Supplementary Appendix This appendix has been provided by the authors to give readers additional information about their work. Supplement to: Sherman SI, Wirth LJ, Droz J-P, et al. Motesanib diphosphate in progressive differentiated thyroid cancer. N Engl J Med 2008;359:31-42.
Motesanib Diphosphate in Progressive Differentiated Thyroid Cancer Steven I. Sherman, M.D., Lori J. Wirth, M.D., Jean-Pierre Droz, M.D., Michael Hofmann, M.D, Ph.D., Lars Bastholt, M.D., Renato G. Martins, M.D., Lisa Licitra, M.D., Michael J. Eschenberg, M.S., Yu-Nien Sun, Ph.D., Todd Juan, Ph.D., Daniel E. Stepan, M.D., Martin J. Schlumberger, M.D., on behalf of the study group Supplementary Appendix 1 Sherman et al 1
TUMOR GENOTYPING METHODS Patients who participated in the genotyping analysis signed a separate informed consent. Prestudy, paraffin-embedded tumor biopsy tissue was submitted to the Amgen central laboratory. Mutational Analysis of Tumor Samples Tumor areas were identified and grossly dissected for genomic DNA preparation from slides containing hematoxylin and eosin-stained tumor sections. Genomic DNA was prepared using the Pinpoint Slide DNA Isolation System (Zymo Research Corporation, Orange, CA) following the manufacturer's instructions. Mutational analysis was performed using a combination of polymerase chain reaction (PCR) and sequencing. PCR primers were designed from intronic regions to amplify separately exons 9 to 17 of human RET, exons 9 and 20 of human PIK3CA, exons 11 and 15 of human BRAF, and exons 2 and 3 of human HRAS, KRAS, and NRAS, respectively. PCR amplicons were purified using the QiaQuick PCR purification kit (Qiagen, Valencia, CA), and sequenced using nested primers and a standard DNA sequencing protocol on an ABI 3700 or ABI 3730 genetic analyzer (Applied Biosystems, Foster City, CA). Data were analyzed using SeQuencher software (Gene Codes Corporation, Ann Arbor, MI) and were compared against respective wild-type exon sequences for presence of mutation. Primer pairs and GenBank accession numbers for reference sequences are listed in Table 1. All PCR experiments were performed in triplicate and each PCR amplicon was sequenced 4 times using 2 forward and 2 reverse primers. Sherman et al 2
Table 1. Primer Pairs Used for Mutational Analysis of Tumor Samples From Patients With Differentiated Thyroid Cancer Gene (GenBank Accession Number) Forward Primer Reverse Primer PIK3CA (NM_006218) Exon 9 5'-CTG TAA ATC ATC TGT GAA TCC AGA GGG G-3' 5'-GTA AAT TCT GCT TTA TTT ATT CCA ATA GGT ATG G-3' Exon 20 5'-CTA ATT TTG TGA CAT TTG AGC AAA GAC CTG AAG G-3' 5'-CAT GGA TTG TGC AAT TCC TAT GCA ATC GG-3' BRAF (NM_004333) Exon 11 5'-GGG GAT CTC TTC CTG TAT CCC TCT CAG GC-3' 5'-GTT TAT TGA TGC GAA CAG TGA ATA TTT CC-3' Exon 15 5'-CAT AAT GCT TGC TCT GAT AGG-3' 5'-GTA ACT CAG CAG CAT CTC AG-3' KRAS (NM_004985) Exon 2 5'-AAG GTA CTG GTG GAG TAT TTG-3' 5'-GTA CTC ATG AAA ATG GTC AGA G-3' Exon 3 5'-CCT TTT TTG AAG TAA AAG GTG CAC TGT AAT AAT CCA G-3' 5'-CAA GAA CTT CAT TTA TAA AAC AGG GAT ATT ACC TAC CTC-3' HRAS (NM_005343) Exon 2 5'-GGC TGA GCA GGG CCC TCC TTG GCA GG-3' 5'-GCC CTA TCC TGG CTG TGT CCT GGG C-3' Exon 3 5'-GGT ACC AGG GAG AGG CTG GCT GTG TGA AC-3' 5'-CAG CGG CAT CCA GGA CAT GCG CAG-3' NRAS (NM_002524) Exon 2 5'-GCT TTA AAG TAC TGT AGA TGT GGC TCG CC-3' 5'-CCT TTA ATA CAG AAT ATG GGT AAA GAT GAT CCG AC-3' Exon 3 5'-GGC TTG AAT AGT TAG ATG CTT ATT TAA CCT TGG C-3' 5'-GCT CTA TCT TCC CTA GTG TGG TAA CCT C-3' RET (NM_020975) Exon 9 5'-GCT TCC GCT GGC AAG GCT CTG TAT ATG G-3' 5'-GGA GGC TCA GCT TGA TGC ATA GAA CTG AC-3' Exon 10 5'-GGC TTC ACC CAT GGC TTC AGA AAG GCA C-3' 5'-GCA ATT TCC TCC CTT GTT GGG ACC TCA GAT G-3' Exon 11 5'-CAT ACG CAG CCT GTA CCC AGT GG-3' 5'-GGA AAT GGG GGC AGA ACA CAG GCC TCG TC-3' Exon 12 5'-GTG ACC ACA CCT GTC ATG TAG CAG CTT TC-3' 5'-GTT TAA AGC TTA GAT CTT CAG TGG CAC TGG C-3' Exon 13 5'-GAG GCC CCT GTC CAC TGA TCC CAA AGG C-3' 5'-CAT GGC AGT GTC ACA CCA GAG ACC TCT G-3' Exon 14 5'-TGG TTC AAG AGA AAG CTG AGG CTT CAA GG-3' 5'-CAG AGC CAT ATG CAC GCA CCT TCA TC-3' Exon 15 5'-CTG CTG GTC ACA CCA GGC TGA GCC AGT G-3' 5'-GGG CTA TAA AAA GCT TAG AGC TCC ACT AAT C-3' Exon 16 5'-GGA GCT CCA GCC CCT TCA AAG ATG TGT G-3' 5'-CAG GTG CTC AGG GCC AGT GCA ATT CCC-3' Exon 17 5'-GCA GCC AGA CCC AGG CTG ACA TCT GTG-3' 5'-GAG CTT GCA TTG CAC CAG TCT CTG CTC-3' Sherman et al 3
Detection of Gene Rearrangements in Tumor Samples Tumor areas were identified and grossly dissected for RNA preparation from slides containing hematoxylin and eosin-stained tumor sections. Total RNA was extracted using the High Pure RNA Paraffin Kit (Roche Diagnostics, Indianapolis, IN) following the manufacturer's instructions. Purified total RNA was eluted in low-salt buffer. Reverse transcriptase (RT)-PCR was performed using the Titanium One-Step RT-PCR Kit (Clontech, Palo Alto, CA). In order to avoid amplification of genomic DNA, PCR primers were designed to generate exon-spanning amplicons between 70 and 150 base pairs in length. Primer pairs are shown in Table 2. The PCR products were gel-purified using the Qiagen Gel Extraction Kit (Qiagen, Valencia, CA), and ligated into the pcr2.1 TOPO plasmid (Invitrogen, Carlsbad, CA). The ligated DNA was transformed into E. coli and DNA was prepared for sequencing as described above. Sherman et al 4
Table 2. Primer Pairs Used for Gene Rearrangement Analysis of Tumor Samples From Patients With Differentiated Thyroid Cancer Rearrangement Forward Primer Reverse Primer Wild-type RET Exons 11-12 5'-AAC CAG GTC TCC GTG GAT GCC TTC AAG-3' 5'-TTC GCC TTC TCC TAG AGT TTT TCC AAG-3' Wild-type PAX8 Exons 7-8 5'-AAC CTC TCG ACT CAC CAG ACC TAC-3' 5'-TAG AAC TGG ACA CCT CGG GGG TTT C-3' Exons 8-9 5'-ATG CTG CCT CCG TGT ACG GGC AGT TC-3' 5'-ATG TGG GGT GGG TAT CCG GGC AG-3' Exons 9-10 5'-TAT GCC TCC TCT GCC ATC GCA GGC ATG-3' 5'-CTG TAG GAG GAG TAG GGG GTG TG-3' Wild-type PPARγ1 Exons 1-2 5'-CAC GCC GCC GTG GCC GCA GA-3' 5'-GAT CCA CGG AGC TGA TCC CAA AGT TGG-3' RET/PTC1 5'-GCT GGA GAC CTA CAA ACT GAA GTG C-3' 5'-TTC GCC TTC TCC TAG AGT TTT TCC AAG-3' RET/PTC3 5'-AAG CTC CTT ACA TAC CCA GCA CCG AC-3' 5'-TTC GCC TTC TCC TAG AGT TTT TCC AAG-3' PAX8 PPARγ1 Exon 7 5'-AAC CTC TCG ACT CAC CAG ACC TAC-3' 5'-GAT CCA CGG AGC TGA TCC CAA AGT TGG-3' Exon 8 5'-ATG CTG CCT CCG TGT ACG GGC AGT TC-3' 5'-GAT CCA CGG AGC TGA TCC CAA AGT TGG-3' Exon 9 5'-TAT GCC TCC TCT GCC ATC GCA GGC ATG-3' 5'-GAT CCA CGG AGC TGA TCC CAA AGT TGG-3' Sherman et al 5
TUMOR GENOTYPING RESULTS Table 3. Tumor Genotype and Best Response for Individual Patients with Differentiated Thyroid Cancer Unique Tumor Histology Best Response per BRAF HRAS KRAS NRAS Patient Independent Review * Mutation Mutation Mutation Mutation 1 Papillary PR V600E 2 Papillary PR V600E 3 Papillary PR G12C 4 Papillary PR 5 Papillary PR 6 Papillary Durable SD V600E 7 Papillary Durable SD V600E 8 Papillary Durable SD V600E 9 Papillary Durable SD V600E 10 Papillary Durable SD Q61R 11 Papillary Durable SD 12 Papillary SD V600E 13 Papillary SD V600E 14 Papillary SD Q61K 15 Papillary SD 16 Papillary SD 17 Papillary PD V600E 18 Papillary PD 19 Papillary PD 20 Papillary Unevaluable 21 Papillary Unevaluable 22 Papillary Not performed Q61R 23 Follicular Durable SD 24 Follicular SD Q61K 25 Follicular SD Sherman et al 6
Unique Tumor Histology Best Response per BRAF HRAS KRAS NRAS Patient Independent Review * Mutation Mutation Mutation Mutation 26 Follicular SD 27 Follicular SD 28 Hürthle cell SD Q61R 29 Hürthle cell SD 30 Hürthle cell Not performed 31 Follicular var. of papillary SD V600E 32 Follicular var. of papillary SD 33 Follicular var. of papillary PD Durable SD=stable disease of 24 weeks; PD=progressive disease; PR=partial response; SD=stable disease. * Best on-study response to motesanib diphosphate per central radiographic review and per RECIST. Lesion was present but could not be evaluated. Computed tomography scans were not available. No mutations in RET or PlK3CA were detected, and no RET/PTC1, RET/PTC3, or PAX8-PPARγ1 rearrangements were found. Sherman et al 7