Genome wide single cell analysis of chemotherapy resistant metastatic cells in a case of gastroesophageal adenocarcinoma
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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.
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.
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.
KeywordsTrastuzumab Bone Metastasis Gastroesophageal Junction KRAS Gene Bone Marrow Metastasis
comparative genomic hybridization
cancer antigen 125
x-ray computed tomography
v-erb-b2 erythroblastic leukemia viral oncogene homolog 2 (gene) alias HER2/NEU
epithelial specific antigen i.e. EpCAM: epithelial cell adhesion molecule
fluorescence in situ hybridization
v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (gene)
mucin 1 cell surface associated
magnetic resonance imaging.
The incidence of adenocarcinomas of the gastric cardia, the gastroesophageal junction (GEJ) and esophagus has had a sharp rise in the past few decades, in particular among white males in the United States and western Europe, whereas the incidence of gastric cancer of the corpus or pyloric region has declined in many western countries [1, 2, 3]. In contrast to squamous cell carcinomas of the esophagus, which are strongly associated to alcohol and tobacco consumption, adenocarcinomas of the lower esophagus, GEJ and proximal stomach are typically associated with factors like Barrett's esophagus, gastroesophageal reflux, asthma medications, cholecystectomy, and obesity [4, 5, 6, 7, 8, 9]. The dismal outcome of these patients, often due to the advanced disease stage at diagnosis, underlines the need for new treatment options, as well as better understanding of the biology of the disease. Improving therapy of metastatic gastroesophageal adenocarcinoma is warranted. A landmark study in this regard was the recently published randomized phase III ToGA study , establishing c-erbB2 (HER-2/neu) as a target for therapy in advanced gastric and gastroesophageal junction cancer, with clinically significant improved overall survival for patients treated with trastuzumab in a first line regimen of 5-fluorouracil/capecitabine and cisplatin. As gastric cancer reveals more heterogeneity in the expression of erbB2 than breast cancer, a modified scoring system for gastric cancer was agreed upon at an international consensus meeting , and used by the panel of pathologists in the ToGA study. Patients having tumors with high expression of erbB2 (IHC3+) and ERBB2 FISH positive revealed a particular benefit of the trastuzumab treatment, whereas the benefit was less for the patients with lower expression levels of erbB2 or with negative FISH. However, in light of these exciting data, the clinical significance of intrapatient erbB2 heterogeneity in metastatic gastroesophageal cancer at diagnosis and during ongoing chemotherapy is less well known. Different metastatic tumor cell clones may show different sensitivity to chemotherapy, or change during disease progression, due to metastatic evolution . In this case of adenocarcinoma of the gastroesophageal junction, we examined the erbB2 expression in multiple metastatic lesions as well as the interlesional heterogeneity in response to chemotherapy. The chemotherapy resistant metastatic cells in a progressing metastatic bone lesion after the initial cycles of chemotherapy were analyzed genome-wide by screening of DNA copy number changes to identify oncogenic aberrations and potential targets of therapy.
Biopsies from the gastroesophageal tumor and metastases in the liver and bone marrow were sampled. The histopathology revealed a moderately differentiated adenocarcinoma, with glandular structures formed by atypical columnar epithelium, with unevenly and aberrantly distributed chromatin structures within the cell nuclei, and tumor infiltration of normal squamous epithelium. Immunohistochemical staining was positive for cytokeratin (CK) 7, caudal related homeobox-2 (CDX-2), CEA, and negative for CK20, prostate specific antigen, synaptophysin, chromogranin A, and thyroid transcription factor TTF-1, thus supporting the diagnosis of a neoplasm of the gastroesophageal junction as the primary tumor.
Chemotherapy was continued, and after two more cycles of EOX, CT scan evaluation showed a further reduction of the lung, liver and lymph nodes metastases, serum CEA was further reduced to 39 μg/L. CT evaluation also showed bone metastasis progressing steadily and unaffected of the palliative chemotherapy given. Due to increased pain in his lower back and the pelvic area, the patient received palliative radiotherapy to 30 Gy in two weeks to the affected area. The EOX chemotherapy was continued and he received zoledronic acid due to lytic bone lesions. Two additional cycles of EOX was given, but due to general disease progression the treatment was discontinued. He experienced increased pain, had decreased appetite and had lost an additional 5 kgs of weight. Shortly after, he was admitted to hospital because of acute neurological symptoms with pareses and paralysis of left cranial nerves (i.e. the trigeminal (V) and abducens (VI) nerves). An MRI scan revealed multiple meningeal metastatic deposits and disseminated pathologic lesions in the bone marrow of the scull, facial bones and spine. High-dose glucocorticosteroid treatment relieved the symptoms, and he was given palliative radiotherapy (to 30 Gy in two weeks) towards the whole brain, including the meninges and base of the skull. His health condition did not allow further treatment with any second-line chemotherapy regimen, but a regimen of trastuzumab monotherapy (4 mg/kg i.v. followed by 2 mg/kg week i.v.) was initiated. Six weeks thereafter he was admitted to the local hospital because of acute respiratory distress and pneumonia. One week after the hospital admission, the patient deteriorated and died.
A main challenge in metastatic cancer treatment is the development or enrichment of therapy-resistant tumor cells. In this young patient with stage IV gastroesophageal adenocarcinoma, we performed global gene analysis of DNA copy number changes in selected tumor cells sampled from a chemotherapy-resistant bone marrow metastasis. The gene analysis showed amplification of KRAS and ERBB2 oncogenes as possible molecular targets of therapy in tumor cells. However, after the discontinuation of EOX therapy, erbB2 targeted therapy with trastuzumab monotherapy was unable to provide substantial disease control, since his general health condition deteriorated rapidly. Other factors in addition to erbB2 receptor expression and signaling could also be important to sustain disease progression. In the case of cetuximab treatment of colorectal cancer, which targets another receptor of the ERBB-family (the epidermal growth factor receptor) the expression of receptor ligands (i.e. amphiregulin and epiregulin), as well KRAS mutation, predict treatment response . For our patient, we did not detect any mutation in the KRAS gene, but amplification of the gene was detected. Potentially, high expression of the GTPase Kras oncoprotein, due to gene amplification, could sustain oncogenic signaling despite erbB2 inhibition by trastuzumab treatment. Other mechanisms of trastuzumab resistance could also be involved . Immunohistochemical analysis showed heterogeneous expression of erbB2, with distinct erbB2-positive tumor cell populations before chemotherapy in the primary tumor, but negative cells in the liver and bone marrow, with cells staining strongly positive for erbB2 in bone marrow after the three initial cycles of chemotherapy. These results were furthermore supported by the finding of a similar pattern for ERBB2 gene amplification, as shown by the FISH results. Heterogeneous gene amplification was found both for the ERBB2 and the KRAS genes, but particularly enrichment of ERRB2 amplified cells was found in the biopsy from the bone metastasis after the initial three courses of chemotherapy. The clinically validated c-erbB2 immunohistochemistry analyses and ERBB2 FISH analyses supported and served as controls for the array CGH results of ERBB2 gene amplification found in the tumor cells harvested from the separate bone marrow aspirate, indicating an enrichment of such cells in the bone marrow metastasis which clearly progressed during the initial three courses of chemotherapy as shown on the computer tomography radiographs. A decreased sensitivity to the EOX treatment in the ERBB2 amplified cells could be one explanation for the enrichment of such cells during chemotherapy.
The patient died 9.5 months after onset of first line treatment with EOX chemotherapy. Median overall survival for patients with advanced gastroesophageal cancer receiving EOX chemotherapy in the REAL2 study was reported to be 11.2 months . As in breast carcinomas, amplification of ERBB2 is found in 20-30% of adenocarcinomas of the esophagus, gastroesophageal junction and stomach, and is associated with poorer survival [19, 20]. Interventional protocols targeting erbB2 in gastroesophageal adenocarcinoma have been conducted . Data from the large phase III ToGA trial was recently published , where stage IV c-erbB2 positive gastric or esophageal adenocarcinomas were randomized to receive cisplatin and 5-FU or capecitabine ± trastuzumab, showing a significantly prolonged survival for the trastuzumab arm. These results have introduced c-erbB2 targeted therapy in the first line treatment for selected patients with metastatic gastroesophageal adenocarcinomas. Among several genetic abnormalities, both KRAS and ERBB2 amplification have been indicated as early events in adenocarcinoma development of Barrett's Esophagus . Amplification of wild-type KRAS has been implicated in therapy resistance in vitro, but the clinical importance is yet poorly described. Analysis of aberrations at the molecular level found in each patient's tumor cells can identify new therapeutic options. Such molecular characterization of tumors to identify potential therapeutic targets has been performed in pilot studies .
Global analysis of genetic aberrations, as illustrated in this case by performing array-CGH analysis on genomic DNA from only a few selected tumor cells of interest, can by way of identifying resistance markers obtain information on potential therapeutic targets responsible for malignant progression, as well as potential predictors of treatment response, thus emphasizing the importance of this powerful tool on the road to more personalized cancer therapies in the future.
Written informed consent was obtained for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.
Genomic microarrays were provided by the Norwegian Microarray Consortium at the national technology platform financed by the Functional Genomics Program of the Research Council of Norway.
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