Supplemental Material Supplemental Methods 1)Sequencing: Hybridization-based capture of 3320 exons from 182 cancer- related genes and 37 introns of 14 genes commonly rearranged in cancer (previous version of the test performed for nine patients) and 3769 exons from 236 cancer-related genes and 47 introns of 19 genes commonly rearranged in cancer (performed for 338 patients) was applied to ≥ 50 ng of DNA extracted from 347 tumor specimens and sequenced to high, uniform coverage with a mean sequencing depth of 714× as previously described 35 . Consistent median sequencing depth was achieved by processing specimens according to optimized, locked down, standard operating procedures (SOP) on automated liquid handlers in a Clinical Laboratory Improvement Act (CLIA)-certified laboratory as previously described 35 . Genomic alterations (base substitutions, small indels, rearrangements, copy number alterations) were determined and then reported for these patient samples. Six or seven copy numbers are reported as equivocal and > 8 are definitive; for ERBB2, equivocal amplification was 5 to 7 copies; all (equivocal or definitely 1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
17
Embed
mct.aacrjournals.org · Web viewP-values were two-sided and considered significant if ≤0.05. Statistical analysis were performed by author MS with SPSS version 22.0. Supplemental
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
Supplemental Material
Supplemental Methods
1)Sequencing:
Hybridization-based capture of 3320 exons from 182 cancer-related genes and 37
introns of 14 genes commonly rearranged in cancer (previous version of the test performed
for nine patients) and 3769 exons from 236 cancer-related genes and 47 introns of 19
genes commonly rearranged in cancer (performed for 338 patients) was applied to ≥ 50 ng
of DNA extracted from 347 tumor specimens and sequenced to high, uniform coverage
with a mean sequencing depth of 714× as previously described35. Consistent median
sequencing depth was achieved by processing specimens according to optimized, locked
down, standard operating procedures (SOP) on automated liquid handlers in a Clinical
Laboratory Improvement Act (CLIA)-certified laboratory as previously described35. Genomic
alterations (base substitutions, small indels, rearrangements, copy number alterations)
were determined and then reported for these patient samples. Six or seven copy numbers
are reported as equivocal and > 8 are definitive; for ERBB2, equivocal amplification was 5
to 7 copies; all (equivocal or definitely amplified) were designated as positive for
Treatment was considered “matched” if at least one agent in the treatment regimen targeted at least one aberration or pathway component harbored in a patient’s molecular profile or a functionally active protein preferentially expressed in the tumor (e.g. estrogen receptor (ER) or HER2, assessed by standard of care testing other than NGS) with a half inhibitory concentration (IC50) in the low nM range. Examples of matched therapy included, but were not limited to: anti-EGFR drugs in the presence of EGFR anomalies, mTOR inhibitors for alterations in the PTEN/PIK3CA/Akt/mTOR pathway, BRAF or MEK inhibitors for RAF or RAS aberrations. More specifically, we defined “matched-direct” if at least one drug directly impacted the gene product of the molecular alteration or a differentially expressed protein [e.g. an EGFR inhibitor in a patient with an EGFR alteration (direct effect on the molecular aberration) or hormonal manipulation in patients with over-expressed estrogen or androgen receptors (proteins preferentially expressed on tumor cells were targeted)]. “Matched-indirect” was the term used for a drug that affects a target removed from the molecular aberration (e.g. mTOR inhibitor administered to patient with a PIK3CA mutation). Matched-direct therapy would include small molecule inhibitors with an IC50 ≤ 100 nM for the target, as well as antibodies whose primary target was the aberrant protein or a differentially expressed protein. Small molecule inhibitors that directly impacted a target, but had an 100 nM < IC50 ≤ 250 nM for that target were considered matched-indirect treatments. Matching designation was confirmed by the senior investigator (RK), who was blinded at the time of designation to the outcomes.
3)Matching ScoreIt is now well known that advanced tumors have multiple aberrations and that
combination therapy is likely to be better than monotherapy. Therefore, an exploratory scoring system (“Matching Score”) was developed that divided the number of matched drugs by the number of aberrations. Under this system, the higher the Matching Score, the better the match. The score for each match (direct or indirect) was assigned a 1; no match was a zero. If a drug directly impacted two targets present in the tumor, a 2 was given (example, a multikinase inhibitor with potent activity against more than one target present in a tumor); if two drugs each impacted a target directly in a patient, a 2 was also given. If two drugs were given that impacted directly (or indirectly) the same target in a patient, the number 2 was still given. The Matching Score was calculated by dividing the number derived from the direct and indirect matches in each tumor (numerator) by the number of aberrations (denominator). For instance, if a patient who tumor harbored six genomic aberrations received two drugs, the Matching Score would be 2/6 or 0.33. The cut-off of 0.2 for the OS analysis was chosen according to the minimum p-value criteria (Mazumdar and Glassman21).
4)Statistical Analysis 1. Patient’s characteristicsPatient characteristics were summarized using descriptive statistics. A diagram
displays the data availability for the matched and unmatched patients; patients who were lost to follow up or were still on prior therapy or on observation were considered unevaluable (see Figure 1).
2. Study endpoints and definitions4
37
3839404142434445464748495051525354555657
58596061626364656667686970717273
74757677787980
The following clinical endpoints were considered: (i) rate of [stable disease (SD)≥6 months/partial response (PR)/complete response (CR)]; (ii) progression-free survival (PFS) of the first line of therapy given after molecular profile results (PFS2); (iii) PFS2 versus PFS1 (immediate prior line of therapy), i.e., using patients as their own controls22,23; (iv) percent of patients with a PFS2/PFS1 ratio ≥ 1.322; and (v) overall survival (OS). SD, PR, or CR was determined per the assessment of the treating physician. PFS was defined as the time from the beginning of therapy to progression or the time to last follow up for patients that were progression-free (patients that were progression-free on the date of last follow up were censored on that date). OS was defined as the time from the beginning of therapy to death or last follow-up date for patients who were alive (the latter were censored on that date). The cut-off date for the analysis was April 1st 2015; all patients that were progression-free (for PFS) or alive (for OS) as of date of analysis were censored on that date unless their date of last follow up was earlier, in which case that was the date of censoring.
3. Analyses performedWhenever appropriate, Chi-Square tests were used to compare categorical
variables and the non-parametric Mann Whitney-U test to compare two groups on one continuous variable. Binary logistic regressions were performed for categorical endpoints. PFS and OS were analyzed by the Kaplan-Meier method24 and the log-rank test was used to compare variables. Cox regression models were used as multivariable analysis, when appropriate for survival endpoints. The importance of a prognostic factor was assessed by the Chi-Square and Wald-type test statistics (for the log-rank test and logistic regression/Cox regression models, respectively). The higher the Chi-square or Wald, the stronger the association.
3.1 Variables assessedThe main variables analyzed in this study were “matched versus unmatched”, the
primary diagnosis, the number of prior therapy lines (in advanced/metastatic setting), if the therapy was single agent or a combination, the total number of alterations, the presence of metastasis at diagnosis, the presence of metastasis at biopsy date (of tissue used for molecular testing).
3.2 Propensity score for being matched vs. notGiven the retrospective nature of our study, and to account for imbalances between
patients who were “matched” versus not, the “propensity” to receive a matched therapy for each patient was determined by using a multivariable logistic regression with “matched or not” as the outcome26–28. Variables included in the final propensity score model were “breast or not” cancers, “gastrointestinal or not” cancers, “skin/melanoma or not” cancers, “first line of treatment or not”, and “number of alterations”. This propensity score was used as a covariate in multivariable models or in the inverse probability of matched treatment weighting method. In the latter method, “matched” patients were given [1/Propensity score] as weight and “unmatched” patients were given [1/(1-Propensity score)] as weight. P-values were two-sided and considered significant if ≤0.05. Statistical analysis were performed by author MS with SPSS version 22.0.
75 Breast PIK3CA amplification, SOX2 amplification, TP53 G302fs*42, FLT3 L260* faslodex for ER+ NO76 Breast AKT1 (E17K) casodex for AR+ NO77 Breast GATA3 *445fs*2+ tamoxifen for ER+ NO
11
78 Breast CCND1 amplification, MCL1 amplification, CDH1 P127fs*89 faslodex for ER+ YES79 Breast MYC amplification, ARID1A R1461*, PALB2 G808fs*43, PALB2 Y1183* estradiol for ER+ NO
Abbreviations: GI: gastrointestinal; GU: genitourinary; ER+: estrogen receptor positive; HER2+: human epidermal growth factor receptor 2 positive; AR+: androgen receptor positive; SD ≥ 6 months or PR or CR: Stable disease ≥ 6 months or partial response or complete response of any duration. N=87 patients were matched; N=30/87 (34.5%) achieved SD ≥ 6 months/PR/CR. 13 of the 87 patients (15%, see patients number 75-87) were matched on basis of non-NGS marker only (these patients were considered matched as next-generation sequencing results were used in context of standard of care). aESR1 is generally a resistance mutation, but patients with such alterations may respond to certain hormonal modulators, depending on the mutation36(p1),37,38(p1)
12
132133134135136
Supplemental Figure 1. Overall survival analysis
13
Overall survival (Months)
Overall survival (Months)
Overall survival (Months)
A. “matched” versus “non-matched”
B. Matching-scores comparison
C. “responders” versus “non-responders”
Overall survival analysis (Kaplan Meier method). Refer to Table 3 for complete analysis. The P-value for the “matching-score” was P = 0.04 in a Cox regression model (backward conditional model).