Genome wide single cell analysis of chemotherapy resistant metastatic cells in a case of gastroesophageal adenocarcinoma.
BACKGROUND
Metastatic progression due to development or enrichment of therapy-resistant tumor cells is eventually lethal. Molecular characterization of such chemotherapy resistant tumor cell clones may identify markers responsible for malignant progression and potential targets for new treatment. Here, in a case of stage IV adenocarcinoma of the gastroesophageal junction, we report the successful genome wide analysis using array comparative genomic hybridization (CGH) of DNA from only fourteen tumor cells using a bead-based single cell selection method from a bone metastasis progressing during chemotherapy.


METHODS
In a case of metastatic adenocarcinoma of the gastroesophageal junction, the progression of bone metastasis was observed during a chemotherapy regimen of epirubicin, oxaliplatin and capecitabine, whereas lung-, liver and lymph node metastases as well as the primary tumor were regressing. A bone marrow aspirate sampled at the site of progressing metastasis in the right iliac bone was performed, and single cell molecular analysis using array-CGH of Epithelial Specific Antigen (ESA)-positive metastatic cells, and revealed two distinct regions of amplification, 12p12.1 and 17q12-q21.2 amplicons, containing the KRAS (12p) and ERBB2 (HER2/NEU) (17q) oncogenes. Further intrapatient tumor heterogeneity of these highlighted gene copy number changes was analyzed by fluorescence in situ hybridization (FISH) in all available primary and metastatic tumor biopsies, and ErbB2 protein expression was investigated by immunohistochemistry. ERBB2 was heterogeneously amplified by FISH analysis in the primary tumor, as well as liver and bone metastasis, but homogenously amplified in biopsy specimens from a progressing bone metastasis after three initial cycles of chemotherapy, indicating a possible enrichment of erbB2 positive tumor cells in the progressing bone marrow metastasis during chemotherapy. A similar amplification profile was detected for wild-type KRAS, although more heterogeneously expressed in the bone metastasis progressing on chemotherapy. Correspondingly, the erbB2 protein was found heterogeneously expressed by immunohistochemical staining of the primary tumor of the gastroesophageal junction, while negative in liver and bone metastases, but after three initial cycles of palliative chemotherapy with epirubicin, oxaliplatin and capecetabine, the representative bone metastasis stained strongly positive for erbB2.


CONCLUSIONS
Global analysis of genetic aberrations, as illustrated by performing array-CGH analysis on genomic DNA from only a few selected tumor cells of interest sampled from a progressing bone metastasis, can identify relevant therapeutic targets and genetic aberrations involved in malignant progression, thus emphasizing the importance and feasibility of this powerful tool on the road to more personalized cancer therapies in the future.
Oslo University Hospital, Division for Cancer and Surgery, Department of Oncology, The Norwegian Radium Hospital, Nydalen, N-0424 Oslo, Norway. Geir.Olav.Hjortland@oslo-universitetssykehus.no
Geir Olav
GO
Hjortland
Leonardo A
LA
Meza-Zepeda
Klaus
K
Beiske
Anne H
AH
Ree
Siri
S
Tveito
Hanne
H
Hoifodt
Per J
PJ
Bohler
Knut H
KH
Hole
Ola
O
Myklebost
Oystein
O
Fodstad
Sigbjorn
S
Smeland
Eivind
E
Hovig
D000230:Adenocarcinoma / Q000188:drug therapy / Q000235:genetics* / Q000473:pathology; D000328:Adult; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000494:pharmacology / Q000627:therapeutic use; D014408:Biomarkers, Tumor / Q000235:genetics; D055028:Comparative Genomic Hybridization; D019008:Drug Resistance, Neoplasm / Q000187:drug effects; D004938:Esophageal Neoplasms / Q000188:drug therapy / Q000235:genetics* / Q000473:pathology; D004943:Esophagogastric Junction / Q000473:pathology; D017809:Fatal Outcome; D006801:Humans; D008297:Male; D009362:Neoplasm Metastasis; D010166:Palliative Care; D059010:Single-Cell Analysis; D013274:Stomach Neoplasms / Q000188:drug therapy / Q000235:genetics* / Q000473:pathology; D016896:Treatment Outcome
D014408:Biomarkers, Tumor