Reprinted with permission of Oxford University Press, Oxford, England and the European Society of Human Reproduction and Embryology.

As published in Molecular Human Reproduction vol. 4, no. 1 pp. 83-86, 1998 

Cytogenetic abnormalities in uterine myomas are associated with myoma size



Mitchell S. Rein, M.D. 1,3, W. Lee Powell2, Frederick C. Walters2, Stanislawa Weremowicz, Ph.D. 2,3, Rita M. Cantor, Ph.D. 4, Robert L. Barbieri, M.D. 1,3, Cynthia C. Morton, Ph.D. 2,3,5

1Departments of Obstetrics, Gynecology, and Reproductive Biology and 2Pathology, Brigham and Women's Hospital, 3Harvard Medical School, Boston, MA 02115 and 4Division of Medical Genetics, Cedars-Sinai Medical Center, Los Angeles, CA 90048

5To whom correspondence should be addressed at: Departments of Obstetrics, Gynecology and Reproductive Biology and Pathology, Brigham and Women's Hospital, 75 Francis Street, Boston, MA 02115.

Uterine leiomyomata (myomas) are associated with a variety of characteristic cytogenetic abnormalities. The significance of these chromosomal aberrations in the pathobiology of myomas remains to be determined. The present study investigated the relationship between myoma cytogenetic abnormalities and size. A total of 114 myoma specimens were obtained from 92 patients undergoing myomectomy or hysterectomy. The maximum diameter of each myoma was measured and a portion of each myoma obtained for cytogenetic analysis. Karyotypes were analyzed and categorized as normal, abnormal (non-mosaic) or mosaic. Cytogenetic analyses revealed 73 (64%) normal, 20 (18%) abnormal (non-mosaic), and 21 (18%) mosaic karyotypes. Mean myoma diameter was 6.5 cm 0.44 with a range of 0.4 to 27 cm. Differences between the mean myoma diameter of specimens with normal versus abnormal karyotypes was determined by the Kruskal Wallis test. The mean myoma diameter among specimens with abnormal (non-mosaic) karyotypes was significantly > myomas with normal karyotypes (10.2 5.9 versus 5.9 4.2 cm; P < 0.001). The proportion of abnormal (non-mosaic) karyotypes in myomas > 6.5 cm was compared to myomas < 6.5 cm by c2-analysis; myomas > 6.5 cm demonstrated a significantly higher proportion of abnormal (non-mosaic) karyotypes when compared to myomas < 6.5 cm (75% versus 34%; P < 0.02). In summary, a significant relationship exists between clonal cytogenetic abnormalities and myoma size suggesting that chromosomal abnormalities associated with individual myomas enhance myoma growth.
Key words: chromosomes/cytgenetics/fibroids/leiomyomata/uterine myomas


________________________________________________________________________________________


Introduction

Symptomatic uterine leiomyomata are the most common indication for hysterectomy in the United States (Gambone et al., 1990). Although these neoplasms pose a major public health problem for women of reproductive age, their biology remains poorly understood. Uterine myomas are independent, monoclonal tumours derived presumably from a single mutated myometrial cell (Townsend et al., 1970; Mashal et al., 1994). They are associated with a variety of non-random cytogenetic abnormalities; rearrangements of chromosome regions 12q14-15 and 7q22 are the most frequently observed (Mark et al., 1990; Nilbert et al., 1990; Rein et al., 1991). In an effort to understand the significance of chromosomal aberrations in the pathobiology of myomas, we tested the hypothesis that the karyotype of a uterine myoma is associated with tumour size.


Materials and methods

A total of 114 myoma specimens were obtained from 92 patients undergoing myomectomy or hysterectomy. Patients pretreated with a gonadotropin releasing hormone analog prior to surgery were excluded from the analyses. The maximum diameter of each myoma was measured. Myoma volume was estimated based on the formula for a sphere, 4/3pr3. Tissue was collected in Hanks' balanced salt solution without calcium and magnesium (Gibco, Grand Island, NY) and transported directly to the Cytogenetics Laboratory for short term culture and cytogenetic analysis as previously described (Rein et al., 1991). Karyotypes were analyzed and each myoma was categorized as cytogenetically normal (46,XX) or abnormal; abnormals were subdivided further into those which were mosaic (i.e., any mixture of 46,XX and chromosomally abnormal cells) and those which were non-mosaic.

Statistical analyses included a multigroup, nonparametric analysis, the Kruskal Wallis test, to compare myoma diameters of normal versus abnormal (mosaic) versus abnormal (non-mosaic) myoma karyotypes. Multiple comparisons of myoma diameters between groups were performed using the Mann-Whitney Rank Sum test. The Mann-Whitney test was also used to compare the median diameter of myomas with rearrangements of chromosome regions 12q14-15 versus 7q22. Nonparametric tests were used because of the non-normality of distributions of tumour sizes. Both mean and median tumour diameters are reported; the measure of dispersion of mean myoma diameters is a standard deviation. A c2-analysis with Yates' correction was used to test differences in the proportion of myomas above the mean myoma diameter for all groups as well as the proportion of abnormal (non-mosaic) karyotypes for myomas greater versus less than the mean myoma diameter. Differences in the means of the patient ages were compared by an analysis of variance.

 
Table I. Summary of myoma size and cytogenetic analysis
Cytogenetic analysis Myoma diameter (cm)

Mean SD

Myoma diameter (cm)

Median

Calculated myoma volume (cm3)

Mean SD

Normal (n = 73) 5.9 4.2a 5.4 334 1123c
Abnormal (n = 41) 7.6 5.3 7.0 642 1638
Abnormal (mosaic)
(n= 21)
5.0 3.0b 4.5 136 165d
Abnormal (non-mosaic)
(n = 20)
10.2 5.9a,b 10.0 1173 2246c,d

a,b,c,d Values with the same superscripts were significantly different, P<0.01.


Results

Patient age ranged from 29 to 56 years with a mean of 41.6 0.49. There were no significant differences in the means of patient ages between groups (P = 0.42). The mean myoma size for all specimens was 6.5 cm 0.44 with a range from 0.4 cm to 27.0 cm. Cytogenetic analysis revealed 73 (64%) normal and 41 (36%) abnormal myoma karyotypes. Among the 41 abnormal karyotypes, there were 21 (18%) abnormal (mosaic), and 20 (18%) abnormal (non-mosaic) karyotypes. Table I summarizes the mean and median myoma size for karyotypically normal and abnormal specimens, and for abnormal (mosaic) and abnormal (non-mosaic) samples. A statistically significant difference in the mean myoma diameters (P = 0.002) was noted among the groups. The mean myoma diameter of specimens with abnormal karyotypes was not significantly different than specimens with normal karyotypes. However, the myoma diameters of specimens with abnormal (non-mosaic) karyotypes were significantly larger than specimens with normal karyotypes (10.2 5.9 versus 5.9 4.2 cm; P = 0.001) and abnormal (mosaic) karyotypes (10.2 5.9 versus 5.0 3.0 cm; P = 0.004). Similarly, the calculated mean myoma volume of specimens with abnormal (non-mosaic) karyotypes was significantly larger than specimens with normal karyotypes (1173 versus 344 cm3; P = 0.007) and abnormal (mosaic) karyotypes (1173 versus 136 cm3; P = 0.004).

Myomas with abnormal (non-mosaic) karyotypes demonstrated a significantly higher proportion of tumours > 6.5 cm when compared to myomas with normal karyotypes (75% versus 34%; P <0.02) and abnormal (mosaic) karyotypes (75% versus 38%; P <0.02). Moreover, myomas > 6.5 cm demonstrated a significantly higher proportion of abnormal (non-mosaic) karyotypes when compared to myomas < 6.5 cm (31% versus 8%; P <0.002).

The karyotype and myoma size for specimens with an abnormal (non-mosaic) and abnormal (mosaic) cytogenetic analysis are reported in Tables II and III, respectively. Chromosome rearrangements involving chromosome 12 were identified in 16 specimens; 10 with abnormal (non-mosaic) and 6 with abnormal (mosaic) karyotypes. Chromosome rearrangements involving chromosome 7 were also identified in 16 specimens; 5 with abnormal (non-mosaic) and 11 with abnormal (mosaic) karyotypes. The diameter of myoma specimens with chromosome 12 abnormalities appears to be larger than the diameter of myomas with chromosome 7 abnormalities (8.5 versus 5.0 cm; P = 0.09). Similarly, the calculated myoma volume of specimens with chromosome 12 abnormalities appears to be larger than the volume of myomas with chromosome 7 abnormalities (1069 versus 168 cm3; P = 0.08). However, these differences did not reach statistical significance.

 
Table II. Abnormal (non-mosaic) karyotype and myoma size
Accession number Myoma karyotype Myoma diameter (cm)
ST89-099a

42,XX,der(1)(?::p33->qter),-2,der(3)(?::p11->q26::?), dic(5)(?::cen->qter),der(6)(?::p11->qter),inv(6)(p21q26), der(?)(7qter->7p11::?),-8,-10,-13,der(19)(pter->q13::?)

11.0
ST89-171b 46,XX,der(14)t(12;14)(q14-15;q2324), der(22)(pter->q11::?) 9.0
ST89-232b 46,XX,t(1;13)(q31;q21),?inv(1)(q31q43),del(3)(q21) 15.0
ST89-250b 46,XX,der(1)(?::1p33->q22::1p13->p34::1q23->qter),t(2;6) (q11;q24),-5,+mar 15.0
ST89-262b 46,XX,del(7)(q22q31) 7.0
ST90-021b 46,XX,r(?1)(?p32q21),t(12;14)(q14-15;q23-24), der(16)(pter->q12::?) 7.5
ST90-194b 46,XX,t(12;14)(q14-15;q23-24) 27.0
ST90-436 46,XX,-8,der(12)?dup(12)(q14->q24),?i(12p),+mar 15.0
ST92-119 46,XX,t(3;19)(q22;p13),t(10;17)(q24;q24) 12.0
ST93-055c 45,XX,t(1;7)(q31;q22),-2,-3,-6,add(11)(q21),-18,+mar1,+mar2, +mar3 3.5
ST93-165d 46,XX,del(1)(q42),t(12;14)(q14;q24) 7.5
ST93-220d 46,XX,t(12;14)(q13;q32) 3.0
ST93-397e 46,XX,inv(6)(p21q15),inv(9) 13.5
ST93-470c 46,XX,del(7)(q22q32) 1.3
ST93-590 46,XX,add(2)(q36),?inv(12)(p13q21) 4.5
ST93-738d 46,XX,t(12;14)(q14-15;q23-24) 14.5
ST94-008e 45,XX,del(1)(p22p35),-6,add(10)(q22),-19,+mar1 9.0
ST94-114d 46,XX,del(7)(q22q32),t(12;14)(q14-15;q23-24) 13.0
ST95-101 46,XX,t(12;14)(q15;q?22) 11.0
ST95-539f 46,XX,del(7)(q22q32) 4.5

Karyotypes previously reported: aFletcheret al. (1990), bReinet al. (1991), cSargentet al. (1994), dSchoenberg Fejzo et al. (1996), eWilliamset al. (1997), fXinget al. (1997).


Table III. Abnormal (mosaic) karyotype and myoma size
Accession number Myoma karyotype Myoma diameter (cm)
ST89-186b 50,XX,dup(12)(q14>q24),+21,+21,+21,+21[1]/46,XX[65] 1.5
ST89-205c

46,XX,del(7)(q22q23)[10]/46,XX, inv(12)(q15q24)[2]/46,XX[77]

0.4

ST89-233b 46,XX,t(2;12)(q13;q15)[1]/46,XX[43] 3.0
ST89-234b 46,XX,t(7;9)(q22;q22)[8]/46,XX[33] 4.0
ST91-328c 43,X,-X,del(1)(p32->pter),-6,del(7)(q22q32),-19[16]/46,XX[7] 4.0
ST93-147c 46,XX,del(7)(q22q32)[10]/46,XX[92] 10.0
ST93-399 46,XX,add(2)(q31),der(3)t(2;3)(q31;q13.2), add(16)(p)[12]/46,XX,add(2)(q31),-3,+mar[5]/46,XX[6] 9.5
ST93-481 46,XX,add(12)(q)[11]/46,XX[6] 7.5
ST93-591 46,XX,t(12;14)(q14-15;q23-24)[16]/46,XX[4] 3.0
ST93-733c 46,XX,del(7)(q22q32)[13]/46,XX[2] 1.5
ST94-145f 46,XX,del(7)(q22)[2]/46,XX[58] 7.0
ST94-146f 46,XX,del(7)(q11),der(14)t(7;14)(q22;q32)[11]/46,XX[6] 7.0
ST94-193f 46,XX,del(7)(q22),9qh+[3]/46,XX,9qh+[17] 2.0
ST94-203f 46,XX,del(3)(q23q26)[15]/46,XX,del(7)(q22q32)[1]/46,XX[4] 7.5
ST94-532f

46,XX,del(7)(q21q31)[13]/46,XX[2]

1.0

ST94-611 46,XX,11p+[4]/46,XX[9] 4.0
ST95-008 47,XX,r(1),+r(1),der(2)t(1;2)(q31;p22)[4]/46,XX,r(1), der(2)t(1;2)(q31;p22)[5]/46,XX[6] 8.0
ST95-479f 46,XX,del(7)(q22q32)[6]/46,XX,?inv(7)(q21q22)[2]/46,XX[10] 6.0
ST95-586 46,XX,t(12;14)(q14-15;q23-24)[2]/44,XX,der(1)t(1;2)(p32;q22), -2,t(12;14)(q14-15;q23-24),-13,der(21)t(13;21)(q12;q22)[12] 9.5
ST95-605 46,XX[14]/39-45,X,-X[2],-1[5],der(5)[5],-19[4][cp5] 5.0
ST95-610 46,XX,add(1)(p36)[13]/46,XX[9] 4.5

Karyotypes previously reported: aReinet al. (1991), bSargentet al. (1994), cXinget al. (1997).


Discussion

The present study demonstrates a significant relationship between clonal cytogenetic abnormalities and myoma size. The calculated volume of myomas with abnormal (non-mosaic) karyotypes was greater than three times the calculated volume of myomas with normal karyotypes. Our data support the hypothesis that chromosomal aberrations in individual myomas may influence myoma growth. The precise molecular mechanisms by which chromosomal abnormalities regulate myoma growth remain to be determined. The wide variety of cytogenetic abnormalities associated with myomas may represent the biologic basis for the differential growth responsiveness of individual tumours.

Our data also suggest that specific cytogenetic abnormalities may have different effects on myoma growth. The frequent observation of cytogenetic deletions of 7q in a mosaic state (69% of chromosome 7 abnormalities in this series) and our finding that mosaic karyotypes are associated with myomas smaller than karyotypically normal specimens, suggest that loss of a gene product at 7q22 results in a negative effect on myoma growth. Comparing the two most common chromosome aberrations, specimens with chromosome 12 rearrangements were larger than specimens with chromosome 7 rearrangements; the absence of a statistically significant difference may be due to sample size. The fundamental importance of the 12q14-15 region in benign neoplasia is supported by the occurrence of consistent rearrangements in numerous other solid benign tumours including lipoma, pleomorphic adenoma of the salivary gland, pulmonary chondroid hamartoma, endometrial polyps and epithelial breast tumours (Bullerdiek et al., 1987; Mandahl et al., 1987). Our laboratory and others identified DNA clones containing the 12q14-15 breakpoint region in uterine myomas (Schoenberg Fejzo et al., 1995; Schoenmakers et al., 1995) and hypothesized dysregulation of the high mobility group protein gene HMGI-C in the pathobiology of myomas (Schoenberg Fejzo et al., 1996). HMGI-C regulates transcription and appears to have a fundamental role in growth and differentiation. HMGI-C has been found to be disrupted in lipomas such that DNA-binding AT hook motifs are fused to distinct transcriptional regulatory domains (Ashar et al., 1995; Schoenmakers et al., 1995). Fusion transcripts in uterine leiomyomata between HMGI-C and mitochondrial aldehyde dehydrogenase (Kazmierczak et al., 1995) and RTLV-H-related sequences (Kazmierczak et al., 1996a) have been identified. In addition, HMGI(Y), another high mobility group protein gene, assigned to 6p21, has been observed to be expressed at a very high level in some uterine leiomyomata (Williams et al., 1997) and to be present in DNA clones which span rearrangements of 6p21 in uterine leiomyomata (Kazmierczak et al., 1996b; Williams et al., 1997). It is clear now that rearrangements of high mobility group protein family genes participate in the molecular genetic events underlying uterine leiomyomata and other benign mesenchymal tumours (Hennig et al., 1996). Future studies will address the relationship between altered HMGI-C and HMGI(Y) expression and abnormal uterine smooth muscle proliferation.

In summary, a significant relationship exists between cytogenetic abnormalities and myoma size indicating that chromosomal aberrations associated with individual myomas affect myoma growth. The present study also supports our hypothesis that development and growth of myomas involves a multistep cascade of tumour initiators and promoters (Rein et al., 1995). Chromosomal abnormalities may induce a local growth promoting environment via aberrant expression of growth factors and/or growth factor receptors or, alternatively, repression of a tumour suppressor factor. Although the molecular mechanisms remain to be elucidated, identification of additional myoma genes will clearly improve our understanding of the biology of these common tumours.


Acknowledgements

These studies were supported in part by NIH HD-30498 (to C.C.M.).


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