DNA ANALYSIS OF CARDIAC MYXOMAS:
FLOW CYTOMETRY AND IMAGE ANALYSIS.
Jeffrey D. Seidman, MD. [1]
Jules J. Berman, PhD, MD. [1,2]
C. L. Hitchcock, MD.
R. L. Becker, MD.
Wolfgang Mergner, MD. [1]
G. William Moore, MD, PhD. [1,2,3]
Renu Virmani, MD.
R. A. Yetter, PhD.
1/26/2008.
http://www.netautopsy.org/camyxoma.htm
From the Pathology and Laboratory Medicine Service,
Veterans Affairs Maryland Health Care System, Baltimore, Maryland [1];
Department of Pathology, University of Maryland Medical System,
Baltimore, Maryland [2]; and
Department of Pathology, The Johns Hopkins Medical Institutions,
Baltimore, Maryland [3].
Send comments and correspondence to:
George.Moore4@va.gov
Seidman JD, Berman JJ, Hitchcock CL, Becker RL, Mergner W,
Moore GW, Virmani R, Yetter RA.
DNA analysis of cardiac myxomas: flow cytometry and image analysis.
Hum Pathol. 1991 May;22(5):494-500.
PMID: 2032696.
PubMed Entry
Full Text of Article:
http://www.netautopsy.org/camyxoma.htm
Key words: Myxoma, cardiac neoplasms, aneuploidy, flow cytometry,
image analysis.
Running Title: Cardiac Myxoma DNA Analysis
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ABSTRACT.
Cardiac myxoma is the most common primary tumor of heart, but there
is a long-standing controversy over whether it is a true neoplasm
or a reactive lesion. We analyzed 24 cardiac myxomas from 22 patients:
22 by DNA flow cytometry and 5 by image analysis. Two myxomas were aneuploid;
one by flow cytometry, and another by image analysis. Proliferative
fractions (S+G2/M) were high in three tumors from patients with multiple
myxomas (mean 15.9% SD 4.0%) as compared to 12 solitary uncomplicated myxomas
(mean 7.7% SD 6.0%). S-phase and proliferative fractions were low
in embolic, recurrent, and solitary myxomas. The presence of aneuploidy
in some myxomas supports a neoplastic origin for this tumor.
INTRODUCTION.
Cardiac myxoma is the most common primary tumor of the heart
[1]. Although its neoplastic nature has been
questioned [2], and its histogenesis uncertain,
many pathologists currently believe that myxomas are neoplastic and that they
are derived from a primitive mesenchymal cell [1,3,4] . Myxomas are almost
always benign; complications are related to their effect on blood flow
through the heart. Occasionally, tumor fragments may embolize. Recurrence
after surgical resection is rare. The reported cases of malignant or
metastatic cardiac myxomas are of uncertain validity [4] .
The DNA content and proliferative characteristics of myxomas have been
studied to a limited extent. [5,6] DeWald and coworkers [5] performed
chromosomal analysis on six tumors and found chromosomally abnormal clones
in two cases (45,X,-Y,+7,-18 and 45,XY). The difficulties of obtaining fresh
myxoma tissue precludes using karyotypic analysis to study a significant
number of myxomas. Flow cytometry, however, can be performed
on paraffin-embedded tissue blocks and permits evaluation of a statistically
representative number of cases collected from tumor registries. We retrieved
the paraffin blocks from 24 cardiac myxomas and performed flow cytometry on
22 cases. Image analysis, an alternative method of studying DNA ploidy, was
performed on five selected cases. We included tumors from patients with a
variety of clinical manifestations of myxomas, including recurrence,
embolism, and multiple tumors. In addition to analyzing DNA content,
we also set out to determine whether detectable and biologically significant
differences in DNA ploidy and proliferative activity exist in the different
clinical forms of this rare tumor.
MATERIALS AND METHODS.
Twenty-four cardiac myxomas from 22 patients with available paraffin
blocks were retrieved from the files of the Department of Pathology,
University of Maryland Medical System (8 cases), and the Armed Forces
Institute of Pathology (16 cases). A single hematoxylin and eosin-stained
slide was prepared from each block to confirm the presence of myxoma.
The paraffin-embedded tissue was prepared for flow cytometry by the Hedley
method [7] , with modifications as described by Hitchcock et al [8],
and additional modifications as follows. Preliminary studies were performed
to optimize the technique regarding section thickness, number of sections,
and conditions for hyaluronidase and pepsin digestion. Testicular
hyaluronidase was chosen as the optimal reagent to digest the extracellular
matrix because it digests both hyaluronic acid and chondroitin sulfate,
the two major glycosaminoglycans of the tumor's extracellular matrix [9].
Five to seven 70 micron thick sections were cut on a standard microtome, deparaffinized with HistoSolv (Biochemical Sciences Inc., Bridgeport, NJ) and rehydrated with Flex (Richard Allan Medical Industries, Richland, MI). Hyaluronidase digestion with 0.1 mg/ml bovine testicular hyaluronidase (type IV; Sigma Chemical Co., St. Louis, MO) in phosphate buffered saline (PBS) for 3 hours at 37 [o] C was followed by pepsin digestion with 0.1% pepsin (Sigma P6887) in normal saline, pH 1.5, for 1 hour at 37 [o] C. Nuclei were counted on a Model ZF Coulter counter (Coulter Electronics, Hialeah, FL). The hyaluronidase, pepsin, ribonuclease A (Sigma), propidium iodide (Sigma), and PBS contained 3% polyethylene glycol-8000 (Sigma). The 0.1% Triton X-PBS solution contained 1 mg/ml bovine serum albumin (Sigma). To confirm the presence of bare nuclei, filtered samples from three representative cases were air-dried on glass slides and stained with hematoxylin and eosin.
Samples were analyzed using an Epics Profile flow cytometer (Coulter). The test reported by Givan et al [10] to correct for the zero point of unstained particles was performed; no correction was necessary. The number of nuclei analyzed per case ranged from 4,628 to 71,392. Histograms were analyzed using the Cytologics program, Version 2.1, Coulter Electronics, model of broadened rectangles). Debris was subtracted using a "split nuclei" routine supplied with the program. Proliferative fraction was calculated as (G2] /M+S)/(G0] /G1] +S+G2] /M). All cases with a high G2] /M fraction (greater than 10%) were re-analyzed after vigorous aspiration through a syringe to minimize doublets. After vigorous aspiration in these cases, the G2+M peak was minimally reduced (by less than 3%).
For image analysis, the blocks were sectioned at 6 microns. One slide was stained with hematoxylin and eosin for histologic reference, and the adjacent section was stained according to the Feulgen procedure [11] . A single area representative of each tumor was selected from the hematoxylin and eosin-stained section and marked for cytophotometric analysis. In case 23, the cytologically atypical, mitotically active area was analyzed; cells in mitosis were excluded. DNA absorbance measurements were made using a computer-controlled microspectrophotometric data acquisition and display system (E. Leitz, Rockleigh, NJ; DADS model 560). A tungsten light source was used for all measurements in conjunction with a 560-nm narrow bandpass filter. For each specimen, total absorbance measurements were obtained from 100 nuclei of tumor cells, and from ten mature lymphocytes, which were measured as controls. For each case, the mean absorbance of the tumor cell population divided by the mean absorbance of the control lymphocytes provided a DNA quotient. Lymphocyte nuclei typically have a slightly lower absorbance than other cell types. Therefore, a DNA quotient greater than 1.0 does not necessarily imply aneuploidy.
RESULTS.
Twenty-four cardiac myxomas from 22 patients were analyzed; 19 by flow cytometry only, two by image analysis only, and three by both flow cytometry and image analysis. Clinical histories on the patients indicated that three had embolic phenomena, three had multiple myxomas, one had two recurrences (all three tumors were analyzed), and one patient had a clinically aggressive myxoma.
Demographic characteristics of the patients and locations of the myxomas are shown in Table 1. The average age at first surgical resection was 56 years (range 21 to 81). There were 12 males and 10 females; nine were white, one Asian, one black, and the race of 11 patients was unknown. All blocks were from surgical specimens, with the exception of one autopsy case (cases 2). The average age of the blocks was 11 years (range 0.5 to 25). Twelve patients had solitary left atrial myxomas (one with two recurrences), two patients had biatrial tumors and one had multiple left atrial myxomas. Two patients had solitary right atrial tumors. The location of the cardiac tumor in five patients was not designated (two were said to be `atrial').
The results of flow cytometric analysis are shown in Table 2. The presence of a significant population of inflammatory cells within all of the tumors served as an internal diploid control. Flow cytometry on paraffin-embedded material requires an internal control because of the wide variation in peak position depending on variables such as the quality and type of fixation, duration of storage, and other variables [10,11] . Twenty-one tumors were DNA diploid. The average S-phase fractions were 1.9%, 2.3%, 3.2%, and 2.7%, and the average proliferative fractions were 7.9%, 7.6%, 5.4%, and 15.9%, for solitary, embolic, recurrent, and multiple tumors, respectively (Table 3). Sample histograms from flow analysis are shown in Figs 1,2 and 3.
One tumor (case 16; Fig 3) was DNA aneuploid by flow cytometry. The DNA index was 1.4. Re-examination of histologic sections of this tumor showed no significant atypia, mitotic activity or other histologic features that were different from the other myxomas studied (Fig 4). Repeat flow cytometric analysis from a different block of the same tumor did not show a DNA aneuploid population. Image analysis of this tumor showed one main peak and occasional cells in the region corresponding to the aneuploid peak detected by flow cytometry (Fig 5). We thus interpret the image analysis as consistent with the histogram from flow cytometric analysis, but not diagnostic of aneuploidy by itself. This patient, a 21-year old man with a left atrial myxoma, was lost to follow-up.
The summary of results of image analysis is shown in Table 4. One of these cases showed an aneuploid population with a DNA index of approximately 1.6 (case 23; Fig 6). This patient is an 81-year-old black man with a left atrial myxoma. Computerized tomography showed multiple brain lesions that were believed to be "metastatic" or embolic myxoma. Histologic examination of the cardiac tumor disclosed a cellular subendocardial population of cytologically atypical spindle cells with high mitotic activity (Fig 7). This area was selected for image analysis. The single available paraffin block in this case did not contain sufficient material for flow cytometric analysis. Two additional cases studied by both flow cytometry and image analysis (cases 3 and 13) showed probable DNA diploidy by image analysis. One additional case studied by image analysis alone showed DNA diploidy (case 24).
COMMENT.
For many years, the question of whether the cardiac myxoma
is a true neoplasm or an exuberant reactive lesion has been debated [2] .
The weight of the evidence now strongly indicates that the myxoma is neoplastic [1] . The best evidence to date is the cytogenetic study by DeWald and associates that demonstrated karyotypic aneuploidy in two myxomas [5] . Also strongly supportive of this conclusion is the occurence of myxomas in families [12,13] , particularly those families with the recently described Carney complex [14-17] .
Immunohistochemical studies support the concept of a primitive, multipotential mesenchymal cell as the cell of origin of myxomas [18-20] . However, they do not provide information to support or refute whether it is neoplastic, nor do they give insight into the cytogenetic characteristics of the tumor. The cytogenetics of tumors is important information in the understanding of both their origin and biologic behavior.
Flow cytometry permits rapid analysis of thousands of cells from individual lesions, and thus makes feasible the study of a series of cases from paraffin embedded archival material. A wide variety of neoplastic and dysplastic lesions have been examined by flow cytometry to determine whether abnormalities in nuclear DNA content are present, and whether prognostically useful information can be obtained [21,22] . Image analysis is an alternative method of DNA quantitation that allows assessment of visually chosen cells on histologic sections on a cell-by-cell basis [23] . Ploidy and cell cycle fractions can be estimated using flow cytometry; image analysis can also analyze ploidy. One advantage of image analysis is that it may allow identification of small aneuploid, or near-diploid aneuploid populations, both of which are frequently below the limit of resolution of flow cytometry [23,24] . Bauer et al. [25] compared these two methods on 92 tumors of several different types and found excellent correlation of ploidy in 80 cases. In 9 cases, an aneuploid population was identified by image analysis but not by flow cytometry, and in three cases, an aneuploid population was identified by flow cytometry but not by image analysis.
Flow cytometry and image analysis are sensitive techniques, however, both rely on proper tissue sampling. With respect to ploidy analysis, false negative diploid results can occur with either method due to sampling error, i.e., a small aneuploid population can be missed. False positive aneuploid results are rare if one adheres to strict criteria for histogram interpretation. Poor tissue fixation or poor sample preparation would yield wider peaks and higher background debris, rather than artifactual aneuploid peaks. Although Alanen et al [26] showed `false aneuploid peaks' in flow-cytometric histograms of autolyzed normal tissues, the histograms shown in this study would not be accepted as aneuploid by strict criteria because there is too much overlap between peaks. The reproducibility of flow-cytometric ploidy measurements has been shown to be better than that for cell cycle fractions from paraffin-embedded tissue, and it has been suggested that cell cycle fractions be compared only among samples prepared by the same procedure [27] .
We analyzed 24 cardiac myxomas from 22 patients, several of whom had unusual clinical features. By including patients with multiple, recurrent, and embolic myxomas, in addition to the solitary uncomplicated myxomas, we have adequately represented the broad range of clinical appearances of this rare tumor.
Two myxomas showed convincing evidence of DNA aneuploidy: one by flow cytometry and one by image analysis. The presence of an aneuploid population of cells in a tumor is generally regarded as evidence that the lesion is neoplastic [28] . Although rare reactive lesions may contain aneuploid cells, these cells are considered to represent a minor subpopulation that has sustained genetic injury and that is incapable of further cell replication.
Furthermore, some of the rare reports of aneuploidy in non-neoplastic lesions are open to question. For example, the published histograms of lichen sclerosus et atrophicus [29] and chronic atrophic gastritis [30] that purport to show aneuploidy might not stand up to strict criteria, because there is too much overlap between peaks. In addition, some of these histograms are similar to those generated by autolyzing normal tissues, shown by Alanen et al (26), who demonstrated `false aneuploid peaks'.
Only two of 24 myxomas (8%) showed DNA aneuploidy. Although this is a small fraction, the percentages of specific benign and malignant neoplasms that show aneuploidy vary widely and only occasionally approach 100% [21,31] . In addition, both of the aneuploid myxomas had very small aneuploid populations. It is possible that some of the DNA diploid myxomas had aneuploid populations that were too small to be detected or focal and not sampled. The correlation in one of these two DNA aneuploid myxomas with focal highly atypical histologic features (mitoses, high cellularity and cytologic atypia) and clinical evidence of aggressive behavior, underscores the possible significance of aneuploidy in this tumor. Whether there is such an entity as a "malignant" cardiac myxoma is open to question. The intracardiac location of these tumors provides embolic access to the general circulation. Is embolic tumor spread the same biologic process as metastasis? This question, with particular reference to cardiac myxomas, has been rarely mentioned.
The S-phase and proliferative fractions determined by flow cytometry (Table 3) show that most myxomas are slowly proliferating. This is what one would expect based on their slow growth and benign biologic behavior. Although myxomas in patients with multiple such tumors showed a proliferative fraction more than twice that of solitary uncomplicated tumors, the number of tumors studied is too small to draw any conclusions from these data. Furthermore, although a high G2/M fraction was found in some of these tumors, using these data alone one cannot be certain whether these cells are actively cycling or are arrested in G2 or M phase.
There has been renewed interest in studies of the cardiac myxoma since the recent flurry of literature on Carney's complex, a rare autosomal dominant disorder characterized by spotty facial lentigines, pigmented nodular adrenocortical hyperplasia, soft tissue myxomas, cardiac myxomas, and occasionally other rare tumors [14-17] . The occurrence of cardiac myxomas in the same patients with multiple soft tissue myxomas suggests a common neoplastic origin. On the other hand, the term `myxoma' is used for the cardiac and soft tissue lesions because it is descriptive of their loose paucicellular appearance and glycosaminoglycan-rich matrix; it is not necessarily implied that they have a common histogenesis. In fact, the spindled endothelial-like cells of cardiac myxomas are morphologically different from the stellate mesenchymal cells of soft tissue myxomas [32] . The possibility that aneuploid cardiac myxomas are yet another separate entity is also worthy of consideration. However, the aneuploid myxomas in this study did not exhibit histologic or clinical features distinctive enough to warrant separate categorization based on these two cases alone.
Our data demonstrate DNA aneuploidy in two cardiac myxomas, and suggest that myxomas in patients with multiple tumors are more proliferative than solitary uncomplicated myxomas. Since familial cardiac myxomas are often multiple, future DNA and cytogenetic studies on myxomas should include patients with Carney's complex as well as other examples of familial myxomas to investigate whether these tumors are in any way biologically different from other cardiac myxomas.
LEGENDS
FIGURE 1. Flow cytometric histogram of solitary atrial myxoma (case 14). Mean peak channel numbers are shown. The abscissa represents fluorescence [in arbitary units (channel number)], which is directly proportional to nuclear DNA content. Thus, each peak is a population of cells (nuclei) with a similar DNA content. The leftmost peak represents the diploid population (2n). The G2/M peak (4n) has a channel number approximately twice that of the diploid peak, and the area between the two peaks represents S-phase.
FIGURE 2.] Flow cytometric histogram of a myxoma from a patient with 3 left atrial myxomas (case 9). Mean peak channel numbers are shown.
FIGURE 3.] Flow cytometric histogram of a DNA aneuploid myxoma, the same case as the image analysis histogram shown in Fig 5 and morphology shown in Fig 4 (case 16). Mean peak channel numbers are shown. The DNA index is 1.4.
FIGURE 4.] Histologically typical DNA aneuploid myxoma, showing spindle cells forming small vascular channels in a loose myxoid matrix (Hematoxylin-eosin stain x50). This is the same case as shown by flow cytometric histogram in Fig 3 and image analysis histogram in Fig 5 (case 16).
FIGURE 5.] Image analysis histogram of a DNA aneuploid myxoma, the same case as the flow cytometric histogram shown in Fig 3 with morphology shown in Fig 4 (case 16). As with the flow cytometric histograms, the abscissa is directly proportional to DNA content; the values are grouped in arbitrary intervals for purposes of graph presentation.
FIGURE 6.] Image analysis histogram of aneuploid myxoma with morphology shown in Fig 7 (case 23). The DNA index is approximately 1.6.
FIGURE 7.] Aneuploid myxoma with hypercellular area (Hematoxylin-eosin x100), the same case as demonstrated by image analysis histogram in Fig 6 (case 23).
TABLE 1.] Demographic characteristics of patients with cardiac myxomas.
TABLE 2.] Summary of flow cytometric analyses of cardiac myxomas.
TABLE 3.] Proliferative features of myxomas categorized by clinical type.
TABLE 4.] Image analysis profiles of 5 cardiac myxomas.®PG¯
TABLE 1
Age at first Sex Race Location Comments
resection
1. 62 M U U Embolic (embolus
analyzed)
2. 77 F U L atrium Embolic (primary
analyzed)
3. 25 M W L atrium Embolic (primary
analyzed)
4. 57 M W L atrium Recurrent (original
resection
5. same as #3 First recurrence
6. same as #3 Second recurrence
7. U M U biatrial Biatrial; RA analyzed
8. 74 M U biatrial TIAs
9. 40 F W 3 in L Arose on septum
atrium and posterior wall
10. 47 F W R atrium
11. 55 F O L atrium
12. 67 M W L atrium
13. 57 F W L atrium
14. 58 M U atrium NOS
15. 51 F U U
16. 21 M U L atrium
17. 57 F U R atrium
18. 59 F W atrium NOS
19. 76 F W U
20. 75 M U L atrium
21. 39 M U L atrium
22. 47 F W L atrium
23. 81 M B L atrium clinically aggressive;
multiple CNS lesions
24. 52 M U L atrium
M, male; F, female; U, unknown; W, white; B, black; O, oriental;
L, left; R, right; RA, right atrial tumor; NOS, not otherwise specified;
TIA, transient ischemic attack; CNS, central nervous system.
TABLE 2.
Age of Ploidy CV of S-phase (%) Proliferative
block(yrs) diploid peak(%) Fraction (%)
1. 11 diploid 2.2 3.3 11.9
2. 17 diploid 3.2 2.8 7.3
3. 11 diploid 3.2 0.9 3.5
4. 15 diploid 5.6 2.3 3.2
5. 12 diploid 4.8 2.7 4.0
6. 1 diploid 3.7 4.6 8.9
7. 18 diploid 2.9 2.7 6.5
8. 2 diploid 4.5 1.3 26.6
9. 10 diploid 4.6 4.0 14.5
10. 0.5 diploid 6.7 1.6 5.1
11. 0.5 diploid 4.1 1.1 5.6
12. 17 diploid 3.5 1.8 24.9
13. 25 diploid 3.9 4.2 6.4
14. 12 diploid 4.4 2.4 5.8
15. 18 diploid 3.5 1.9 6.5
16. 15 aneuploid
17. 16 diploid 4.0 0.6 3.3
18. 14 diploid 4.2 1.6 9.6
19. 12 diploid 4.2 1.8 2.3
20. 9 diploid 3.9 1.0 3.7
21. 11 diploid 2.7 2.3 9.1
22. 9 diploid 3.8 2.2 10.2
CV, coefficient of variation ((standard deviation/mean)x100).
TABLE 3
S-phase fraction (%) Proliferative fraction (%)
Embolic 2.3 +- 1.3 7.6 +- 2.8
(n=3)
Recurrent 3.2 +- 1.2 5.4 +- 3.1
(n=3)
Multiple 2.7 +- 1.4 15.9 +- 4.0
(n=3)
Solitary 1.9 +- 0.9 7.7 +- 6.0
uncomplicated
(n=12)
Complicated (embolic, 2.7 +- 1.2 9.6 +- 7.4
recurrent, or multiple)
(n=9)
Values are mean
±SD.
TABLE 4
Case # Lymphs CV(%) Tumor cells CV(%) DNAQ Inter-
(MA) (MA) pretation
3. 10.3 13.9 16.0 19.5 1.6 diploid
13. 6.5 12.6 8.4 24.2 1.3 c/w
diploid
16. 5.3 10.4 9.5 24.2 1.8 c/w FCM
aneuploidy
23. 10.3 10.7 22.6 25.6 2.2 aneuploid
24. 7.8 14.4 11.7 27.2 1.5 diploid
MA = Mean absorbance, in arbitrary units; c/w = Consistent with; FCM=Flow cytometric; DNAQ=DNA quotient.
ACKNOWLEDGMENT
The authors are grateful to Ms. Annette Geisel
for technical instruction in the preparation of the tissues
for flow cytometry.
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Last updated: 1/26/2008, by
G. William Moore, MD, PhD.