Detection of minor, genetically distinct subpopulations within tumors is a key challenge in cancer genomics. Here we report STAR-FISH (specific-to-allele PCR–FISH), a novel method for the combined detection of single-nucleotide and copy number alterations in single cells in intact archived tissues. Using this method, we assessed the clinical impact of changes in the frequency and topology of PIK3CA mutation and HER2 (ERBB2) amplification within HER2-positive breast cancer during neoadjuvant therapy. We found that these two genetic events are not always present in the same cells. Chemotherapy selects for PIK3CA-mutant cells, a minor subpopulation in nearly all treatment-naive samples, and modulates genetic diversity within tumors. Treatment-associated changes in the spatial distribution of cellular genetic diversity correlated with poor long-term outcome following adjuvant therapy with trastuzumab. Our findings support the use of in situ single cell–based methods in cancer genomics and imply that chemotherapy before HER2-targeted therapy may promote treatment resistance.
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We thank E. Winer, I. Krop, B. Vogelstein and members of the Polyak and Michor laboratories for their critical reading of the manuscript and useful discussions. We thank A. Marusyk and D. Tabassum for their help with the xenograft assays, R. Witwicki for help with data processing, L. Cameron in the Dana-Farber Cancer Institute Confocal Microscopy center for her technical support, A. Richardson (Dana-Farber Cancer Institute) for providing slides from a human breast tumor with known status for the PIK3CA mutation encoding p.His1047Arg, and H. Russness and I. Rye (Oslo University Hospital) for providing the BAC probe for HER2. This work was supported by the Dana-Farber Cancer Institute Physical Sciences–Oncology Center (U54CA143798 to F.M.), the European Molecular Biology Organization (EMBO; M.J.), the Swiss National Science Foundation (M.J.), the American Cancer Society (CRP-07-234-06-COUN to C.L.A.) and the Breast Cancer Research Foundation (K.P.).
The authors declare no competing financial interests.
Integrated supplementary information
(a) Primers designed to have a single mismatch at the 3′ end efficiently discriminate between wild-type and mutant alleles as the presence of a double mismatch at the 3′ end significantly inhibits primer annealing and the efficiency of the PCR reaction; green, wild-type nucleotide; red, mutant nucleotide; blue, mismatched nucleotide. (b–d) Testing the sensitivity of mutation-specific STAR-FISH primers by performing PCR using defined mixtures of genomic DNA from cell lines with known mutation status. Total of 10 ng of genomic DNA was used for each PCR reaction; separate reactions were performed using WT and MUT primers. Full-length gels are presented in Supplementary Figure 2. (b) PIK3CA His1047Arg–specific PCR. MDA-MB-231 breast cancer cells are PIK3CA wild type (WT), whereas SUM-185PE cells are homozygous for PIK3CA His1047Arg mutation (MUT). (c) PIK3CA E542K–specific PCR. The BT-483 cell line is homozygous for PIK3CA E542K mutation. (d) TP53 R175H–specific PCR. MDA-MB-231 cells are wild-type, whereas AU565 cells are homozygous for the TP53 R175H mutation. (e–g) Testing the specificity of PIK3CA His1047Arg (e), PIK3CA E542K (f) and TP53 (g) mutation-specific STAR-FISH primers by performing in situ PCR using formalin-fixed, paraffin-embedded (FFPE) tissue sections of xenografts or histogel samples derived from breast cancer cells. (e) Upper panel, only wild-type (WT) primers were used in the first round of in situ PCR reactions, and both wild-type (WT) and mutant (MUT) primers were used in the second round of PCR. (f,g) Both WT and MUT primers were used in both rounds of PCR. Scale bar, 75 μm.
PCR using defined mixtures of genomic DNA from cell lines with known mutation status. A total of 10 ng of genomic DNA was used per PCR reaction; separate reactions were performed using WT and MUT primers. (a) PIK3CA His1047Arg–specific PCR. MDA-MB-231 breast cancer cells are PIK3CA wild type (WT), whereas T-47D cells are heterozygous and SUM-185PE cells are homozygous for the PIK3CA His1047Arg mutation (MUT). WT and MUT PCR reactions were loaded in the same well. Upper band, mutant amplicon; lower band, wild-type amplicon. (b) PIK3CA His1047Arg–specific PCR. MDA-MB-231 breast cancer cells are PIK3CA wild type (WT), whereas SUM-185PE cells are homozygous for PIK3CA His1047Arg mutation (MUT). (c) PIK3CA E542K–specific PCR. The BT-483 cell line is homozygous for PIK3CA E542K mutation. (d) TP53 R175H–specific PCR. MDA-MB-231 cells are wild-type, whereas AU565 cells are homozygous for the TP53 R175H mutation.
(a) Upper panel, PIK3CA His1047Arg–specific STAR-FISH on a xenograft sample obtained by co-injecting MDA-MB-231_mCherry (PIK3CA WT) and SUM-185PE_GFP (PIK3CA His1047Arg MUT) cell lines. Arrows point to mutant cells with red STAR-FISH signal. Lower panel, the same xenograft stained with antibody to GFP to detect SUM-185PE_GFP cells. Scale bar, 75 μm. (b) FACS analysis of a freshly dissociated mixed MDA-MB-231_mCherry and SUM-185PE_GFP cell line xenograft. (c) Comparison of replicates of mutation detection by STAR-FISH, FACS and immunofluorescence. The observed differences are not significant (ns; ANOVA test, two-tailed t test). Error bars, s.d. (d) Numerical summary of the results obtained by the different methods applied to the same xenograft.
(a) Comparison of the percentages of mutant cells detected by MassARRAY and STAR-FISH in a cohort of patient samples with adjacent invasive (IDC) and in situ (DCIS) components (analyzed separately). Green color indicates accordance between the compared methods. (b) Mutant cell percentages detected by STAR-FISH and MassARRAY. Linear regression line and correlation P < 0.0001 (****). (c) STAR-FISH on a single patient sample. Despite a high extracellular autofluorescence background in the DCIS component (upper panel), the STAR-FISH signal can easily be detected. Scale bar, 75 μm.
Timeline and details of tumor sample collection and details of neoadjuvant and adjuvant treatment (patient number in parentheses).
(a,b) The frequency of cells with HER2/CEP17 ratio ≤ 2.2 and no WT or MUT signal (HER2noAmp) (a) and cells without any detectable signal (NA) (b) per each tumor area analyzed (n = 183). The percentage of HER2noAmp and NA cells is variable in different areas of the same tumor, and the distribution is random. The horizontal line in the middle of each box is the median value. The edges of each box are the 25th and 75th percentiles of the data. The lower (upper) whiskers are the maximum (minimum) between the minimum (maximum) value and the 25th (75th) percentile + 1.5 × IQR. (c) Immunohistochemical quantification of the smooth muscle actin (SMA) stromal marker in tumor samples with the highest and lowest frequencies of HER2noAmp cells. To account for variability in total cell numbers per sample, the SMA staining area was normalized to 350 cells. Error bars, s.d. (d) The effect of including taxanes in the neoadjuvant regimen on HER2noAmp cells. Taxane treatment has been shown to increase stromal cell content in post-treatment samples. Thus, if HERnoAmp cells were all stromal cells, an increase in frequency should be observed in the post-treatment samples from patients receiving paclitaxel therapy, but no significant changes were observed. The horizontal line in the middle of each box is the median value. The edges of each box are the 25th and 75th percentiles of the data. The lower (upper) whiskers are the maximum (minimum) between the minimum (maximum) value and the 25th (75th) percentile + 1.5 × IQR. (e) The percentage of NA cells in normal breast tissue is lower than in cancer tissue. The horizontal line in the middle of each box is the median value. The edges of each box are the 25th and 75th percentiles of the data. The lower (upper) whiskers are the maximum (minimum) between the minimum (maximum) value and the 25th (75th) percentile + 1.5 × IQR. (f) Distribution of nuclei size in tumors and normal tissue. Dashed lines represent the distribution limits for the normal tissue and denote the long tails of the tumor nuclei size distribution.
Colors indicate the relative frequency of each cell type within tumors. Each plot represents an individual case before and after treatment, whereas each bar corresponds to one area of the tumor. The y-axis indicates cell frequency (%).
Supplementary Figure 8 Overall changes in the frequency of types and diversity indices due to neoadjuvant chemotherapy.
(a) Frequency of each of the five cell types analyzed. Each mark represents a mean value for an individual patient. Circles and triangles indicate pre- and post-chemotherapy samples, respectively. Colors correspond to cell types, as in Supplementary Figure 2. Differences between pre- and post-treatment for each cell type was tested with unpaired two-tailed t tests: MUT, ***P = 0.0003, MUT + AMP, **P = 0.0018; AMP, *P = 0.0272; WT + AMP, **P = 0.0046. Error bars, s.d. (b) Overall frequency of cells with HER2 amplification irrespective of PIK3CA mutation status in all tumor areas analyzed. Unpaired two-tailed t-test P value = 0.0006. The horizontal line in the middle of each box is the median value. The edges of each box are the 25th and 75th percentiles of the data. The lower (upper) whiskers are the maximum (minimum) between the minimum (maximum) value and the 25th (75th) percentile + 1.5 × IQR. (c,d) Simpson’s index of intratumoral heterogeneity before and after neoadjuvant chemotherapy. Error bars represent two times the standard error obtained from 1,000 bootstrap samples. Simpson’s index was calculated on the basis of the counts of the five cell types by combining all measured areas of each sample (across-species comparison) (c) or across different areas of the same tumor (across-area comparison) (d). Confidence intervals were obtained using nonparametric bootstrap resampling methods. The global difference in diversity before and after treatment for all 22 patients was evaluated using paired Wilcoxon signed rank tests, P value = 0.063 (across species) and 0.156 (across areas).
Supplementary Figure 9 Validation of STAR-FISH–based PIK3CA His1047Arg mutation frequency in a patient cohort for HER2-positive breast tumors by droplet digital PCR.
(a) The order of the slides used for assay comparison. Note the three consecutive slides used for DNA extraction for digital PCR and the location of the slides used for STAR-FISH analysis. (b) Comparison of the percentages of mutant cells detected by digital PCR and STAR-FISH. Green color indicates concordance between the two methods. Two outliers are marked by open circle. (c) Mutant cell percentage detected by STAR-FISH and digital PCR. The linear regression line was plotted with exclusion of the two outliers. ****P < 0.0001. (d) The tumor cell content for the outlier samples is lower than 20% (open circles).
Supplementary Figure 10 The effect of combining targeted therapy with neoadjuvant chemotherapy on intratumoral heterogeneity.
The distribution of diversity indices changes in groups of patients who received cytotoxic drugs (Ctx alone) and a combination of chemotherapy and trastuzumab. The y-axis indicates the proportion of patients in a given treatment group.
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Janiszewska, M., Liu, L., Almendro, V. et al. In situ single-cell analysis identifies heterogeneity for PIK3CA mutation and HER2 amplification in HER2-positive breast cancer. Nat Genet 47, 1212–1219 (2015). https://doi.org/10.1038/ng.3391
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