations from the tumors using a special holder for 10 ml disposable syringes (Fig. 1). This h o l d e r permits a one h a n d grip during the aspiration; thus enabling fixation of the enlarged l y m p h node with the other hand. The needles used had an outer diameter of 0.7 m m (gauge 22) and were 25 or 50 m m in length. The p u n c t u r e s were performed after disinfection of the skin and u n d e r aseptic conditions. Local anesthesia was not used. A s p i r a t i o n was started w h e n the needle had reached the target and was terminated w h e n the needle tip left the object. The cells were i m m e d i a t e l y s u s p e n d e d in 5 - 1 0 ml of McCoy's 5A m e d i u m supplem e n t e d with 20% n e w b o r n calf serum, 2% glntamin, and 2 IU/ml heparin. The cell n u m b e r was counted, and since the best results were obtained if the cell n u m b e r was adjusted to a p p r o x i m a t e l y 1 m i l l i o n cells/ml, the cells, if necessary, were centrifuged at 1100 r p m and r e s u s p e n d e d in the adjusted a m o u n t of the same culture m e d i u m and incubated at 37°C. Normally a culture contained 5 - 1 0 m i l l i o n cells s u s p e n d e d in 5 - 1 0 ml culture m e d i u m . Two to three aspirations were u s u a l l y necessary to obtain this n u m b e r of cells (Table 1). Cultures were incubated for 1 8 - 48 hr w i t h colchicine treatment in most cases carried out overnight at a final concentration of 0.0025 /~g/ml. Preparations were m a d e according to the standard air-drying technique of our laboratory i n c l u d i n g h y p o t o n i c treatment for 20 min in 0.075 M KC1 and fixation with 4 : 1 methanol:acetic acid followed by three changes in 3 : 1 methanol:acetic acid. The c h r o m o s o m e s were stained with the trypsin-Giemsa b a n d i n g technique: slides were treated for 2 - 30 sec in trypsin, d i l u t e d in 0.02% versene in a calcium-magnesium free buffer to a final concentration varying between 0 . 0 4 - 0 . 2 5 % ; different tumors s h o w e d a great variation in sensitivity to trypsin treatment. After rinsing in calc i u m - m a g n e s i u m free buffer, slides were i m m e d i a t e l y stained for 5 - 7 rain in 2% Giemsa in SSrensen p h o s p h a t e buffer at pH 7.0, and after rinsing twice in distilled water, the slides were blotted dry and mounted. Twenty-five consecutive biopsies from non-Hodgkin, non-Burkitt l y m p h o m a s from the files at the Department of Oncology in Lurid have been investigated. Four biopsies from three patients were from subcutaneous l y m p h o m a s and 21 biopsies from 16 patients were from l y m p h node manifestations of l y m p h o m a s .
Fine Needle Aspiration Biopsy
Table 1
55
Results obtained by fine needle aspiration biopsy from 25 consecutive l y m p h o m a biopsies Estimated amount of cells at aspiration
Case 1 2 3 4 5 6a 6b
Tissue"
Adequate (>5 × 106)
In In In In in In In
x x x x × x x
Doubtful (<5 x 106)
Number of analyzable metaphases 0
<15
15-20
>20
X ×
7
sc
X
X
8 9a 9b 10 11a llb 12a 12b 13a 13b 14 15 16 17 18 19a 19b
In In In In In In sc sc In In sc In In In In In In
X
X
x
X
x
X X
X
x x X
X
X
X
X
X
X
X
X
X
x x x x X
X
X
X
"ln = lymph node;sc = subcutaneouslymphoma.
The chromosomes were classified according to the system r e c o m m e n d e d by ISCN (1978) [5]. A clone was defined as two cells with the same extra chromosome or structural rearrangement, or three cells with the same missing chromosame. A stem line was defined as the most frequent chromosome constitution of the cell population. RESULTS Out of the 21 l y m p h node biopsies, analyzable chromosomes were found in 14 biopsies representing 11 cases. The remaining seven l y m p h node biopsies and the four biopsies from subcutaneous ]ymphomas failed owing to an insufficient a m o u n t of cells obtained at aspiration. The results from each biopsy are presented in Table 1. It was possible to obtain complete banded karyotype analyses in 12 out of the 14 biopsies where mitoses were found. Representative karyotypes from three different l y m p h o m a s are shown in Figs. 2 - 4 . As the m a i n purpose of this paper is to evaluate the feasibility of fine needle aspiration biopsy in cytogenetic studies of malignant disorders, neither detailed chromosome analysis nor cytologic classification are presented; these matters will be discussed elsewhere. Also, the present material is too small to permit any conclusions as regards specific or n o n r a n d o m chromosome i n v o l v e m e n t or attempts at clinical/
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Fine Needle Aspiration Biopsy
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cytogenetical correlations. At this stage, only a few points may be worth mentioning. Of the 12 biopsies representing nine patients where complete banding analysis was possible, chromosome #7 was involved in clonal aberrations in five cases, and chromosomes #1, #3, #6, #12, and #14 in three cases each. A 14q÷ marker was found as a clonal aberration in two cases. In addition, both cases where complete banding analysis was not possible presented a Dq÷ marker, which might have originated from a chromosome #14, but this could not be decided unequivocally. In one case (No. 6) the two biopsies from different lymph node regions of the same patient revealed different karyotypes: 46,XY,14q÷ and 46,XY,12q÷, respectively. The origin of the 14q÷ marker was interpreted as a t(14;18) (q32;q22); the extra segment on chromosome #12 could not be identified unequivocally. Metaphases with a completely normal diploid karyotype were found in only one case (No. 4). In all remaining 13 biopsies from ten patients all mitoses analyzed had abnormal karyotypes; clonal changes were identified in all of these biopsies. DISCUSSION
In this series of 25 biopsies from 19 patients we failed to obtain adequate samples for chromosome analysis in 11 biopsies from eight patients. Among these were the three patients with subcutaneous manifestations of the lymphomas; none of these were available for repeated biopsy. Seven lymph node biopsies failed. Two of these were unsuccessful in spite of repeated attempts; the other five could not be repeated because the patients were not available for a new biopsy. In the unsuccessful cases the tumor was either very small or at a site difficult to reach, so no or very few cells could be obtained by aspiration. In three of the successful biopsies the number of countable metaphases was less than 15, but in the remaining 11 biopsies 17 - 50 metaphases were counted and 4 - 1 7 cells could be analyzed in detail by means of G banding. Thus, so far, the success rate qf this technique as regards lymph node biopsies is about 70%; whereas, the results h o , n the few subcutaneous lymphomas studied suggest that the technique may not be suitable for tumors with an infiltrative growth. Only when more tumors have been studied, it will become apparent whether this difficulty can be overcome. The present technique has many advantages in cancer cytogenetic work. The cells are in suspension immediately after biopsy and, therefore, no mechanical mincing is necessary to obtain free cells. The method is very simple to handle, requiring no surgical intervention, and it causes little distress to the patient. Furthermore, it is possible to obtain repeated biopsies from the same lymphoma, biopsies from different sites, and additional biopsies if relapse occurs. Thus, cytogenetic analysis of malignant lymphomas by means of fine needle aspiration biopsy may be useful to 1. study the cytogenetic evolution in various lymphoma types at different sites in relation to clinical parameters, 2. investigate whether the cytogenetic picture will correlate with response to therapy or survival or both, 3. correlate the cytogenetic findings with other biological parameters during the course of the disease. A broad collaborative study covering these aspects is in progress. It may be mentioned that this method h~s been tried also in other types of solid tumors, such as bronchial and breast carcinomas. The results are very promising, but further experience is needed before any firm conclusion can be drawn.
60
U. K r i s t o f f e r s s o n et al.
This study was supported by grants from the Swedish Cancer Society, and the John and Augusta Persson Foundation for Medical Research.
REFERENCES
1. Sandberg AA (1980): The Chromosomes in Human Cancer and Leukemia. Elsevier North Holland, New York. 2. Mitelman F (1980): Cytogenetics of experimental neoplasms and non-random chromosome correlations in man. Clin Haematol 9,195 - 219.
3. Zajicek J (1974): Aspiration Biopsy Cytology. Part I. In: Monographs in Clinical Cytology, vol. 4, GL Wied, ed. S. Karger, Basel, pp. 1 - 29. 4. Mitelman F (1975): Comparative cytogenetic studies of bone marrow and extramedullary tissues in chronic myeloid leukemia. Ser Haemat 8, 113-117. 5. ISCN (1978): An international system for h u m a n cytogenetic nomenclature. Cytogenet Cell Genet 21:309-404.