Circulating KRAS G12D but not G12V is associated with survival in metastatic pancreatic ductal adenocarcinoma

While high circulating tumor DNA (ctDNA) levels are associated with poor survival for multiple cancers, variant-specific differences in the association of ctDNA levels and survival have not been examined. Here we investigate KRAS ctDNA (ctKRAS) variant-specific associations with overall and progression-free survival (OS/PFS) in first-line metastatic pancreatic ductal adenocarcinoma (mPDAC) for patients receiving chemoimmunotherapy (“PRINCE”, NCT03214250), and an independent cohort receiving standard of care (SOC) chemotherapy. For PRINCE, higher baseline plasma levels are associated with worse OS for ctKRAS G12D (log-rank p = 0.0010) but not G12V (p = 0.7101), even with adjustment for clinical covariates. Early, on-therapy clearance of G12D (p = 0.0002), but not G12V (p = 0.4058), strongly associates with OS for PRINCE. Similar results are obtained for the SOC cohort, and for PFS in both cohorts. These results suggest ctKRAS G12D but not G12V as a promising prognostic biomarker for mPDAC and that G12D clearance could also serve as an early biomarker of response.


Supplementary Methods
For the PRINCE trial, peripheral blood was collected into Streck Cell-Free DNA Blood Collection Tubes tubes (Streck #0230470).For the SOC cohort, specimens were collected in K2EDTA Blood Collection Tubes (EMSCO Fisher #0265732) or Streck Cell-Free DNA Blood Collection tubes.K2EDTA collected specimens were processed to plasma within 3 hours of collection while Streck collected specimens were processed within 7 days.Blood processing occurred at room temperature with an initial centrifugation at 1600 g for 10 minutes (centrifuge brake-off).The upper, plasma, layer was transferred to a new tube and centrifuged again (one or two more times) at 3000 g for 10 minutes (EDTA) or 4122 g for 15 minutes (Streck), again with centrifuge brake-off.The final plasma layer was isolated and banked in 1 mL aliquots in 2mL Sarstedt tubes (Sarstedt #72.694.416) at -80 C.
Circulating cell-free DNA (ccfDNA) extraction using the QIAmp Circulating Nucleic Acid Kit (Qiagen #55114) was performed according to the manufacturer's protocol with two modifications: the proteinase K digestion was extended to 1 hour and final elution was performed twice with 30 μL of Buffer AVE (total 60 μL).Extraction with the QIAmp MinElute ccfDNA Mini Kit (Qiagen #55204) was also performed according to the manufacturer's instructions with a single modification: final elution in 30 μL of ultraclean water run through the column twice.Samples were stored at 4 C prior to quantification.
Quantitative PCR for a 115 bp amplicon of human ALU repeat element was used to determine ccfDNA concentration (primers: forward 5'-CCTGAGGTCAGGAGTTCGAG-3' and reverse 5'-CCCGAGTAGCTGGGATTACA-3') 1 .Samples were diluted 1:10 with nuclease free water and a standard curve was generated by serial dilution of a commercial DNA standard (Promega G3041).Power SYBR Green PCR Master Mix (Applied Biosystems #4367659) was used according to the manufacturer's instructions on a ViiA 7 Real-Time PCR System (Applied Biosystems).Results were analyzed using QuantStudio Real-Time PCR Software (Applied Biosystems).
Prior to droplet digital PCR (ddPCR), ccfDNA was pre-amplified for the KRAS G12 locus (primers: forward 5'-AGGCCTGCTGAAAATGACTGAATAT-3' and reverse 5'-GCTGTATCGTCAAGGCACTCTT-3').This PCR was performed using the Q5 Hot Start Hi-Fidelity Master Mix (NEB #M0494), and 0.05 μM primers with either 15 μl of QIAmp Circulating Nucleic Acid Kit ccfDNA or up to 24 μl of QIAmp MinElute ccfDNA Mini Kit ccfDNA (with a maximum of 30 ng).PCR was performed in the Veriti 96 Well Thermal Cycler (Applied Biosystems) with the following program: 98 C for 3 minutes, 9 cycles of 98 C for 10 seconds, 63 C 3 minutes, and 72 C 30 seconds, followed by 72 C for 2 minutes 2 .Pre-amplified material was diluted 1:4 with TE buffer and stored at 4 C in the short-term and -20 C in the long-term storage.
For RainDrop ddPCR, initial characterization was carried out with a multiplex reaction (using the same primers as pre-amplification above) and probes for KRAS G12WT (VIC-TTGGAGCTGGTGGCGT-MGBNFQ), G12D (FAM-TGGAGCTGATGGCGT-MGBNFQ), G12V (FAM-GAGCTGTTGGCGT-MGBNFQ), and G12R (FAM-TTGGAGCTCGTGGCGT-MGBNFQ).Reactions contained 2x TaqMan Genotyping Master Mix (Applied Biosystems #4371355), 25x Droplet Stabilizer (RainDance #30-07026), 200nM primers, G12WT, G12D, and G12V probes at 100 nM, G12R probe at 50 nM and 10 μl of pre-amplified ccfDNA in a total of 30 μl.Final quantification of variant allele fraction (VAF) was carried out in a duplex reaction with the above conditions but only two probes (G12WT and G12Mutant of interest) at 100 nM each.Droplets were generated from 25 μl of reaction mix on the RainDrop Source instrument (RainDance Technologies, Inc.).Next, PCR was performed in a GeneTouch PCR Thermal Cycler (Bioer Technology Co., Ltd.); 0.6 C per second ramp rate with the following cycling conditions, 95 C for 10 minutes, 45 cycles of 95 C for 15 seconds and 60 C for 1 minute, 10 minutes at 5 C, and held at 4 C. Droplets were read on the RainDrop Sense instrument (RainDance Technologies, Inc.) and data analyzed in RainDrop Analyst software (RainDance Technologies, Inc.).Droplet counts were adjusted to copy counts using the Poison correction and background adjusted based on 20 healthy control samples 3 .

4 .
of clinical variable values for G12D-vs G12V-bearing tumors for therapy-naïve patients in PRINCE cohort.Shown is analysis of 33 patients with a G12D-bearing tumor and 23 patients with G12V for all variables except CA19-9, for which there were 26 patients with G12D and 21 patients with G12V.A red asterisk * indicates significance at 0.05.Mann-Whitney test (two-sided) used for all variables other than location of primary tumor, Chi-square test (two-sided).Dotted horizontal lines for clinical laboratory values shown in C indicate upper and lower limits of normal clinical values.Green denotes ctKRAS G12D and dark grey is G12V.Abbreviation ALT (Alanine Transaminase), AST (Aspartate Transaminase), and BUN (Blood Urea Nitrogen) are used.Source data are provided as a Source Data file. of clinical variable values for G12D-vs G12V-bearing tumors for patients in SOC cohort.Not all variables could be collected for all patients.For imaging analysis shown in A and B, measures of pancreatic lesions are shown for 33 patients with a G12D-bearing tumor and for 18 with G12V, all other imaging variables were analyzed for 30 patients with a G12D-bearing tumor and for 12 with G12V.Shown in C, D, and E is analysis of CA19-9 is shown for 31 G12D patients and 19 G12V patients, the remaining variables are shown for 34 patients with a G12D-bearing tumor and for 20 patients with G12V.Mann-Whitney test (two-sided) used for all variables other than location of primary tumor, for which Chi-square test (two-sided) was used.Dotted horizontal lines for clinical laboratory values shown in C indicate upper and lower limits of normal clinical values.Abbreviation ALT (Alanine Transaminase), AST (Aspartate Transaminase), and BUN (Blood Urea Nitrogen) are used.Green denotes ctKRAS G12D and dark grey is G12V.Source data are provided as a Source Data file.Association of tumor KRAS mutation status (G12D-vs G12V-bearing tumors) with overall survival (A-C) and progression-free survival (D-F) for therapy-naive patients enrolled in PRINCE trial or who received standard of care (SOC) therapy, or the pooled therapy-naïve PRINCE + SOC cohorts.Cox regression hazard ratios (HR) and 95% confidence intervals (CI) are shown with log-rank p-values.Source data are provided as a Source Data file.

HR
= Eastern Cooperative Oncology Group Performance Status 2 SOD = Sum of diameters for all target lesions Supplementary Figure 7. Survival association for baseline ctKRAS variant allele fraction (VAF) by variant for therapy-naïve combined PRINCE and standard of care(SOC) patients.Shown are the Kaplan-Meier curves for baseline VAF dichotomized at the median for overall survival (top, A and B) and progression-free survival (bottom, C and D) for patients with G12D-(left, A and C) or G12V-bearing tumors (right, B and D).Cox regression hazard ratios (HR) and 95% confidence intervals (CI) are shown with log-rank p-values.Source data are provided as a Source Data file.Estimated cubic spline functions relating log ctKRAS variant allele fraction (VAF) to results of the Cox models.Results for overall survival (OS) and progression-free survival (PFS) shown for PRINCE (top two rows), standard of care (SOC, middle two rows), and combined PRINCE and SOC patients (bottom two rows).The left column shows results for ctKRAS G12D only, and the right column for ctKRAS G12V only.Red box indicates yaxis maximum for G12V is different than for G12D for the SOC Overall Survival functions.Source data are provided as a Source Data file.
clearance and association with survival for all therapy-naïve PRINCE and standard of care (SOC) patients with any ctKRAS variant detected.Includes (A, C, and E) all PRINCE patients, and (B, D, and F) SOC patients.Shown at top (A and B) is ctKRAS clearance at week 8 on therapy for all patients with oneyear survival data (alive or dead at one year) (Fisher's Exact Test, two-sided), in the middle (C and D) is Kaplan-Meyer analysis for overall survival and on bottom (E and F) is progression-free survival.Cox regression hazard ratios (HR) and 95% confidence intervals (CI) are shown with log-rank p-values.Source data are provided as a Source Data file.

Supplementary Figure 1. Prior Treatment and ctKRAS VAF Levels for PRINCE patients. Among
).

Mann-Whitney Test (two-sided), †Fisher's Exact Test (two-sided), ‡Chi-square Test (two-sided) PRINCE patients SOC patients
Supplementary Table3.Of 67 therapy-naïve PRINCE patients (left) who had a KRAS variant detected in tissue or plasma, 33 had a KRAS G12D and 23 had G12V.Of 54 therapy-naïve SOC patients (right) who had a baseline KRAS variant, 34 had KRAS G12D and 20 had G12V.All patients shown in these tables are therapy-naïve.ECOG PS refers to Eastern Cooperative Oncology Group Performance Status scale.Source data are provided as a Source Data file.

.11 [1.30-3.43] 0.003 2.43 [1.33-4.44] 0.004 3.16 [1.12-8.87] 0.029 4.26 [1.12-16.18] 0.033
SupplementaryFigure 5. Baseline plasma ctKRAS variant allele fraction (VAF) dichotomized at the median and associated with survival for therapy-naïve PRINCE patients (A and D, n=67), SOC patients (B and E, n=69) or combined therapy naïve PRINCE and SOC patients (C and F, N =136) with any ctKRAS variant.Overall survival shown in A, B and C, progression-free survival shown in D, E, and F. Cox regression hazard ratios (HR) and 95% confidence intervals (CI) are shown with log-rank p-values.Source data are provided as a Source Data file.Supplementary Table 4.Cox analysis for baseline plasma log ctKRAS variant allele fraction (VAF) as a continuous variable for therapy-naïve PRINCE (top table), standard of care (SOC, middle table), and combined therapy-naïve PRINCE and SOC (bottom table), with results for ctKRAS G12D on left and for G12V on right.CA19-9 excluded from PRINCE and sum of diameters (SOD) excluded from SOC cohort due to incomplete data.Of the 33 and 31 patients analyzed in the PRINCE and SOC tables respectively for G12D, only the 53 patients with both CA19-9 and SOD values available were included in combined cohort (bottom table).Significant values (without adjustment for multiple testing) indicated by bolded red text.Source data are provided as a Source Data file.

Table 5 . Sensitivity analysis for LoDs (Limits of Detection) of 0.04% (as used for our assay, see Methods), 0.10%, and 0.25% for combined PRINCE and standard of care (SOC) cohorts.
Shown in the top table are survival associations (hazard ratios, HR, with 95% confidence intervals (CI), and log-rank p-values) for baseline ctKRAS variant allele fraction (VAF) by variant (G12D on left and G12V on right) at the three LoDs.In the bottom table, Cox analysis is shown for baseline ctKRAS log of variant allele fraction (VAF) as a continuous variable for overall survival (OS) and progression-free survival (PFS), in each table.Significant values (without adjustment for multiple testing) indicated by bolded red text.Source data are provided as a Source Data file.