Research ArticleCancer

Integrated molecular analysis of tumor biopsies on sequential CTLA-4 and PD-1 blockade reveals markers of response and resistance

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Science Translational Medicine  01 Mar 2017:
Vol. 9, Issue 379, eaah3560
DOI: 10.1126/scitranslmed.aah3560
  • Fig. 1. Genomic landscape of serial tumor biopsies and genomic and immune correlates of treatment response.

    (A) Patients with metastatic melanoma were initially treated with CTLA-4 blockade (n = 56; asterisk indicates that 2 of the 56 patients were CTLA-4 blockade–naïve). Both responded to PD-1 blockade, and only pretreatment samples were available for WES and TCR-seq. Nonresponders (NRs) to CTLA-4 blockade (n = 47) were then treated with PD-1 blockade. Double nonresponders (DNRs) progressed on CTLA-4 blockade first and then progressed on PD-1 blockade. Serial tumor biopsies were collected at multiple time points (pretreatment, early on-treatment, and progression on CTLA-4 blockade and PD-1 blockade) when feasible. WES and TCR-seq were performed on these serial tumor biopsies. The numbers in parentheses indicate the number of samples available for responders (Rs) and nonresponders after quality control of WES and TCR-seq data. (B) For each sample (columns), genomic profiles (rows) were characterized. Column annotations represent biopsy time (pre–αCTLA-4, pre–CTLA-4 blockade samples; pre–αPD-1, pre–PD-1 blockade samples; post–αPD-1, post–PD-1 blockade samples) and response status (red, responders; blue, nonresponders; asterisk indicates failed CTLA-4 blockade but responded to PD-1 blockade) for each sample (sample ID denotes patient ID followed by biopsy time: A, pre–αCTLA-4; C, post–CTLA-4/pre–PD-1; E, post–PD-1). Mutational burden and neoantigen burden for each sample are shown at the top of the panel. Neoantigens were defined as having a median inhibitory concentration (IC50) <500 nM. Color scale shows the range of IC50 from 500 to 50 nM. Synonymous (light) and nonsynonymous (dark) mutations are shown in different shades of blue. Additional genomic profiles included selected somatic point mutations and indels. No indels were found among melanoma driver genes. When multiple mutations were found in one gene, the following precedence rule was applied: nonsense mutation > frameshift indel > splice site mutation > missense mutation > in-frame indel. (C) Boxplots summarize TCR clonality by response status (blue, nonresponders; red, responders) in pre–CTLA-4 blockade samples, pre–PD-1 blockade samples, on–CTLA-4 blockade samples, and on–PD-1 blockade samples; median values (lines) and interquartile range (whiskers) are indicated. P values were calculated using two-sided Mann-Whitney U test (P > 0.05 for TCR clonality in pre–CTLA-4 blockade and on–CTLA-4 blockade samples, P = 0.041 for TCR clonality in pre–PD-1 blockade samples, and P = 0.032 for TCR clonality in on–PD-1 blockade samples).

  • Fig. 2. Copy number loss as a potential resistance mechanism.

    (A) Boxplots summarize burden of copy number gain or loss in five groups of interest: responders to CTLA-4 blockade assessed before treatment, pre–CTLA-4 blockade DNRs, responders to PD-1 blockade assessed before treatment, pre–PD-1 blockade DNRs, and post–PD-1 blockade DNRs; median values (lines) and interquartile range (whiskers) are indicated. P values were calculated using two-sided Mann-Whitney U test (P = 0.042 for pre–CTLA-4 blockade responders versus DNRs, P = 0.029 for pre–CTLA-4 blockade responders versus post–PD-1 blockade DNRs, and P > 0.05 for all others). The pre–CTLA-4 blockade and post–PD-1 blockade DNR groups are highlighted in bold. (B) Segment gain or loss (SGOL) scores were calculated for each copy number segments as sum of log2 copy ratios (tumor/normal) of each copy number segment across all DNR samples with copy number loss burden higher than 2000 (n = 9). Higher positive SGOL scores indicate higher copy number gain of copy number segments, and lower negative SGOL scores indicate higher copy number loss of copy number segments. Tumor suppressor genes with recurrent copy number loss are indicated in chromosomes 6q, 10q, and 11q23.3.

  • Fig. 3. Copy number loss as a potential resistance mechanism in an independent cohort.

    (A) Boxplots summarize burden of copy number gain or loss in three patient subgroups from the Van Allen cohort: clinical benefit, long-term survival with no clinical benefit, and minimal or no clinical benefit; median values (lines) and interquartile range (whiskers) are indicated. P values were calculated using two-sided Mann-Whitney U test (P = 0.016 for burden of copy number loss in clinical benefit versus minimal or no clinical benefit and P > 0.05 for all others). (B) Gene set enrichment analysis (GSEA) results show top enriched KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways from down-regulated genes (blue bars) and up-regulated genes (red bars) in high burden of copy number loss group versus low burden of copy number loss group (false discovery rate–adjusted P < 0.001). (C) Proportions of patients with clinical benefit, long-term survival with no clinical benefit, and minimal or no clinical benefit were calculated within each of the four patient subgroups: high mutational load and low burden of copy number loss, high mutational load and high burden of copy number loss, low mutational load and low burden of copy number loss, and low mutational load and high burden of copy number loss. The numbers in parentheses indicate the number of patients with different levels of response (clinical benefit, long-term survival, and no clinical benefit) out of the total number of patients in each of the four patient subgroups.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/9/379/eaah3560/DC1

    Fig. S1. Workflow diagram of multidimensional profiling analysis.

    Fig. S2. Distribution of sequencing coverage and tumor purities across samples.

    Fig. S3. Mutational load, neoantigen load, and clinical response.

    Fig. S4. ITH and clinical response.

    Fig. S5. The effects of previous CTLA-4 blockade exposure on the baseline TCR clonality of pre–PD-1 blockade samples.

    Fig. S6. Burden of CNAs in responders versus nonresponders.

    Fig. S7. Copy number profiles of responders and DNRs.

    Fig. S8. Correlation between burden of copy number loss and mutational load.

    Fig. S9. Correlation between burden of copy number loss and mutational load in the Van Allen cohort.

    Fig. S10. SGOL scores in three patient subgroups from the Van Allen cohort.

    Fig. S11. Burden of recurrent CNAs in patients from the Van Allen cohort.

    Fig. S12. Correlation between burden of copy number loss and immune scores.

    Fig. S13. Up- and down-regulated pathways in patients with high copy number loss versus low copy number loss in the Van Allen cohort.

    Fig. S14. Patient stratification based on mutational load and burden of copy number loss in the Van Allen cohort.

    Table S1. Clinical and demographic characteristics of patient cohort.

    Table S2. Sample information, tumor purity, mutational load, neoantigen load, number of clones per tumor, and burden of CNAs.

    Table S3. Summary measure of the data across clinical subgroups.

    Table S4. List of somatic point mutations and indels.

    Table S5. List of HLA somatic mutations.

    Table S6. List of neoantigens and predicted major histocompatibility complex binding affinity.

    Table S7. TCR clonality.

    Table S8. Log2-transformed NanoString normalized count data in our cohort.

    Table S9. Immune scores (NanoString) in our cohort.

    Table S10. Log2 copy number ratio segment file by Sequenza in our cohort.

    Table S11. Recurrent CNAs in DNRs with copy number loss burden higher than 2000.

    Table S12. Log2 copy number ratio segment file by Sequenza in the Van Allen cohort.

    Table S13. Burden of CNAs in the Van Allen cohort.

    Table S14. Recurrent CNAs in the Van Allen cohort.

    Table S15. Relative contribution of copy number loss burden on chromosome 10 in CTLA-4 blockade resistance.

    Table S16. Immune scores (RNA-seq) in the Van Allen cohort.

    Table S17. Preranked gene list for GSEA, KEGG pathway, and GO analysis in the Van Allen cohort.

    Table S18. Additive effects of high mutational load and low burden of copy number loss on response to CTLA-4 blockade.

  • Supplementary Material for:

    Integrated molecular analysis of tumor biopsies on sequential CTLA-4 and PD-1 blockade reveals markers of response and resistance

    Whijae Roh, Pei-Ling Chen, Alexandre Reuben, Christine N. Spencer, Peter A. Prieto, John P. Miller, Vancheswaran Gopalakrishnan, Feng Wang, Zachary A. Cooper, Sangeetha M. Reddy, Curtis Gumbs, Latasha Little, Qing Chang, Wei-Shen Chen, Khalida Wani, Mariana Petaccia De Macedo, Eveline Chen, Jacob L. Austin-Breneman, Hong Jiang, Jason Roszik, Michael T. Tetzlaff, Michael A. Davies, Jeffrey E. Gershenwald, Hussein Tawbi, Alexander J. Lazar, Patrick Hwu, Wen-Jen Hwu, Adi Diab, Isabella C. Glitza, Sapna P. Patel, Scott E. Woodman, Rodabe N. Amaria, Victor G. Prieto, Jianhua Hu, Padmanee Sharma, James P. Allison, Lynda Chin, Jianhua Zhang, Jennifer A. Wargo,* P. Andrew Futreal*

    *Corresponding author. Email: afutreal{at}mdanderson.org (P.A.F.); jwargo{at}mdanderson.org (J.A.W.)

    Published 1 March 2017, Sci. Transl. Med. 9, eaah3560 (2017)
    DOI: 10.1126/scitranslmed.aah3560

    This PDF file includes:

    • Fig. S1. Workflow diagram of multidimensional profiling analysis.
    • Fig. S2. Distribution of sequencing coverage and tumor purities across samples.
    • Fig. S3. Mutational load, neoantigen load, and clinical response.
    • Fig. S4. ITH and clinical response.
    • Fig. S5. The effects of previous CTLA-4 blockade exposure on the baseline TCR clonality of pre–PD-1 blockade samples.
    • Fig. S6. Burden of CNAs in responders versus nonresponders.
    • Fig. S7. Copy number profiles of responders and DNRs.
    • Fig. S8. Correlation between burden of copy number loss and mutational load.
    • Fig. S9. Correlation between burden of copy number loss and mutational load in the Van Allen cohort.
    • Fig. S10. SGOL scores in three patient subgroups from the Van Allen cohort.
    • Fig. S11. Burden of recurrent CNAs in patients from the Van Allen cohort.
    • Fig. S12. Correlation between burden of copy number loss and immune scores.
    • Fig. S13. Up- and down-regulated pathways in patients with high copy number loss versus low copy number loss in the Van Allen cohort.
    • Fig. S14. Patient stratification based on mutational load and burden of copy number loss in the Van Allen cohort.
    • Legends for tables S1 to S18

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). Clinical and demographic characteristics of patient cohort.
    • Table S2 (Microsoft Excel format). Sample information, tumor purity, mutational load, neoantigen load, number of clones per tumor, and burden of CNAs.
    • Table S3 (Microsoft Excel format). Summary measure of the data across clinical subgroups.
    • Table S4 (Microsoft Excel format). List of somatic point mutations and indels.
    • Table S5 (Microsoft Excel format). List of HLA somatic mutations.
    • Table S6 (Microsoft Excel format). List of neoantigens and predicted major histocompatibility complex binding affinity.
    • Table S7 (Microsoft Excel format). TCR clonality.
    • Table S8 (Microsoft Excel format). Log2-transformed NanoString normalized count data in our cohort.
    • Table S9 (Microsoft Excel format). Immune scores (NanoString) in our cohort.
    • Table S10 (Microsoft Excel format). Log2 copy number ratio segment file by Sequenza in our cohort.
    • Table S11 (Microsoft Excel format). Recurrent CNAs in DNRs with copy number loss burden higher than 2000.
    • Table S12 (Microsoft Excel format). Log2 copy number ratio segment file by Sequenza in the Van Allen cohort.
    • Table S13 (Microsoft Excel format). Burden of CNAs in the Van Allen cohort.
    • Table S14 (Microsoft Excel format). Recurrent CNAs in the Van Allen cohort.
    • Table S15 (Microsoft Excel format). Relative contribution of copy number loss burden on chromosome 10 in CTLA-4 blockade resistance.
    • Table S16 (Microsoft Excel format). Immune scores (RNA-seq) in the Van Allen cohort.
    • Table S17 (Microsoft Excel format). Preranked gene list for GSEA, KEGG pathway, and GO analysis in the Van Allen cohort.
    • Table S18 (Microsoft Excel format). Additive effects of high mutational load and low burden of copy number loss on response to CTLA-4 blockade.

    [Download Tables S1 to S18]

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