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Functional Characterization and Drug Response of Freshly Established Patient-Derived Tumor Models with CpG Island Methylator Phenotype.

Maletzki C, Huehns M, Knapp P, Waukosin N, Klar E, Prall F, Linnebacher M - PLoS ONE (2015)

Bottom Line: Initial DNA fingerprint analysis confirmed identity with the patient in all four cases.These freshly established cells showed characteristic features associated with the CIMP-phenotype (HROC40: APCwt, TP53 mut, KRAS mut; 3/8 marker methylated; HROC43: APC mut, TP53 mut, KRAS mut; 4/8 marker methylated; HROC60: APCwt, TP53 mut, KRASwt; 4/8 marker methylated; HROC183: APC mut, TP53 mut, KRAS mut; 6/8 marker methylated).Of note, most cell lines were sensitive towards "non-classical" CRC standard drugs (sensitivity: Gemcitabin > Rapamycin > Nilotinib).

View Article: PubMed Central - PubMed

Affiliation: Molecular Oncology and Immunotherapy, University of Rostock, Rostock, Germany.

ABSTRACT
Patient-individual tumor models constitute a powerful platform for basic and translational analyses both in vitro and in vivo. However, due to the labor-intensive and highly time-consuming process, only few well-characterized patient-derived cell lines and/or corresponding xenografts exist. In this study, we describe successful generation and functional analysis of novel tumor models from patients with sporadic primary colorectal carcinomas (CRC) showing CpG island methylator phenotype (CIMP). Initial DNA fingerprint analysis confirmed identity with the patient in all four cases. These freshly established cells showed characteristic features associated with the CIMP-phenotype (HROC40: APCwt, TP53 mut, KRAS mut; 3/8 marker methylated; HROC43: APC mut, TP53 mut, KRAS mut; 4/8 marker methylated; HROC60: APCwt, TP53 mut, KRASwt; 4/8 marker methylated; HROC183: APC mut, TP53 mut, KRAS mut; 6/8 marker methylated). Cell lines were of epithelial origin (EpCAM+) with distinct morphology and growth kinetics. Response to chemotherapeutics was quite individual between cells, with stage I-derived cell line HROC60 being most susceptible towards standard clinically approved chemotherapeutics (e.g. 5-FU, Irinotecan). Of note, most cell lines were sensitive towards "non-classical" CRC standard drugs (sensitivity: Gemcitabin > Rapamycin > Nilotinib). This comprehensive analysis of tumor biology, genetic alterations and assessment of chemosensitivity towards a broad range of (chemo-) therapeutics helps bringing forward the concept of personalized tumor therapy.

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Related in: MedlinePlus

SNP Array 6.0 for assessment of CIN in CIMP+ cell lines.Analysis was performed according to manufacturer’s instructions. Data shown here result from three out of four CIMP+ cell lines. (A) HROC40 cells, (B) HROC43 cells, and (C) HROC60 cells. SNP Array on HROC183 did not yield evaluable data due to quality control failure.
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pone.0143194.g004: SNP Array 6.0 for assessment of CIN in CIMP+ cell lines.Analysis was performed according to manufacturer’s instructions. Data shown here result from three out of four CIMP+ cell lines. (A) HROC40 cells, (B) HROC43 cells, and (C) HROC60 cells. SNP Array on HROC183 did not yield evaluable data due to quality control failure.

Mentions: A consensus panel for determining cytosine methylation at promoter CpG islands of tumor suppressor genes is only starting to take shape. We decided to use a combined panel covering eight tumor-specific methylation markers [16–18]. Hence, three cell lines (HROC40 (3/8 methylated promoters), HROC43 and HROC60 (each 4/8 methylated promoters)) were defined as CIMP-L and the remaining cell line HROC183 (6/8 methylated promoters) exhibited a CIMP-H phenotype. MSI was excluded by using the advanced Bethesda panel (0/6 marker). Additionally, instability on the chromosomal level was analyzed by SNP 6.0 arrays (Fig 4). Cell lines exhibited complex chromosomal aberrations, present as both deletions and insertions. Besides, typical molecular characteristics associated with the CIMP phenotype were present, i.e. KRAS mutations (codon 12 or 13 of exon 2 [19]), and BRAFV600 wildtype. By contrast, conflicting results have been reported for the TP53 gene [8, 20]. Here, mutations were evident in all four cases.


Functional Characterization and Drug Response of Freshly Established Patient-Derived Tumor Models with CpG Island Methylator Phenotype.

Maletzki C, Huehns M, Knapp P, Waukosin N, Klar E, Prall F, Linnebacher M - PLoS ONE (2015)

SNP Array 6.0 for assessment of CIN in CIMP+ cell lines.Analysis was performed according to manufacturer’s instructions. Data shown here result from three out of four CIMP+ cell lines. (A) HROC40 cells, (B) HROC43 cells, and (C) HROC60 cells. SNP Array on HROC183 did not yield evaluable data due to quality control failure.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4664421&req=5

pone.0143194.g004: SNP Array 6.0 for assessment of CIN in CIMP+ cell lines.Analysis was performed according to manufacturer’s instructions. Data shown here result from three out of four CIMP+ cell lines. (A) HROC40 cells, (B) HROC43 cells, and (C) HROC60 cells. SNP Array on HROC183 did not yield evaluable data due to quality control failure.
Mentions: A consensus panel for determining cytosine methylation at promoter CpG islands of tumor suppressor genes is only starting to take shape. We decided to use a combined panel covering eight tumor-specific methylation markers [16–18]. Hence, three cell lines (HROC40 (3/8 methylated promoters), HROC43 and HROC60 (each 4/8 methylated promoters)) were defined as CIMP-L and the remaining cell line HROC183 (6/8 methylated promoters) exhibited a CIMP-H phenotype. MSI was excluded by using the advanced Bethesda panel (0/6 marker). Additionally, instability on the chromosomal level was analyzed by SNP 6.0 arrays (Fig 4). Cell lines exhibited complex chromosomal aberrations, present as both deletions and insertions. Besides, typical molecular characteristics associated with the CIMP phenotype were present, i.e. KRAS mutations (codon 12 or 13 of exon 2 [19]), and BRAFV600 wildtype. By contrast, conflicting results have been reported for the TP53 gene [8, 20]. Here, mutations were evident in all four cases.

Bottom Line: Initial DNA fingerprint analysis confirmed identity with the patient in all four cases.These freshly established cells showed characteristic features associated with the CIMP-phenotype (HROC40: APCwt, TP53 mut, KRAS mut; 3/8 marker methylated; HROC43: APC mut, TP53 mut, KRAS mut; 4/8 marker methylated; HROC60: APCwt, TP53 mut, KRASwt; 4/8 marker methylated; HROC183: APC mut, TP53 mut, KRAS mut; 6/8 marker methylated).Of note, most cell lines were sensitive towards "non-classical" CRC standard drugs (sensitivity: Gemcitabin > Rapamycin > Nilotinib).

View Article: PubMed Central - PubMed

Affiliation: Molecular Oncology and Immunotherapy, University of Rostock, Rostock, Germany.

ABSTRACT
Patient-individual tumor models constitute a powerful platform for basic and translational analyses both in vitro and in vivo. However, due to the labor-intensive and highly time-consuming process, only few well-characterized patient-derived cell lines and/or corresponding xenografts exist. In this study, we describe successful generation and functional analysis of novel tumor models from patients with sporadic primary colorectal carcinomas (CRC) showing CpG island methylator phenotype (CIMP). Initial DNA fingerprint analysis confirmed identity with the patient in all four cases. These freshly established cells showed characteristic features associated with the CIMP-phenotype (HROC40: APCwt, TP53 mut, KRAS mut; 3/8 marker methylated; HROC43: APC mut, TP53 mut, KRAS mut; 4/8 marker methylated; HROC60: APCwt, TP53 mut, KRASwt; 4/8 marker methylated; HROC183: APC mut, TP53 mut, KRAS mut; 6/8 marker methylated). Cell lines were of epithelial origin (EpCAM+) with distinct morphology and growth kinetics. Response to chemotherapeutics was quite individual between cells, with stage I-derived cell line HROC60 being most susceptible towards standard clinically approved chemotherapeutics (e.g. 5-FU, Irinotecan). Of note, most cell lines were sensitive towards "non-classical" CRC standard drugs (sensitivity: Gemcitabin > Rapamycin > Nilotinib). This comprehensive analysis of tumor biology, genetic alterations and assessment of chemosensitivity towards a broad range of (chemo-) therapeutics helps bringing forward the concept of personalized tumor therapy.

Show MeSH
Related in: MedlinePlus