1 CLINICAL VALUE OF DNA COPY NUMBER CHANGES IN SOLID TUMORS — DATA EXTRACTED FROM OUR ONLINE CGH DATA COLLECTION http://www.helsinki.fi/cmg/CHG-data.htm

Sakari Knuutila, Departments of Pathology and Medical Genetics, Haartman Institute and Helsinki University Central Hospital, University of Helsinki, Helsinki, Finland

CGH DATA COLLECTION ON OUR WEB SITE http://www.helsinki.fi/cmg The comparative genomic hybridization (CGH) method was developed and documented in 1992 by Anne Kallioniemi and coauthors (Kallioniemi et al., 1992). It turned out to be a powerful means for screening DNA copy number changes in solid tumors. We have collected CGH findings from 16,530 documented cases of some 80 tumor entities. The diagram (Fig. 1) shows that year by year the number of publications has increased almost exponentially. Figure 2 indicates the total number of cases in the main tumor categories.

FROM COPY NUMBER CHANGES TO GENES As a screening method, CGH has revealed an enormous amount of DNA copy number losses, gains and amplifications that have pinpointed chromosomal areas with tumor suppressor genes and oncogenes. Table 1 shows examples of genes that we have characterized subsequent to a copy number change found by the CGH technique.

TABLE 1. From DNA copy number change to gene: examples from our studies.

CHANGE TUMOR GENE REFERENCE (Hemminki et al., 1997; dim(19p) Peutz-Jeghers LKB1 Hemminki et al., 1998) amp(18q) Diffuse large B-cell BCL2 (Monni et al., 1997) lymphoma amp(1q21) Osteosarcoma COAS1, 2 and 3 (Meza-Zepeda et al., 2002) amp(17q12q21) Gastric carcinoma DARPP-32 (Kokkola et al., 1997; El-Rifai et al., 2002; Varis et al., 2002) 2 COPY NUMBER CHANGES TELL TUMOR PROGRESSION AND MOLECULAR PATHOGENESIS, AND HELP DIFFERENTIAL DIAGNOSES In a great variety of malignancies, especially in carcinomas, gains are more frequent than losses. The malignant nature of a tumor is usually indicated by numerous changes (Knuutila et al., 1998; Knuutila et al., 1999; Knuutila et al., 2000). Nevertheless, lack of changes does not rule out malignancy. Ewing's sarcoma, as an example, may not show any changes (Armengol et al., 1997). In many tumors, such as colorectal and cervical carcinomas, the number of changes, especially amplifications, increase during tumor progression (Ried et al., 1999).

Are there any copy number profiles in tumor that help in routine pathologic diagnosis? Differential diagnosis between mesothelioma and adenocarcinoma of the lung is difficult but prognostically and therapeutically important. Our previous studies demonstrated that different profiles in these tumors help in differential diagnosis (Björkqvist et al., 1998). DNA copy number profile in leiomyosarcoma differs from that in gastrointestinal stromal tumor (El-Rifai et al., 1998). Furthermore, copy number profiling has increased the insight into the biological pathogenesis of tumors. For example, in gynecological tumors a relevant question is whether serous tumors in the ovary, uterus and fallopian tube have similar pathogenesis. We have showed that the profiles were similar in serous tumors but distinct from those in other histological subtypes (Pere et al., 1998). This suggests that serous ovarian, uterine and tubal carcinomas may have a common molecular pathogenesis.

DNA COPY NUMBER CHANGES AS PROGNOSTIC AND PREDICTIVE MARKERS Data extracted from our compilation of documented CGH findings indicate numerous copy number changes that seem to have clinical value in establishing prognosis and selecting therapy. Table 2 is by no means a comprehensive presentation of all changes that have clinical value. Numerous findings, also those included in the table, need to be verified on larger patient material. According to my critical evaluation, it is fully justified to perform CGH analyses for clinical purposes, and this is also a practice that has been adopted in our laboratory for glial tumors, neuroblastoma, Ewing's sarcoma, mesothelioma/adenocarcinoma of the lung, and lipoma/lipoma-like liposarcoma. To conclude, I should say that this listing is just the top of an iceberg, and many more well- conducted clinicopathological/CGH studies are needed to identify all clinically relevant DNA copy number changes. Moreover, CGH array is an important novel technology which is able to show the biologically essential target genes from the copy number changes (Varis et al., 2002). In the future, the target genes can most probably be 3 studied by techniques other than CGH, such as FISH or PCR (see the Poster Abstract by Varis et al.: Gene amplification and overexpression at 17q in gastric cancer). 4

TABLE 2. Clinical value of DNA copy number changes by CGH.

TUMOR* CHANGE CLINICAL SIGNIFICANCE REFERENCE

BONE AND SOFT TISSUE Leiomyoma vs. leiomyosarcoma Normal vs. chaotic Benign vs. malignant (El-Rifai et al., 1998; Sarlomo-Rikala et al., 1998; Levy et al., 2000) Uterine leiomyosarcoma dim(10q) High grade, recurrent (Hu et al., 2001) Lipoma vs. lipoma-like liposarcoma Normal vs. amp(12q) Benign vs. malignant; (Szymanska et al., differential diagnosis 1997; Pedeutour et al., 1999) GIST No gains vs. numerous gains Benign vs. malignant (El-Rifai et al., 2000) Chondroma vs. chondrosarcoma Normal vs. numerous changes Benign vs. malignant (Larramendy et al., 1997; Larramendy et al., 1999) Chondrosarcoma dim(13q) as the sole change Longer survival (Hu et al.,2001; enh(8q24qter) Shorter overall survival (Larramendy et al.,1999) Ewing's enh(1q) Adverse overall survival (Hattinger et al., dim(16q) Event-free survival 2002) PNST enh(7p15p21, 17q22qter) Poor overall survival (Schmidt et al., 1999) MFH enh(7q32) Poor overall survival, worse (Skytting et al., 1999) metastasis-free survival Fibrosarcoma enh(12q14q22) Poor overall survival (Schmidt et al., 2002)

NEUROGENIC TUMORS Astrocytic tumor enh(7p) vs. end(8q) Higher survival rate vs. (Nishizaki et al., Lower survival rate 2000) Oligodendroglial tumor 5

Anaplastic dim(1p,19q), no TP53 mutation PCV treatment sensitive (+) (Weber et al., 1996; Bigner et al., 1999) Well-differentiated dim(9p,10), enh(7) No PCV+ TP53 pos., no dim(1p,19q) Mixed gliomas dim(1p,19q), no TP53 Oligodendroglial origin, (oligodendrocytomas); astrocytic PCV+ and oligodendroglial elements TP53 pos., no dim(1p,19q) Astrocytic origin, no PCV+ Neuroblastoma enh(17q21qter) Poor overall survival (Bown et al., 2001; Brinkschmidt et al., 2001) enh(17q12),proximal to ERBB2 Suppresses progression (Lastowska et al., enh(17q23.1qter), distal to Promotes progression; 2002) ERBB2 poorer survival RESPIRATORY TRACT Mesothelioma vs. adenocarcinoma of dim(10,14) (loss profiling) vs. Differential diagnosis (Björkqvist et al., the lung gain profiling 1998)

DIGESTIVE TRACT Esophageal squamous cell carcinoma enh(5p) Unfavorable prognosis; (Yen et al., 2001; independent prognostic Ueno et al., 2002) marker Hepatoblastoma enh(8q,20) Unfavorable prognosis (Weber et al., 2000) enh(2q24) Unfavorable prognosis, (Kumon et al., 2001) independent marker Hepatocellular carcinoma dim(8p,13q), amp(11q13) Unfavorable prognosis (Kusano et al., 2002)

FEMALE GENITALS Endometrioid carcinoma dim(9q32q34,11q23,Xq12q24) Lymph. node metastasis, (Suehiro et al., 2000) cervical involvement Cervical squamous cell carcinoma dim(9p) Lymph. node metastasis, (Dellas et al., 1999) dim(11p,18p) poor prognosis PCV: procarbazine, cyclophosphamide, vincristine; * tumors in bold: CGH analyses performed in our laboratory for clinical purposes 6 7

Figure 1

1992 1993 1994 1995 1996 1997 1998 8

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Figure 2 Barcelona, September 2002 10

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