Genetic Characterization of a Human Skin Carcinoma Progression Model: from Primary Tumor to Metastasis  Susanne Popp, Stefan Waltering, Petra Boukamp  Journal of Investigative Dermatology  Volume 115, Issue 6, Pages 1095-1103 (December 2000) DOI: 10.1046/j.1523-1747.2000.00173.x Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Summary of chromosomal gains and losses (CGH) in the four different tumors. Recurrent tumors 2, 3, and X, and metastatic tumor 4 (solid lines) and four corresponding cell lines: MET-1 established from primary tumor, MET-2, and MET-3 from recurrent tumors 2 and 3, and MET-4 from metastatic tumor (dotted lines). Gains are shown on the right and losses on the left side of the chromosome idiogram. Journal of Investigative Dermatology 2000 115, 1095-1103DOI: (10.1046/j.1523-1747.2000.00173.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Characterization of chromosomal aberrations in the MET lines. (A–C) M-FISH analysis; (D) painting of individual chromosome arms; (E) CGH analysis for specific chromosomes. (A) Multicolor karyotype of MET-1 cells (hypodiploid) showing several rearrangements: t(3;11) (arrow), t(5p;7p), del(7p), der(8) containing only 8q material, t(5p;10q), t(3;11) (arrowhead), t(15q;21q), dup(17)(q22q25), and t(17;19). (B) MET-2 cells (hyperdiploid) exhibit all but two aberrations detected in MET-1 [t(15;21) and del(7p) are missing] but in addition contain t(5;22) and del(4q);i(5p) is present in only few metaphases. (C) Multicolor karyotype of MET-4 cells (hypotetraploid) shows all but two aberrations described for MET-1 cells [t(3;7) and i(10q) likely derived from t(3;11) and t(5p;10q)] in addition to one new aberration, t(X;2) present in all metaphases and translocations occurring in individual metaphases only such as t(1q;15q), t(4;5), t(9;14), t(9;15), and t(9;15;21). (D) Partial metaphase spreads from MET-1 after three-color painting with microdissected probes for chromosome arms 3p, 3q, 11p, and 11q. In the first setting chromosome 3p is painted in green, 3q in red, and 11 in yellow (left). Under these conditions two normal chromosomes 3 and 11 and two different translocation chromosomes are seen (arrow and arrowhead). In the second setting chromosome 3 is painted in yellow, 11p in green, and 11q in red (right). This combination shows a t(3q;11q) (arrow) and a complex translocation composed of 11q, 11p, 11q, and 3q (arrowhead). Below: the DAPI-banded chromosomes and respective color schemes are given for the two hybridizations showing four chromosomes. The vertical bars show the order of rearranged chromosome material. (E) Selected CGH profiles for chromosomes 3 and 11 from MET-1 to MET-4 cells and tumor 4 (TU-4), the metastasis. The three vertical lines in the ratio profiles represent the balanced state (middle), upper (right), and lower (left) thresholds. All cell lines and the tumor show loss of 3p (exceed left threshold) and gain of 3q material (exceed right threshold) corresponding to the rearranged chromosome 3 (compare with D, left). For chromosome 11, all CGH profiles reveal a peak of amplified material at q13 corresponding to overrepresented parts of 11q in both translocation chromosomes (compare with D, right). As MET-3 and MET-4 cells only contain the complex translocation, gain of 11q13 is caused by the smaller 11q region in this chromosome (D, asterisk). Journal of Investigative Dermatology 2000 115, 1095-1103DOI: (10.1046/j.1523-1747.2000.00173.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Semiquantitative reverse transcribed–PCR analysis of FHIT transcripts. (A) Using primers spanning the entire FHIT open reading frame (819 bp FHIT fragment) only MET-4 cells give rise to the right-size transcript. GAPDH (397 bp fragment) was coamplified in the same reaction tube. (B) Nested PCR demonstrates the presence of a normal 550 bp transcript also in MET-1 cells. mRNAs from primary keratinocytes and a human SCC line served as the control. Journal of Investigative Dermatology 2000 115, 1095-1103DOI: (10.1046/j.1523-1747.2000.00173.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Hypothetical scheme of MET development. Starting from a common precursor population containing a complex t(3;11) t(5p;7p), t(5p;10q), der(8), t(3;11), t(17;19), and dup(17)(q22q25), MET-1 and MET-2 differ in two maker chromosomes each: t(15;21) and del(7p) in MET-1 cells; t(5;22) and del(4q) in MET-2 cells. Also MET-4 cells contain the markers of the precursor population, although two have changed [t(3;7) and i(10q)]. In addition, MET-4 cells contain the two markers of MET-1 but none of MET-2 cells suggesting that MET-4 cells have evolved from MET-1 cells whereas MET-2 cells developed in parallel. Journal of Investigative Dermatology 2000 115, 1095-1103DOI: (10.1046/j.1523-1747.2000.00173.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions