Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Clinical Study

Whole-exome sequencing of long-term, never relapse exceptional responders of trastuzumab-treated HER2+ metastatic breast cancer

Abstract

Trastuzumab has significantly improved the overall survival of patients with HER2+ metastatic breast cancer (MBC). However, outcomes can vary, with patients progressing within 1 year of treatment or exceptional cases of complete response to trastuzumab for ≥10 years. Identification of the underlying genomic aberrations of “exceptional responders (ExRs)” compared to “rapid non-responders (NRs)” increases our understanding of the mechanisms involved in MBC progression and identification of biomarkers of trastuzumab response and resistance. Whole-exome sequencing was performed on six ExRs compared to five NR. The overall fraction of genome copy number alteration (CNA) burden was higher in NR patients (P = 0.07), while more significantly pronounced in copy number gains (P = 0.03) in NR compared to ExRs. Delineation of the distribution of CNA burden across the genome identified a greater degree of CNA burden in NR within Chr8 (P = 0.02) and in Chr17 (P = 0.06) and conferred a statistically significant benefit in overall survival. Clinical trial number: NCT01722890 [ICORG 12/09].

Background

Historically, HER2+ metastatic breast cancer (MBC) was designated as an incurable disease. The introduction of anti-HER2 therapies such as trastuzumab has markedly improved survival1 and extended to 56.5 months with the combination of trastuzumab plus pertuzumab plus docetaxel.2 We and others3, 4 have reported cases of long-term durable complete response to trastuzumab in HER2+ MBC. However, to date only clinical and molecular analysis of this “exceptional” cohort exists. Somatic copy number alterations (CNAs) alter a significant proportion of the cancerous genome. CNAs dominate the breast cancer genome, with somatic mutations in breast cancer genes at low frequencies and mainly characterised in driver genes by high-throughput targeted mutation profiling.5 However, somatic single-nucleotide variants (SNVs) and insertions/deletions in driver genes do contribute to tumour biology.6 It remains unclear as to the extent in which genomic CNA burden can act as a prognostic measure of predicting response to trastuzumab in long-term, never relapse exceptional responders (ExRs) compared to rapid non-responders (NRs). To investigate this hypothesis, we present the first study of whole-exome sequencing (WES) analysing the genome CNA burden of six ExRs compared with five NRs with HER2+ MBC patients treated with trastuzumab.

Methods

A retrospective, single institution review from 2000 to 2018 identified 295 HER2+ MBC who received treatment with trastuzumab in the metastatic setting. Informed consent was obtained and approved by St. Vincent’s University Hospital Ethics Committee. HER2+ immunohistochemical status was confirmed by pathologist (C.Q.). Analysis of patients with never relapse and >3-year overall survival follow-up data identified 40 patients. A further refined analysis revealed 11 patients with a minimum overall survival of 10 years (range 10–19 years). We performed WES on 6/11 of these patients and 5 corresponding NRs (median relapse-free survival (RFS) ≤14 months) (Supplementary Table 1). DNA from tumour and adjacent normal tissue (where available) underwent WES (Agilent SureSelect Human All Exon V4) using an Illumina HiSeq (2 × 100 bp) at a mean depth of 56×. Reads were trimmed and aligned to the hg38 reference genome using BWA7 and duplicate reads were marked. Base recalibration was conducted with GATK.8 Variant calling was performed using Mutect2 from GATK (4.1.3). CNAs were identified by EXCAVATOR2.9 Variant statistics and tumour mutational burden (TMB) were calculated by maftools.10,11,12

Two-sample t test with unequal variances was used to evaluate total genome CNA burden between ExRs and NRs, and across individual chromosomes, two-sided P value < 0.05 was considered statistically significant. Somatic CNA burden was used to stratify patients into high and low CNA burden groups based on the median CNA burden observed across each chromosome. The binary CNA stratification groups were further assessed using Kaplan–Meier survival estimation; P value < 0.05 log rank was considered statistically significant.

Results

We estimated the fraction of the genome amplified/deleted and present CNA burden as a measure of genome instability. We observed that the overall fraction of genome CNA burden was higher in NR patients (P = 0.07; Fig. 1a), while more significantly pronounced in copy number gains (P = 0.03) in NRs compared to ExRs (Fig. 1b), with non-significant copy number losses (Fig. 1c). We further delineated the distribution of CNA burden across the genome and identified a greater degree of CNA burden in NRs in chromosome 8 (P = 0.02) and in chromosome 17 (Fig. 1d). Kaplan–Meier survival analysis highlighted the extended survival of ExRs compared to NRs (P = 0.0007, log rank; Fig. 1e). Further analysis highlighted that dichotomisation into low versus high CNA burden does not significantly affect overall survival (P = 0.389, log rank); however, it does stratify patients (Fig. 1f). Subanalysis of the CNA burden at specific chromosomes revealed that low total CNA burden at Chr8 and Chr17 conferred a statistically significant benefit in overall survival (P = 0.02 and P = 0.02, log rank; Fig. 1g, h).

Fig. 1: Results of CNA burden on ExRs and NRs and overall survival outcome.
figure1

a Total overall genome-wide CNA burden. b Fraction of genome gained. c Fraction of genome lost. d Individual chromosome CNA burden in Chr8 and Chr17 in non-responder (NR) and exceptional responder (ExRs) genomes. e Kaplan–Meier overall survival curves of cases dichotomised by exceptional responder and non-responder. f Total CNA burden, low CNA (total CNA burden < median) and high CNA (total CNA ≥ median). g Overall CNA burden on Chr8, low CNA (Chr8 total CNA burden < median) and high CNA (Chr8 total CNA burden ≥ median). h Overall CNA burden on Chr17, low CNA (Chr17 total CNA burden < median) and high CNA (Chr17 total CNA burden ≥ median).

To provide additional insight into the somatic mutation landscape at variant, gene, and biological pathway levels, we refined our study to include only normal–tumour matched pairs of ExR (n = 5; 3 never relapse, 2 RFS >96 months) and NR (n = 4) cases. Of our small cohort, we observed a higher median of SNV in ExRs (median = 1621) compared to NRs (median = 638); albeit this large variance was mostly driven by an unusual SNV in ExR patient 1 (Supplementary Fig. 1a, b). Mutations were observed in TTN (80% of ExR and 75% NR samples), HSPG2 (60% of ExR and 75% NR samples), SYNE1 (40% of ExR and 75% NR samples), SYNE2 (80% of ExR and 50% NR samples) and MACF1 (40% of ExR and 75% NR samples) (Supplementary Fig. 1c). We used the mutant-allele tumour heterogeneity (MATH) score to quantify intratumour heterogeneity based on the variation in variant’s allele frequencies of all mutations in a tumour. MATH scores were computed for the ExR and NR tumours separately. MATH scores from ExR (mean = 47, median = 41, interquartile range (IQR) = 16–46) and NR (mean = 35, median = 31, IQR = 23–102) and TMB was insignificantly different between both cohorts (Supplementary Fig. 1d, e).

Discussion

Identification of genomic alterations associated with exceptional response and survival may improve risk assessment and treatment strategies for HER2+ MBCs. This is the first study to propose that CNA burden in HER2+ MBC ExRs may represent a novel prognostic predictor to trastuzumab response. Despite the limited sample size, we observed a trend in which the overall CNA burden was lower in ExRs compared to NRs; moreover, individual CNA analysis per chromosome revealed that specific chromosomes 8 and 17 were more altered in NR genomes compared to ExRs, and stratified analysis revealed significantly poorer overall survival. CNA burden was previously shown to be associated with overall survival and disease-specific survival in breast cancer, with chromosome 8 along with chromosomes 1 and 16 carrying the highest CNA burden, suggesting a further role of chromosome 8 in prognosis.13 Key genes in our analysis such as TTN, HSPG2, SYNE1, SYNE2 and MACF1 are frequently altered in breast and other cancers but their roles in HER2+ breast cancer tumorigenesis and trastuzumab response/resistance are as yet uncertain. Our investigation of genome-wide CNA burden offers the potential to gain insight into the underlying genetic landscape of long-term, never relapse exceptional response to trastuzumab. Recent analysis of the SAKK 22/99 trial identified that a subset of advanced HER2+ patients displayed long-term disease control with trastuzumab monotherapy;14 however, this study was unable to identify any meaningful clinical predictive markers to characterise these patients. Therefore, our preliminary study supports our hypothesis that CNA burden may account for exceptional response to trastuzumab. Particularly as MBC is generally termed incurable, this study presents a paradigm shift in the conventional ideology of oncology therapeutics that is unexplored and clearly warrants further investigation. The contribution of the immune system to the therapeutic effect of trastuzumab and other HER2 antibodies has been established, and further investigation into immune-related markers may provide predictive information for increased clinical activity in combination with genomic CNA data. Therefore, an extended analysis of 40 HER2+ MBC ExRs and NRs is now underway to validate our findings; furthermore, we intend to characterise the role of CNAs and immune signatures that may contribute to long-term trastuzumab (and other HER2 therapies) therapeutic response in the metastatic setting.

References

  1. 1.

    Slamon, D. J., Leyland-Jones, B., Shak, S., Fuchs, H., Paton, V., Bajamonde, A. et al. Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2. N. Engl. J. Med. 344, 783–792 (2001).

    CAS  Article  Google Scholar 

  2. 2.

    Swain, S. M., Baselga, J., Kim, S. B., Ro, J., Semiglazov, V., Campone, M., Ciruelos, E. et al. Pertuzumab, trastuzumab, and docetaxel in HER2-positive metastatic breast cancer. N. Engl. J. Med. 372, 724–734 (2015).

    CAS  Article  Google Scholar 

  3. 3.

    Gullo, G., Zuradelli, M., Sclafani, F., Santoro, A. & Crown, J. Durable complete response following chemotherapy and trastuzumab for metastatic HER2-positive breast cancer. Ann. Oncol. 23, 2204–2205 (2012).

    CAS  Article  Google Scholar 

  4. 4.

    Gámez-Pozo, A., Pérez Carrión, R. M., Manso, L., Crespo, C., Mendiola, C., López-Vacas, R. et al. The Long-HER Study: clinical and molecular analysis of patients with HER2+ advanced breast cancer who become long-term survivors with trastuzumab-based therapy. PLoS ONE 9, e109611 (2014).

    Article  Google Scholar 

  5. 5.

    Pereira, B., Chin, S. F., Rueda, O. M., Moen Vollan, H. K., Provenzano, E., Bardwell, H. A. et al. The somatic mutation profiles of 2,433 breast cancers refines their genomic and transcriptomic landscapes. Nat. Commun. 7, 11479 (2016).

    CAS  Article  Google Scholar 

  6. 6.

    Ciriello, G., Miller, M. L., Aksoy, B. A., Senbabaoglu, Y., Schultz, N., Sander, C. et al. Emerging landscape of oncogenic signatures across human cancers. Nat. Genet. 45, 1127–1133 (2013).

    CAS  Article  Google Scholar 

  7. 7.

    Li, H. & Durbin, R. Fast and accurate long-read alignment with Burrows-Wheeler transform. Bioinformatics 26, 589–595 (2010).

    Article  Google Scholar 

  8. 8.

    Depristo, M. A., Banks, E., Poplin, R., Garimella, K. V., Maguire, J. R., Hartl, C. et al. A framework for variation discovery and genotyping using next-generation DNA sequencing data. Nat. Genet. 43, 491–501 (2011).

    CAS  Article  Google Scholar 

  9. 9.

    D’Aurizio, R., Pippucci, T., Tattini, L., Giusti, B., Pellegrini, M., Magi, A. et al. Enhanced copy number variants detection from whole-exome sequencing data using EXCAVATOR2. Nucleic Acids Res. 44, e154 (2016).

    PubMed  PubMed Central  Google Scholar 

  10. 10.

    Mayakonda, A., Lin, D. C., Assenov, Y., Plass, C. & Koeffler, H. P. Maftools: efficient and comprehensive analysis of somatic variants in cancer. Genome Res. 28, 1747–1756 (2018).

    CAS  Article  Google Scholar 

  11. 11.

    Mroz, E. A. & Rocco, J. W. MATH, a novel measure of intratumor genetic heterogeneity, is high in poor-outcome classes of head and neck squamous cell carcinoma. Oral. Oncol. 49, 211–215 (2013).

    CAS  Article  Google Scholar 

  12. 12.

    Yarchoan, M., Hopkins, A. & Jaffee, E. M. Tumor mutational burden and response rate to PD-1 inhibition. N. Engl. J. Med. 377, 2500–2501 (2017).

    Article  Google Scholar 

  13. 13.

    Zhang, L., Feizi, N., Chi, C. & Hu, P. Association analysis of somatic copy number alteration burden with breast cancer survival. Front. Genet. 9, 421 (2018).

    CAS  Article  Google Scholar 

  14. 14.

    Schmid, S., Klingbiel, D., Aebi, S., Goldhirsch, A., Mamot, C., Munzone, E. et al. Long-term responders to trastuzumab monotherapy in first-line HER-2+ advanced breast cancer: characteristics and survival data. BMC Cancer 19, 902 (2019).

    Article  Google Scholar 

Download references

Acknowledgements

We thank the patients of this study, their family members and the nurses and medical specialists for their contribution to this study. The authors acknowledge the translational study ICORG 12/09, Cancer Trials Ireland (formerly known as the Irish Clinical Oncology Research Group [ICORG]).

Author information

Affiliations

Authors

Contributions

J.C. and G.G. conceived the study. J.C., G.G. and N.W. supervised the project. N.W. designed and performed the experiments and wrote the manuscript. A.M. performed experiments. S.J.F., P.O. and C.A. analysed and presented the data. S.J.F. assisted and supervised the data analysis. G.G. and C.Q. provided pathological support and database curation.

Corresponding authors

Correspondence to Naomi Walsh or John Crown.

Ethics declarations

Ethics approval and consent to participate

Ethical approval for this study was granted by St. Vincent University Hospital Ethics and Medical Committee, Dublin, Ireland. Informed consent was sought and granted by all exceptional responders for participation in the study. The study was performed in accordance with the Declaration of Helsinki.

Consent to publish

Not applicable.

Data availability

Whole-exome sequencing and clinical data from this project is available from EGA (accession number: EGAS00001004486). Archive for academic research within the constraints of the consent given from patients. All bioinformatics tools are fully described in “Methods”.

Competing interests

The authors declare no competing interests.

Funding information

Funding for this study was provided by Cancer Clinical Research Trust (CCRT) (to N.W.). We would like to thank the Irish Centre for High End Computing (https://www.ichec.ie/) for the use of HPC infrastructure. S.J.F. acknowledges support from the Royal College of Surgeons in Ireland StAR programme and the Irish Cancer Society CCRC BREAST-PREDICT (CCRC13GAL). The funders had no role in the design and conduct of the study; collection, management, analysis and interpretation of the data; preparation, review or approval of the manuscript and decision to submit the manuscript for publication.

Additional information

Note This work is published under the standard license to publish agreement. After 12 months the work will become freely available and the license terms will switch to a Creative Commons Attribution 4.0 International (CC BY 4.0).

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Rights and permissions

This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Walsh, N., Andrieu, C., O’Donovan, P. et al. Whole-exome sequencing of long-term, never relapse exceptional responders of trastuzumab-treated HER2+ metastatic breast cancer. Br J Cancer 123, 1219–1222 (2020). https://doi.org/10.1038/s41416-020-0999-z

Download citation

Search

Quick links