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Massively parallel sequencing fails to detect minor resistant subclones in tissue samples prior to tyrosine kinase inhibitor therapy.

Heydt C, Kumm N, Fassunke J, Künstlinger H, Ihle MA, Scheel A, Schildhaus HU, Haller F, Büttner R, Odenthal M, Wardelmann E, Merkelbach-Bruse S - BMC Cancer (2015)

Bottom Line: Fresh-frozen samples showed the same range of artificially mutated allele frequencies as the FFPE material.This supports the theory that secondary KIT resistance mutations develop under treatment by "de novo" mutagenesis.Alternatively, the detection limit of two mutated clones in 10,000 wild-type clones might not have been high enough or heterogeneous tissue samples, per se, might not be suitable for the detection of very small subpopulations of mutated cells.

View Article: PubMed Central - PubMed

Affiliation: Institute of Pathology, University Hospital Cologne, Kerpener Str. 62, 50937, Cologne, Germany. carina.heydt@uk-koeln.de.

ABSTRACT

Background: Personalised medicine and targeted therapy have revolutionised cancer treatment. However, most patients develop drug resistance and relapse after showing an initial treatment response. Two theories have been postulated; either secondary resistance mutations develop de novo during therapy by mutagenesis or they are present in minor subclones prior to therapy. In this study, these two theories were evaluated in gastrointestinal stromal tumours (GISTs) where most patients develop secondary resistance mutations in the KIT gene during therapy with tyrosine kinase inhibitors.

Methods: We used a cohort of 33 formalin-fixed, paraffin embedded (FFPE) primary GISTs and their corresponding recurrent tumours with known mutational status. The primary tumours were analysed for the secondary mutations of the recurrences, which had been identified previously. The primary tumours were resected prior to tyrosine kinase inhibitor therapy. Three ultrasensitive, massively parallel sequencing approaches on the GS Junior (Roche, Mannheim, Germany) and the MiSeq(TM) (Illumina, San Diego, CA, USA) were applied. Additionally, nine fresh-frozen samples resected prior to therapy were analysed for the most common secondary resistance mutations.

Results: With a sensitivity level of down to 0.02%, no pre-existing resistant subclones with secondary KIT mutations were detected in primary GISTs. The sensitivity level varied for individual secondary mutations and was limited by sequencing artefacts on both systems. Artificial T > C substitutions at the position of the exon 13 p.V654A mutation, in particular, led to a lower sensitivity, independent from the source of the material. Fresh-frozen samples showed the same range of artificially mutated allele frequencies as the FFPE material.

Conclusions: Although we achieved a sufficiently high level of sensitivity, neither in the primary FFPE nor in the fresh-frozen GISTs we were able to detect pre-existing resistant subclones of the corresponding known secondary resistance mutations of the recurrent tumours. This supports the theory that secondary KIT resistance mutations develop under treatment by "de novo" mutagenesis. Alternatively, the detection limit of two mutated clones in 10,000 wild-type clones might not have been high enough or heterogeneous tissue samples, per se, might not be suitable for the detection of very small subpopulations of mutated cells.

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Related in: MedlinePlus

Visual depiction of the different experiments and workflows performed on the GS Junior (Roche) (A) and the MiSeq™ (Illumina) (B) with FFPE and fresh-frozen material.
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Fig1: Visual depiction of the different experiments and workflows performed on the GS Junior (Roche) (A) and the MiSeq™ (Illumina) (B) with FFPE and fresh-frozen material.

Mentions: Six sections of 10 μm thickness were cut from FFPE tissue blocks. After deparaffinisation, tumour areas were macrodissected from unstained slides. The tumour area was marked on a haematoxylin-eosin (H&E) stained slide by a senior pathologist (E. W., H.-U. S.). DNA was extracted with the MagAttract® DNA Mini M48 Kit (Qiagen, Hilden, Germany) on the BioRobot® M48 (Qiagen). Samples collected before the year 2010 were extracted manually with the QIAamp® DNA Mini Kit (Qiagen). DNA extraction of the subregions were performed with the Maxwell® 16 FFPE Plus Tissue LEV DNA Purification Kit (Promega, Mannheim, Germany) on the Maxwell® 16 (Promega). Fresh-frozen tissues were extracted with the DNeasy® Blood & Tissue Kit (Qiagen) (Figure 1). All extraction procedures were performed following the manufacturers’ instructions.Figure 1


Massively parallel sequencing fails to detect minor resistant subclones in tissue samples prior to tyrosine kinase inhibitor therapy.

Heydt C, Kumm N, Fassunke J, Künstlinger H, Ihle MA, Scheel A, Schildhaus HU, Haller F, Büttner R, Odenthal M, Wardelmann E, Merkelbach-Bruse S - BMC Cancer (2015)

Visual depiction of the different experiments and workflows performed on the GS Junior (Roche) (A) and the MiSeq™ (Illumina) (B) with FFPE and fresh-frozen material.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4404105&req=5

Fig1: Visual depiction of the different experiments and workflows performed on the GS Junior (Roche) (A) and the MiSeq™ (Illumina) (B) with FFPE and fresh-frozen material.
Mentions: Six sections of 10 μm thickness were cut from FFPE tissue blocks. After deparaffinisation, tumour areas were macrodissected from unstained slides. The tumour area was marked on a haematoxylin-eosin (H&E) stained slide by a senior pathologist (E. W., H.-U. S.). DNA was extracted with the MagAttract® DNA Mini M48 Kit (Qiagen, Hilden, Germany) on the BioRobot® M48 (Qiagen). Samples collected before the year 2010 were extracted manually with the QIAamp® DNA Mini Kit (Qiagen). DNA extraction of the subregions were performed with the Maxwell® 16 FFPE Plus Tissue LEV DNA Purification Kit (Promega, Mannheim, Germany) on the Maxwell® 16 (Promega). Fresh-frozen tissues were extracted with the DNeasy® Blood & Tissue Kit (Qiagen) (Figure 1). All extraction procedures were performed following the manufacturers’ instructions.Figure 1

Bottom Line: Fresh-frozen samples showed the same range of artificially mutated allele frequencies as the FFPE material.This supports the theory that secondary KIT resistance mutations develop under treatment by "de novo" mutagenesis.Alternatively, the detection limit of two mutated clones in 10,000 wild-type clones might not have been high enough or heterogeneous tissue samples, per se, might not be suitable for the detection of very small subpopulations of mutated cells.

View Article: PubMed Central - PubMed

Affiliation: Institute of Pathology, University Hospital Cologne, Kerpener Str. 62, 50937, Cologne, Germany. carina.heydt@uk-koeln.de.

ABSTRACT

Background: Personalised medicine and targeted therapy have revolutionised cancer treatment. However, most patients develop drug resistance and relapse after showing an initial treatment response. Two theories have been postulated; either secondary resistance mutations develop de novo during therapy by mutagenesis or they are present in minor subclones prior to therapy. In this study, these two theories were evaluated in gastrointestinal stromal tumours (GISTs) where most patients develop secondary resistance mutations in the KIT gene during therapy with tyrosine kinase inhibitors.

Methods: We used a cohort of 33 formalin-fixed, paraffin embedded (FFPE) primary GISTs and their corresponding recurrent tumours with known mutational status. The primary tumours were analysed for the secondary mutations of the recurrences, which had been identified previously. The primary tumours were resected prior to tyrosine kinase inhibitor therapy. Three ultrasensitive, massively parallel sequencing approaches on the GS Junior (Roche, Mannheim, Germany) and the MiSeq(TM) (Illumina, San Diego, CA, USA) were applied. Additionally, nine fresh-frozen samples resected prior to therapy were analysed for the most common secondary resistance mutations.

Results: With a sensitivity level of down to 0.02%, no pre-existing resistant subclones with secondary KIT mutations were detected in primary GISTs. The sensitivity level varied for individual secondary mutations and was limited by sequencing artefacts on both systems. Artificial T > C substitutions at the position of the exon 13 p.V654A mutation, in particular, led to a lower sensitivity, independent from the source of the material. Fresh-frozen samples showed the same range of artificially mutated allele frequencies as the FFPE material.

Conclusions: Although we achieved a sufficiently high level of sensitivity, neither in the primary FFPE nor in the fresh-frozen GISTs we were able to detect pre-existing resistant subclones of the corresponding known secondary resistance mutations of the recurrent tumours. This supports the theory that secondary KIT resistance mutations develop under treatment by "de novo" mutagenesis. Alternatively, the detection limit of two mutated clones in 10,000 wild-type clones might not have been high enough or heterogeneous tissue samples, per se, might not be suitable for the detection of very small subpopulations of mutated cells.

Show MeSH
Related in: MedlinePlus