Simple and effective clinical testing protocol using ICE COLD- PCR across targeted cancer gene panels using the Veriti Thermal Cycler & Sanger Sequencing Benjamin Legendre, Jr. PhD Vice President, Laboratory Operations Overview: Comprehensive testing of specific amplicons for targeted treatment and monitoring of cancer is becoming incorporated into standard clinical practice to enable Precision Medicine through the use of non-invasive liquid biopsy analysis. The R family genes affect signal transduction downstream of and play a critical role in determining response to targeted therapies. Primary resistance to -specific tyrosine kinase inhibitors (TKIs) and other targeted therapies, including cetuximab and panitumumab, in colorectal cancer (CRC) is often due to the presence of R mutations 1,2. and mutations are found in ~25% of human tumors and lead to constitutive activation of Ras proteins 3. About 35-45% of colorectal tumors carry Exon 2 (codons and 13) mutations that constitutively activate signaling downstream of thereby nullifying the effect of a TKI or other targeted therapies 1,2,4. The presence of Exon 2 mutations therefore predicts response to anti- therapy. However, ~15% of patient tumors without mutations in Exon 2 have recently been found to harbor mutations in Exons 3 or 4 or Exons 2, 3 or 4 4. In addition, V600E mutations in patients with metastatic colorectal cancer have been shown to be prognostic for patient outcome with regards to survival, but this mutation is not reliably predictive of the outcome treatment with anti- agents 4. Similarly, several studies have suggested that mutations in Exons 9 and 20 may be associated to anti- therapy resistance 5. In the absence of response to therapy in Exon 2 wild type patients, several retrospective studies have shown that the presence of mutations is predictive of resistance to such therapies 5. High sensitivity detection of cancer genetic biomarkers from cell-free DNA (cfdna) has the potential to contribute to cancer diagnosis by clinical oncologists, providing (a) guidance for personalized precision cancer treatment in the circumstances where there is no or inadequate available tumor tissue and (b) a process for monitoring treatment effectiveness and the emergence of drug resistance 6. Patients genetic profiles derived from liquid biopsies contribute to real-time treatment decisions or support use of other diagnostic tools such as CAT scans. Use of liquid biopsies offers several advantages over analysis of traditional solid tumor samples: (a) reduced cost of collection, (b) the non-invasive and facile nature of collection provides both large cancer centers as well as localized clinics the ability to screen patients as they undergo therapy and (c) enables analysis where there is either insufficient solid tumor to sample or dispersed tumor tissue due to metastasis. 1
ICE COLD-PCR (ICP) preferentially enriches DNA sequence alterations when they are present in an excess of wild-type DNA. The use of an oligonucleotide complementary to wild-type sequence (-Oligo) is the key element in the process which prevents PCR amplification of wild-type sequences while allowing amplification of DNA containing any altered sequence in the region covered by the -Oligo (Figure 1). Additionally, ICP allows the quantification of the mutant fraction within the starting sample when comparing the mutation % observed after ICP to a semi-log plot of the LOD for a given assay (input DNA % vs. observed mutation %). In order to increase throughput (turn-around time) and decrease costs and resources required for ICP analysis in a clinical environment, we have been investigating a multiplex approach for ICP (MX-ICP TM ). Given that optimal thermal parameters for individual ICP reactions are different for the initial offering of our ICEme Kits, we investigated the use of a Veriti thermal cycler (Thermo Fisher Scientific, Waltham, MA) to simultaneously amplify multiple ICP reactions with different temperature profiles. In this proof of concept study, we performed a multiplex ICP analysis of a series of samples focusing on ICP assays associated with CRC analysis. Figure 1. ICP process overview. 95 C 95 C Tc 1. All DNA is denatured into single strands 2. Reduce Temperature: Cross-Hybridize The -oligobinds to one strand of the wild-type and altered sequences: mutant:-oligoforms a heteroduplex. 3. Selectively Mismatched Sequences The reaction is incubated at the critical temperature (Tc) and the altered:-oligo denatures but the wildtype:-oligo remains bound Thermal Cycling Cycling Complete 6. Perform Reactions for Standard Sanger Sequencing, NGS, or other detection platform 7. Analyze data to determine sequence alteration 5. Amplification Extension of the PCR primers along the altered and wildtype DNA sequences. The sequence containing any alteration will undergo exponential amplification while the amplification of the wild-type sequence will be linear. 4. Reduce Temperature for Primer Annealing to Both Strands of Any Altered Sequences The forward and reverse PCR primers will bind to both strands of the mutant DNA, but only one strand of the wild-type. - Mutant Type - Wild Type - Reference Sequence Oligo Tc - Critical Temperature ICP Primers ICE COLD-PCR using the Veriti Thermal Cycler: The Veriti thermal cycler allows six zones of varying temperature profile within a single thermal cycler run. By harmonizing the number of cycles as well as the times of the cycles, multiple ICP reactions for different amplicons possessing different thermal profiles can be performed simultaneously on a single thermal cycler. Figure 2 shows an example of the Veriti thermal cycler setup for MX-ICP. Figure 2. Overview of the Veriti thermal cycler workflow for MX-ICP. The Veriti thermal cycler contains 6 separate Peltier heat blocks, allowing precise temperature control for 6 independent thermal cycler reactions provided that (1) the number of cycles are constant, (2) the cycling times are constant and (3) the temperature difference at each step is within 5 C of the adjacent block. Figure 2A shows a graphic representation of the thermal cycler conditions of two separate ICP analyses. Figure 2B is a representative example of six separate ICPs being run on the Veriti thermal cycler for up to 16 samples/icp. 2
A. B. ICP1 ICP2 ICP3 ICP4 ICP5 ICP6 98 98 Tc Tc1 Tc Tc2 72 67 - Anneal Extend 72 67 - Anneal Extend P ICP1 Primer Anneal 1 P ICP2 Primer Anneal 2 Time Time Transgenomic offers numerous ICP panels which allow detection of actionable and resistance mutations for high prevalence cancers such as non small cell lung cancer (NSCLC), melanoma and colorectal cancer (CRC) [Table 1]. Table 1. Summary of ICE COLD-PCR panels for Veriti thermal cycler. Panel Veriti Plate 1 Veriti Plate 2 # of Samples Exon 20 Exon 19 Exon 18 Exon 21 Exon 20 NSCLC Exon 15 Exon 3 Exon 19 Exon 18 Exon 9 Exon 21 Exon 20 Exon 2 R CRC Melanoma Metastatic Breast Cancer Exon 4A Exon 3 Exon 4B Exon 4A Exon 4B Exon 9 Exon 20 Exon 4A Exon 20 Exon 3 Exon 15 Exon 4B Exon Exon 4A Exon 3 Exon 15 Exon 2 Exon 20 Exon 9 AKT Exon 3 Exon 2 Exon 2 Exon 3 Exon 4B Exon 9 Exon 3 Exon 2 Exon 2 36 4 24 The Transgenomic MX-ICP CRC panel includes Exons 2, 3, & 4; Exons 2, 3, & 4; Exon 15; Exon ; and Exons 9 & 20. Using the Veriti thermal cycler, all reactions can be performed with only 2 thermal cycler runs for 4+ samples (depending on controls desired) using the plate layouts shown in Figure 3. In addition, the addition of a standard M13 tail to one of the ICP primers in the ICP assay enables Sanger sequencing with a single primer/sequencing master mix. 3
Figure 3. Veriti plate layouts for MX-ICP CRC analysis. Plate 1. Tc = 69.5, P = 61.0 Tc = 69.7, P = 61.0 Tc = 69.0, P = 60.0 Tc = 69.5, P = 58.0 Tc = 70.3, P = 55.0 Tc = 70.5, P = 55.0 Column 1 Column 2 Column 3 Column 4 Column 5 Column 6 Column 7 Column 8 Column 9 Column 10 Column 11 Column E4A_S1 E20_S1 E3_S1 E15_S1 E4B_S1 E_S1 E4A_S1 E4A_S2 E20_S2 E3_S2 E15_S2 E4B_S2 E_S2 E4A_S2 E4A_S3 E20_S3 E3_S3 E15_S3 E4B_S3 E_S3 E4A_S3 E4A_S4 E20_S4 E3_S4 E15_S4 E4B_S4 E_S4 E4A_S4 NTC1 NTC1 NTC1 NTC1 NTC1 NTC1 NTC1 NTC2 NTC2 NTC2 NTC2 NTC2 NTC2 NTC2 (+) CTRL (+) CTRL (+) CTRL (+) CTRL (+) CTRL (+) CTRL (+) CTRL Plate 2. Tc = 70.8, P = 55.0 Tc = 71.5, P = 56.0 Tc = 76.4, P = 52.0 Tc = 75.5, P = 57.0 Tc = 75.5, P = 62.0 Column 1 Column 2 Column 3 Column 4 Column 5 Column 6 Column 7 Column 8 Column 9 Column 10 Column 11 Column E4B_S1 E9_S1 E3_S1 E2_S1 E2_S1 E4B_S2 E9_S2 E3_S2 E2_S2 E2_S2 LEAVE OPEN BUT SET E4B_S3 E9_S3 E3_S3 TEMP FOR TC TO E2_S3 E2_S3 75.5C, Primer OPEN E4B_S4 E9_S4 E3_S4 Annealing to 57C E2_S4 E2_S4 NTC1 NTC1 NTC1 NTC1 NTC1 NTC2 NTC2 NTC2 NTC2 NTC2 (+) CTRL (+) CTRL (+) CTRL (+) CTRL (+) CTRL The workflow for the ICE COLD-PCR analysis using the Veriti thermal cycler for the CRC Panel is as follows (Figure 4). Figure 4. Workflow for the MX-ICP CRC analysis. Sample Receipt DNA Extraction and QC MX PCR MX-ICP Sanger Sequence/ Analysis Dilution Report/ Signoff X 1 X 2 X 1 DAY 1 DAY 2 DAY 3 DAY 4 4
Synopsis of Results: A subset of samples was tested using the Veriti thermal cycler to investigate the effectiveness of multiplexed ICE COLD-PCR CRC Analysis. The results are shown below in Figure 5. Samples RD15-221 and RD15-222 were extracted from plasma samples from late stage, chemonaive CRC patients. The mutation control (Mut CTRL) was a cell line mixture prepared by Horizon Diagnostics (Cambridge, UK) and the Wild-Type control ( CTRL) was a commercially available K562 cell line (Promega, Madison, WI). For these samples, the MX PCR was performed on a Bio-Rad (Hercules, CA) C1000 thermal cycler followed by MX-ICP on the Veriti thermal cycler. The results for these 2 samples matched the results obtained on the C1000 running the ICE COLD-PCRs individually. Figure 5. ICE COLD-PCR Sanger sequencing traces for two plasma samples amplified on the Veriti thermal cycler. E20 E3 E15 E RD15-221 RD15-222 Mut CTRL CTRL E9 E3 E2 E2 RD15-221 RD15-222 Mut CTRL CTRL Conclusions: We have demonstrated the clinical utility of multiplexing ICE COLD-PCR amplifications using the Veriti thermal cycler in order to expedite testing from liquid biopsy samples. With the Veriti thermal cycler, ICE COLD-PCR amplifications can be grouped based upon critical temperature and primer annealing thermal cycler parameters to enable multiple amplicon enrichment within a single thermal cycler run. The combination of the flexibility of the Veriti thermal cycler, the superior enrichment power of ICP, and the quick turnaround time of Sanger sequencing provide a distinct advantage for routine clinical testing and patient monitoring from liquid biopsy samples. The use of ICE COLD-PCR in conjunction with the Veriti thermal cycler and Sanger sequencing enables very rapid turnaround of liquid biopsy samples in as little as 4 days compared to ~4 weeks (28 days) for standard NGS analysis. We feel that this methodology of coupling ICE COLD-PCR chemistries with the Veriti thermal cycler and Sanger sequencing analysis platforms enables molecular diagnostic labs to reevaluate the use of Sanger platforms for use in diagnostics labs as a rapid and cost effective standalone mutation detection platform and, in addition, allows Sanger confirmation of low level mutations (<5%) originally detected by NGS platforms that would have been traditionally missed by Sanger alone. 5
References: 1 T. Brand and D. L. Wheeler, " mutant colorectal tumors-past and Present," Small GTPases 20 3(1) 34-39. 2 W. Shaib, R. Mahajan and B. El-Rayes, Markers of resistance to anti- therapy in colorectal cancer, J Gastrointest Oncol. 2013 4(3) 308 318. 3 J. Downward, "Targeting R signaling pathways in cancer therapy," Nat Rev Cancer. 2003 3(1) 11 22. 4 J. Douillard et al, Panitumumab FOLFOX4 Treatment and R Mutations in Colorectal Cancer, N Engl J Med. 2013 369(11) 1023-1034. 5 H-Y Lou et al, Predictive and prognostic biomarkers with therapeutic targets in advanced colorectal cancer, World J Gastroenterol. 2014 20(14) 3858 3874. 6 G. Francis and S. Stein, "Circulating Cell-Free Tumour DNA in the Management of Cancer," Int J Mol Sci. 2016 16(6) 142-14142. About Transgenomic: Transgenomic, Inc. is a global biotechnology company advancing personalized medicine through the use of cutting-edge molecular technologies designed to improve medical diagnoses and patient outcomes. Transgenomic develops highly-innovative mutation enrichment technologies for determination of drug effectiveness. Transgenomic has exclusively licensed ICE COLD-PCR (Improved and Complete Enrichment CO-amplification at Lower Denaturation) from the Dana- Farber Cancer Institute and continues to refine the technology for analysis of the genes implicated in oncology and other diseases. Additionally, Transgenomic has expertise and capabilities in high-quality DNA extraction from a variety of key sample types (blood, plasma, FFPE) and a range of detection platforms, widely used in pharmacogenomic applications, including Sanger sequencing, next generation sequencing of targeted or custom panels, RNA sequencing, SNP genotyping, methylation and DNA quantification. Additionally, we have issued and pending patent applications relating to our proprietary improvements and extensions of the ICE COLD-PCR method. Finally, Transgenomic provides clinical and research services to biopharmaceutical companies and academic institutes developing targeted therapies or investigating oncology signaling pathways. Trademarks & Copyright TRANSGENOMIC is a registered trademark and the helix logo and Advancing Personalized Medicine are trademarks of Transgenomic, Inc. All other trademarks are trademarks of their respective holders. 2016 Transgenomic, Inc. All rights reserved. Printed in USA. Document No. 602510-00 6