Cytogenetic and molecular genetic demonstration of polyclonality in an acinic cell carcinoma.

The paradigm that human malignancies are monoclonal has been questioned during recent years by the finding of unrelated, cytogenetically aberrant clones in short-term cultures from certain tumour types, notably carcinomas of the breast, skin and upper aerodigestive tract. In order to analyse whether cytogenetically unrelated clones are also unrelated at the molecular level, we analysed the X-chromosome inactivation status in cell cultures from a cytogenetically highly polyclonal acinic cell carcinoma of the parotid gland. By using cell cultures dominated by a single abnormal clone, obtained through in vitro culturing for 3-5 passages, we showed that the different clones must indeed have originated from different cells.

Summary The paradigm that human malignancies are monoclonal has been questioned during recent years by the finding of unrelated, cytogenetically aberrant clones in short-term cultures from certain tumour types, notably carcinomas of the breast, skin and upper aerodigestive tract. In order to analyse whether cytogenetically unrelated clones are also unrelated at the molecular level, we analysed the Xchromosome inactivation status in cell cultures from a cytogenetically highly polyclonal acinic cell carcinoma of the parotid gland. By using cell cultures dominated by a single abnormal clone, obtained through in vitro culturing for 3-5 passages, we showed that the different clones must indeed have originated from different cells.
Keywords: cytogenetics: molecular genetics: polyclonality; acinic cell carcinoma Todav it is generallv beliexed that most human neoplasms are monocellular in origin. an assumption supported by a wealth of molecular genetic. immunologic and cytogenetic data (Wainscoat and Fey. 1990). However. for some beni'gn tumour types. e.g colonic adenomas in patients w%ith familial adenomatous polyposis and fibroadenomas of the breast. molecular studies have show-n convincincly that they are often polvclonal. argguing for a multicellular oriain of these lesions (Noauchi et al. 1993: Noxelli et al. 1996. Whether human malignancies could also be composed of multiple neoplastic cell populations is less certain. but indirect support for this possibility comes from the cvtogenetic detection of unrelated. karyotypically abnormal clones in tumours that have been analysed after short-term culturing in vitro: such cv'togenetic heterogeneity has been reported particularly often for carcinomas of the breast. skin and upper aerodigestive tract (Jin et al. 1995. 1997a: Heim et al. 1997. The finding, of cytogenetic polvclonality does not. however. proxvide definitive proof that the lesion under study had a multicellular onigin. Auxiliarv hypotheses that may be invoked when arguing for a monocellular origin in such cases would be that the cyitogeneticallv unrelated clones could share a submicroscopic rearrangement. i.e. the' are monoclonal for other genetic markers. e.g. point mutations or X-chromosome inactivation pattern. or that some or all of the abnormal clones represent stromal cells rather than tumour parenchymal cells. Indeed. almost alwavs when unrelated abnormal clones have been detected in short-term cultured carcinomas. they has-e been pseudoor near diploid with relatively few chromosomal aberrations. and similar clones may be detected in. e.g. normal skin and non-neoplastic mesenchymal and epithelial mucosa cells as well (Jin et al. 1997b).
In the present study. w-e combined cytogenetic and molecular aenetic techniques in the analvsis of an acinic cell carcinoma Received 9 October 1997 Revised 27 January 1998 Accepted 28 January 1998 Correspondence to: C Jin (ACC) of the parotid gland to demonstrate. for the first time. that cyvtoeneticallv distinct clones in tumour tissue may also be unrelated at the molecular level.

MATERIALS AND METHODS Clinical and histopathological data
A 72-year-old %voman wvith no prior malignant disease presented 'A ith a 3-month historx of a growing tumour in the right parotid region. Cytological analysis of cells from a fine-needle aspiration biopsy was suaaestixe of ACC. At total parotidectomy. saving the facial nerxe. a 2-cm tumour '-as found in the superficial lobe. The tumour was circumscribed and divided into nodules by fibrous bands. Histologically. neoplastic periodic acid-Schiff (PAS)-positive serous acinar cells 'were found in a microcy-stic pattern x ith moderate lymphocytic infiltrates throughout. but accentuated in the periphery. The diagnosis was low -grade malignant ACC.

Cytogenetic techniques
The sample used for gyenetic analyses wAas taken from the excised ACC: one portion '-as immediately frozen at -80'C and one was used for short-term culturinga and cv-togenetic analysis. as described previously (Jin et al. 1995). The cell suspension obtained after mechanical and enzymatic disaggregation A as subdivided into sex en portions from x-hich primarv cultures LI-L7 w-ere initiated. After 5-7 days. partial har'estingy of the primary cultures '-as performed. The remaininc cells in cultures LI1-L3 were further cultured until 90% confluence (3-5 days). Then. each culture wxas split 1:3: one cell portion was used for DNA extraction. one '-as used for cvtogenetic anal'sis and the remaininc cells A ere plated for further culturing. This scheme w-as repeated until the cells spontaneously stopped dixiding (passages 3-5). The cell culture morphologv assessed bv an inverted microscope was epithelial-like in all primarv cultures and subcultures.

Molecular genetic analysis of X-chromosome inactivation pattem
Total DNA extraction from primary tumour tissue. peripheral blood and cell cultures was performed using standard procedures (Sambrook et al. 1989). An aliquot of 5 gg of DNA was digested with the appropriate enzymes (Abrahamson et al. 1990). blotted on to nylon filters (Genescreen. Dupont) and hybridized with 3Plabeled DXS255 (M27P) DNA (Church et al. 1984;Abrahamson et al. 1990). Hybridizations and washings were as described by Church and Gilbert (1984).

Cytogenetic findings
A total of 1164 cells from the seven primary cultures (Ll-L7) was analysed. Of these. 235 (20%) had a normal chromosome complement 109 (10%) had non-clonal aberrations. and the remaining 820 (70%) karyotypically abnormal metaphase cells gave rise to 47 cytogenetically unrelated clones altogether (Table 1). The number of cells in each clone varied from 2 to 223. Ten of the abnormal clones were found in more than one independent primary culture. The karyotypic changes were diverse. and all chromosomes, except chromosome 21, were involved. Chromosome 6 was involved in eight clones: three had rearrangements of 6p2l and five had structural rearrangements affecting 6q. Cell subcultures from three (LI-L3) of the seven lines were further studied cytogenetically after continued in vitro culturing. In all three lines, the cytogenetic complexity decreased with time. i.e. the initial polyclonality was reduced to near monoclonality with Polychinality in an acnic cell carcinorna 295 one clone making up 82-96%7 of the cells after 3-4 passages ( Figure 1); C48 [46XX.t(15:18) [46,XX.t( 1 ;9Xp34;q22).t(4:9)(pl6;ql3),t(9;13)(p22;ql4)I in L3.
The cell populations that took over the cultures were different in the three lines and were either very small (C21 was found in 12 of 599 cells and C49 was present only as a single cell) or could not be detected at all (C48) in the primary cultures.

Molecular findings
The patient was shown to be heterozygous for the polymorphic marker DXS225 by analysis of DNA from peripheral blood (data not shown). To determine the X-chromosome inactivation status. DNA extracted from lines LI-L3 and frozen tumour tissue was digested with Pst L. giving rise to the polymorphic restriction fragments, and the methylation-sensitive enzyme Hpa II. Analysis of the primary tumour tissue showed one dominating clone with respect to X-chromosome inactivation, with one allele being partially and one completely methylated. whereas cell cultures LI and L3, each consisting of one dominating cytogenetic clone ( Figure 1). showed opposite X-inactivation patterns, one of the two alleles being completely methylated and the other completely unmethylated (Figure 2, lanes 4 and 6). The remaining culture. L2, showed the same methylation pattem as the tumour tissue. All analyses of DNA from cell cultures and tumour tissue were repeated with identical results.

DISCUSSION
ACC is an uncommon, usually low-grade, malignancy of the salivary glands. Including the present case. only 11 ACCs with abnormal karyotypes have been reported (Mitelman, 1998). All of them have displayed pseudoor near-diploid chromosome numbers with simple karyotypic changes and, excluding clones with sex chromosome aberrations as the sole anomaly. four of them have had cytogenetically unrelated clones. The only recurrent changes that have been identified are all numerical, i.e. -Y (six cases). + 8 (three cases) and + 7 (two cases). It may also be noted that four of the ACCs had structural rearrangements of 6q, a chromosome arm frequently involved in other types of salivary gland carcinoma (Mitelman. 1998).
The ACC of the present study was highly polyclonal at cytogenetic analysis of the primary cultures. By obtaining relatively pure subcultures. each containing one dominant abnormal clone, the cell cultures became suitable for analysis of their X-chromosome inactivation pattems. In women, one of the two parental X-chromosomes is randomly inactivated in each somatic cell in early fetal life: the same pattern of inactivation is then stably transmitted to the daughter cells at every cell division. It follows, then. that the finding of completely opposite methylation pattems in cultures LI and L3. each of which had a single abnormal clone (C48 and C49 respectively) making up 96% of the total number of cells, must indicate that the two dominant clones had originated from different cells. The cleavage patterns observed for DNA from primary tumour tissue and culture L2 (Figure 2. lanes 3 and 5) most probably resulted from the combination of sampling from less homogeneous cell populations (the dominating clone in L2 constituted only 82% of the cells) and intercellular variation in the degree of methylation at the CCGG site, recognized by the Hpa H enzyme, on inactive X chromosomes (Hendriks et al, 1992). All the abnormal clones in the ACC. both in primary cultures and after in vitro passaging. had relatively simple karyotypes, and it may be argued that only a few. or even none, of them were representative of the tumour parenchyma. We cannot entirely dismiss this possibility, buts as outlined above, all previously reported cytogenetically abnormal ACCs have had relatively simple, neardiploid karyotypes and have often been polyclonal. The finding of near-diploid clones in the present case is also in agreement with DNA flow cytometry data on a series of 15 ACCs. showing that low malignant cases have a diploid DNA content (el-Naggar et al. 1990). Fmally. it should also be emphasized that the patient had not received any genotoxic treatment and that. although cytogenetically abnormal clones may be found in non-neoplastic short-term cultured mucosa samples, these cultures have never displayed as many abefrations as the present ACC (Jin et al. 1997b).