apparent correlation of sex chromosome loss and disease course in urothelial cancer

Apparent Correlation of Sex Chromosome Loss and Disease Course in Urothelial Cancer

Isaac Powell, Michael Tyrkus, and Edwardo Kleer

ABSTRACT: In recent years, interest in the genetics of various solid tumors has increased dramatically. Over the last several years, our laboratory has pursued genetic studies of transitional cell carcinoma of the bladder. We have studied the cytogenetics and disease course of more than 100 patients, achieving successful cultures in more than 75%. In these patients, we observed the loss of a sex chromosome •either X or Y) with a frequency of 40%. We stratiJled patients with loss of sex chromosomes into three groups: 1) presence of markers or chromosome re- arrahgements, 2) aneuploidy without marker chromosomes, and 3) loss of a sex chromosome only. The relevancy of both the natural history and potential impact of sex chromosome loss to the disease course, including invasive potential, will be discussed in detail for each of the three groups.

INTRODUCTION

In recent years, there has been an increase in the number of investigations dealing with the genetics of solid tumors. A number of these studies have been concerned with the cytogenetics of urothelial tumors. These studies have suggested that specific cytogenetic complements and anomalies are characteristic of particular tumors. How- ever, it has not been determined whether the presence of these cytogenetic changes precedes changes in tumor behavior or disease progression. Over the last several years, we have studied the cytogenetics of over 100 such patients, achieving successful cultures in more than 75%. In general, our findings agree with those previously reported in the literature that the presence of structurally altered "marker" chromosomes is an indicator of an invasive or potentially invasive nature of the tumor [1, 2]. However, in addition to the above, we have observed and evaluated a frequent loss of a sex chromosome in some of these cultures. This article reports the tumors in which this observation was made in terms of their invasiveness and observed potential for disease progression.

At The Third International Workshop on Chromosomes of Solid Tumors, Jenkins et al. [3] reported on the cytogenetics of 72 human gliomas and described a loss of the Y chromosome and other chromosomal changes in 30% of the grade 4 tumors (high grade) studied. In another study, Zanld et al. [4] found the Y chromosome to be missing in 25% of meningiomas of male origin and the verified loss of an X chromosome from tumor tissue in one female patient. In addition, Stenman and Mark [5] observed a secondary loss of the Y chromosome in a cultured human salivary gland

From the Department of Urology (I. P., E. K) Wayne State University, Detroit, Michigan, and Department of Clinical Laboratories (M. T.), The Toledo Hospital, Toledo, Ohio.

Address reprint requests to: Dr. Isaac Powell, Assistant Professor, Wayne State University, Department of Urology, 1017 Harper Professional Building, 4160 John R., Detroit, Mi 48201.

Received February 13, 1990; accepted April 19, 1990.

9 7

© 1990 Elsevier Science Publishing Co., Inc. 655 Avenue of the Americas, New York, NY 10010

Cancer G~net Cytogenet 50:97-101 (1990) 0165-4608/90/~03.50

9 8 I. Powell et al.

Table 1 Group A: Markers and sex chromosome loss

Initial Most recent

Case Date Stage Grade Date Stage Grade Treatment

A1 ° 8/87 B/A 3,CIS 4/88 A 3,CIS

A2 5/84 B2 3 Lost to follow-up

A3 b 5/84 0 3 11/85 B2 3

A4 4/87 0 2 Lost to follow-up A5 12/87 B2 2 Lost to follow-up

A6 4/86 0 1 9/88 No tumor A7 11/87 0 3,CIS 3/88 B1 CIS A8 9/86 B 3 A9 12/83 0 3 10/84 B1 3 A10 1/85 0 3 11/85 B1 3

Turbt/intra vesical RX

Cystectomy recommended



Cystectomy Cystectomy Cystectomy Cystectomy

recommended

Average time to invasion for this group was 11 mo. Q Nephroureterectomy 4/88 with muscle invasion. b Medical contraindication for cystectomy (i.e., too sick).

adenocarcinoma. They started with a karyotypically normal diploid tumor (in culture) that subsequently progressed to an aneuploid status that deviated from the normal only by loss of the Y chromosome. From this hypodiploid structure, other cytogenetic changes o~:curred. Holmes et al. [6] demonstrated the loss of Y chromosome in 13 patients with acute myelogenous leukemia (AML) in 8 years. In nine of these patients, loss of Y chromosome was the only karyotypic change. These observations suggest that the secondary loss of the Y chromosome is a consistent, albeit uncommon, chromosome marker in AML associated with an aggressive clinical course and inter- mediate prognosis. Smeets et al. [7] found the Y chromosome missing in 3 of 10 males with bladder cancer. Finally, Bateman et al. [8] were able to show that the loss of the Y chromosome was confined to malignant cells when normal cells were present. The results of our studies suggest that there is a "subgroup" of urothelial cancers whose behavior, propensity for recurrence, and invasiveness may be related to and predicted by the loss of a sex chromosome in a percentage of their cells.

MATERIALS AND METHODS

Tumor biopsies from patients diagnosed as having urothelial carcinoma were placed in tissue culture. Samples were maintained in nutrient media, supplemented with 15% fetal calf serum and antibiotics. Cultures were maintained in a biological atmo- sphere at 37°C. The length of time in culture varied from 3 days to 4 weeks, averaging 9-10 days. Harvest procedures were initiated when sufficient metaphase cells were available for analysis. Further data submitted elsewhere demonstrate that chromosomal evaluation is not a factor for interpretation in bladder tumors cultured up to 3 months. Chromosomal preparations were prepared with standard methods, and the resultant slides stained with either a GTG or QFQ differential banding procedure.

Patients with a determinable sex chromosome loss were stratified into three groups: 1) those with marker chromosomes, other structural or numeric changes present; 2)

X or Y Loss/Urothelial Cancer Disease Course 9 9

Table 2 Group,B: Markers and no sex chromosome loss (age between 39 and 80 yr)

Initial Most recent


B1 7/86 81 3 B2 4/83 0 2 1/87 B1 3 B3 12/87 0 2 ~/88 No tumor B4 5/86 A 3 8/88 0 2 B5 5/86 0 3 11/88 0 3 B6 ° 6 /85 B 2-3 9/88 No tumor B7 12/87 0 2 9/88 No tumor B8 1/88 A 3 Lost to follow-up B9 5/85 B2 3 9/85

Cystectomy Cystectomy Turbt Turbt Turbt

Turbt

Cystectomy

Average time to invasion 24 mo. o Nephroumterectomy in 6185.

those with numeric changes (including polyploidy) but no evidence of structural changes; 3) and those where the loss of the sex chromosome is the o 1,, observable anomaly.

Patient groups were further subdivided for analysis into 1) those with markers and loss of a sex chromosome; 2) those with markers but no apparent loss of sex chromosome; 3) those with aneuploid complements including polyploidy and a sex chromosome loss; and 4) those with aneuploidy, including polyploidy, but no sex chromosome loss. The disease course of all groups was compared with a group of patients diagnosed as urothelial cancer, but whose tumors exhibited a normal karyotype.

RESULTS

The observations collected on these samples are presented in tabular form (Tables 1-5).

D I S C U S S I O N

There are several reports in the literature equating stage and grade delineations of urothelial tumors with karyotypic changes. These include, but are not limited to the studies of Falor and Ward [1] or Sandbars [2]. The conclusion of these studies is that the presence of marker chromosomes or other identifiable structural rearrangements in such tumors is suggestive of an invasive potential. The results of the present study

Table 3 Group C: Aneuploidy (polyploidy) with sex chromosome loss (age between 62 and 70 yr)

Initial Most recent


C1 1/78 0 2 4/88 No tumor Turbt C2 7/83 A 3 4/88 No tumor Turbt C3 2/85 A 2 4/88 0 1 Turbt

All patients demonstrated frequent recurrences prior to 4/88.

1 0 0 I. Powell et al.

Table 4 Group D: Aneuploidy with no sex chromosome loss (age between 72 and 79 yr)

Initial Most recent


D1 7185 0 2 1/86 0 2 2 Turbt D2 7186 A 2 7/86 Cystectomy

agree with these findings but further suggest that a specific subgroup of patients who are at risk for disease progression may be identified cytogenetically.

The data reported here suggest that patients with a sex chromosome loss (observable in tumor tissue) tend to progress faster than those who retain both sex chromosomes. More specifically, when the lesions are stratified on the basis of more general- ized chromosomal abnormalities (i.e., markers and aneuploidy) and then evaluated in terms of disease progression, there is an apparent trend suggesting that those with a sex chromosomal loss progress faster than those with the same autosomal anomalies but no sex chromosome problems. The present groups of patients with aneuploidy with or without sex chromosome loss are too small to evaluate meaningfully, but those patients in whom the only apparent cytogenetic anomaly is the loss of a sex chromosome [by definition, this is aneuploidy in the strict sense) show a surprising correlation with progression of disease. Six of seven tumors in this group either recurred frequently or progressed during the time of the study. This observation can be contrasted with those tumors in which no anomaly is present (i.e., normal karyotypes) and in those tumors where no progression occurred except in 1 of 27 patients. The presence of a low-frequancy mosalcism for additional cell lines can not be explicitly ruled out for those tumors in which we observed sex chromosome loss as the only anomaly. However, the number of cells evaluated in most of these cases statistically excludes mosalcism below 10%.

Since we determined mosacism at this level or lower for several cell lines containing marker chromosomes, we consider the nondetection of such additional lines unlikely in those tumors interpreted as sex chromosome loss only. Attempts were made to biopsy the most malignant site or the center of the tumor, but there remains the possibility of sampling error.

Table 5 Group E: Sex chromosome loss only [age between 58 and 70 yr)

Initial Most recent

Case D a t e S t a g e Grade Date Stage G r a d e Treatment

E1 2/88 0 2-3,CIS 9/88 B 2-3 Cystectomy recommended

E2 7187 0 3 12187 B1 3,CIS Cystectomy E3 5/86 0 1 10/88 0 2 Turbt/frequent

recurrences E4 9/86 B1 3 9/86 Died Cystectomy

recommended E5 2/86 B2 2 2/86 Cystectomy E6 1/87 0 2-3,CIS 2/88 No tumor Turbt E7 1/85 A 3,CIS 11/88 B1 CIS Turbt/frequent

recurr/cystectomy 2/89

E7 has demonstrated karyotypic evolution to numerous structural abnormalities.

X or Y Loss/Urothelial Cancer Disease Course 1 0 1

Sex chromosome loss in peripheral blood cultures and/or bone marrow cultures is an expected phenomenon in elderly persons. Cytogeneticists have determined that this is an ase-related phenomenon and is most apparent in tissues with a high rate of mitotic activity. The patient age of 54 years is generally accepted as a time when this phenomenon is observable in males and is frequently apparent in bone marrow preparations. This phenomenon has not been reported previously in solid tumors, and it has not been conclusively confirmed in tissues other than blood or marrow. Sandberg [9] studied a series of 20 normal bladder samples derived from patients in whom the loss of a Y chromosome had been diagnosed in the bone marrow. All of the patients in this series exceeded 65 years of age but no loss of the Y was observed in these normal bladder biopsies. Several publications dealing with bladder tumors, including that of Smeets et al. [7], have mentioned the presence of sex chromosomal loss but have not examined its implication clinically.

It is tempting to speculate as to the mechanism, source, and significance of this loss. It is possible, since the sex chromosomes are among the last of the complement to replicate, that their loss in these tumors is inadvertant and merely reflects a biologic change in the tumor cells that alters the rate and sequence of cellular division. This theory would dictate that the loss is through the mechanism of anaphase |ag. Indirect supporting evidence is found through the lack of observations of cells containing extra copies of the sex chromosomes that would have been expected ff the process involved was nondisjunction. It is our current belief that the above process may contribute to the occurrence of this phenomenon. The loss of the sex chromosome may simply reflect changes in the cells that have developed a highly malignant nature or change that is not detectable at this cytogenetic level. However, since, as we have illustrated, there is a lag phase between our observation of sex chromosome loss and the confirmation of progression by histologic means in some patients, it is possible that the loss has a more direct effect on tumor development. Although it is admittedly hi~lly speculative at this point, we offer the suggestion that if, through the course of evolution, nature has permitted man to evolve a "master" anti-oncogene (one that suppresses the action of oncogenes by its presence and/or influence), what better place would there be to place such a locus than on the one mammalian chromosome pair that has not changed appreciably for many millania.

REFERENCES 1. Falor WH, Ward RN (1981) Cytogenetics of bladder carcinoma: A key to prognosis in noninva-

siva and submucosal invasive carcinoma. Cancer Detect Pmv 4:449-453. 2. Sandbar8 AA (1986} Chromosome changes in bladder cancer: Clinical and other correlations.

Cancer Genet Cytnganet 19:163-175. 3. Jenkins R, Kimmel D, Moertei C, Shultz C, Scheithauer B, Kelly P, Dewald G (1989) Cytoge-

netic Studies of 72 Human Giiomas: Abstract from The Third International Workshop on Chromosomes of Solid Tumors, No. 19, p. 25.

4. Zanld H, Seidel H, Zang KD (1975) Cytological and cytogenetical studies on brain tumors. Humangenetik 27:119-128.

5. Stenman G, Mark J (1983) Loss of the Y chromosome in a cultured human salivary gland adanocarcinoma. J Oral pathol 12:458-464.

6. Holmes RI, Keating MJ, Cork A, Trujillo JM, McCredie KB, Freireich EJ (1985) Loss of the Y chromosome in acute myelogenous leukemia: A report of 13 patients. Cancer Genet Cytogenet 17:269-278.

7. Smears W, Pauwels R, Lisette L, Debruyne F, Geraedts J (1987) Chromosomal analysis of bladder cancer. IIL Nonrandom alterations. Cancer Genet Cytogenet 29:29-41.

8. Bateman BJ, Sanger WG, Armita~e JO (1987) Missing Y chromosomes in lymph nodes from patients with non-Hodgkin's lymphoma. Cancer Genet Cytoganet 25:219-225.

9. Sandberg AA (1980) The Chromosomes in Human Cancer and Leukemia. Elsevier North Holland, Inc., New York, 1980, pp. 505-506.

apparent correlation of sex chromosome loss and disease course in urothelial cancer

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