Research ArticleCancer

BRCAness, SLFN11, and RB1 loss predict response to topoisomerase I inhibitors in triple-negative breast cancers

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Science Translational Medicine  19 Feb 2020:
Vol. 12, Issue 531, eaax2625
DOI: 10.1126/scitranslmed.aax2625
  • Fig. 1 Response to irinotecan in TNBC PDXs.

    (A) Waterfall plot representing responses to irinotecan treatment in 40 TNBC PDXs. Each bar represents the median change in tumor volume from baseline in treated xenografts; n = 4 to 13 xenografts per group. (B) Irinotecan response in HBCx-39, HBCx-10, and HBCx-4B. Means ± standard deviation, n = 8 to 13. (C) Response rates (number of PDXs) to irinotecan according to patients’ pretreatment. NAC, neoadjuvant chemotherapy. (D) Response rates to irinotecan according to patients’ distant relapse after surgery for the primary tumor.

  • Fig. 2 Response to irinotecan and BRCAness.

    (A) Waterfall plot representing irinotecan responses in PDXs with (green) and without BRCAness (gray). BRCA1/2 mutations and BRCA1 methylation are indicated with * and M, respectively. (B) Contingency analysis of BRCA1/2 mutations and BRCAness (Fisher’s exact test). R, response; SD, stable disease; PD, progressive disease; wt, wild type. (C) Response to irinotecan in HBCx-60 (BRCAness) and HBCx-106 (no BRCAness). n = 4 to 5, means ± standard deviation. (D) Western blot analysis of γH2AX in HBCx-60 and HBCx-106 xenografts, 4 and 24 hours after a single treatment with irinotecan (n = 3). (E) Percentage of geminin-positive nuclei with more than 10 RAD51 foci in HBCx-106 and HBCx-60 xenografts (control and irinotecan-treated groups, tumors harvested 24 hours after a single treatment); n = 3. ns, not significant. (F) Representative images showing RAD51 foci (green) and geminin (red) immunofluorescence in HBCx-106 and HBCx-60 xenografts harvested 24 hours after a single irinotecan treatment. Scale bars, 10 μm. DAPI, 4′,6-diamidino-2-phenylindole.

  • Fig. 3 Identification of SLFN11 expression and RB1 down-regulation as potential markers of irinotecan response.

    (A) Volcano plot displaying differentially expressed genes between responding PDXs (R, n = 15) as compared to nonresponding PDXs (PD, n = 15). The y axis corresponds to log10 (P value), and the x axis displays the log2 fold change value. X-axis grid cutoff lines are shown for fold change of 1.5 and −1.5 and y-axis grid line at P value of 0.05. (B) Robust Multichip Average (RMA)–normalized expression of SLFN11 and RB1 genes. (C) RT-PCR analysis of SLFN11 and RB1 expression. Results are expressed as n-fold differences in target gene expression relative to the TBP gene. (D) SLFN11 expression in HBCx-39 (negative), HBCx-40 (low expression), and HBCx-14 (high expression) analyzed by IHC. Scale bars, 50 μm. (E) Pearson’s correlation between SLFN11 gene (RT-PCR) and protein (H-score) expression. r = 0.6992; P < 0.0001 (two-tailed). (F) Frequency distribution of SLFN11 H-scores in the whole set of PDXs. (G) SLFN11 H-scores in PD as compared to R and in PD and SD categories as compared to R.

  • Fig. 4 Combination markers correlated with irinotecan response.

    (A) A waterfall plot showing SLFN11 expression and irinotecan response. (B) Combination of SLFN11 expression and BRCAness as potential markers of response to irinotecan. (C) Contingency analysis, Fisher’s exact test. (D) Individual tumor growth curves of HBCx-60, HBCx-145, HBCx-9, HBCx-15, and HBCx-66 xenografts treated with irinotecan (n = 5 to 10) and olaparib for HBCx-15 and HBCx-66 PDXs (n = 3). (E) SLFN11 expression in HBCx-60, HBCx-145, HBCx-9, HBCx-15, and HBCx-66. Scale bars, 50 μm. (F) Waterfall plots showing irinotecan response, RB1 loss determined by IHC, and BRCAness. (G) Contingency analysis, Fisher’s exact test.

  • Fig. 5 Combination of irinotecan with an ATR inhibitor and antitumor activity of indenoisoquinolines.

    (A) Tumor response to irinotecan (40 mg/kg) and the ATR inhibitor VE-822 (VX-870, berzosertib; 50 mg/kg) in the HBCx-1 and HBCx-23 PDXs (BRCAness positive and SLFN11 negative); n = 4 for control, irinotecan, and VE-822–treated groups; n = 7 for the combination group (HBCx-1); and n = 8 for the HBCx-23 xenograft groups. Statistical significance of the difference between irinotecan and irinotecan + VE-822–treated groups was determined by the Mann-Whitney test. (B) Western blot analysis of P-CHK1 (Ser345), P-CHK2, and KU80 or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in treated xenografts after a single dose of irinotecan (tumors harvested 24 hours after irinotecan treatment) alone or associated with two injections of VE-822 (administered at days 1 and 2, tumors harvested 4 hours after the second VE-822 treatment); n = 2 for control, irinotecan, and VE-822 xenografts; n = 3 for irinotecan + VE-822 xenografts (HBCx-1); and n = 3 or 4 xenografts for HBCx-23. (C) Antitumor activity of the indenoisoquinolines LMP400 (indotecan) and LMP776 (indimitecan) in the HBCx-60, HBCx-8, HBCx-10, and HBCx-39 PDXs. Means ± standard deviation, n = 5 to 6. RTV, relative tumor volume.

  • Fig. 6 SLFN11, RB1, and BRCAness and response to AC.

    (A) Response to AC and irinotecan in 39 TNBC PDXs (expressed as a fraction of the total tumor number). (B) Combination of SLFN11 expression, RB1 loss, and BRCAness as correlates of AC response in 39 TNBC PDXs. P values were calculated with the Fisher’s exact test. (C) MFS (metastasis-free survival). and OS of 250 patients with TNBC according to SLFN11 gene expression, determined by RT-PCR analysis. Survival distributions were estimated by the Kaplan-Meier method, and P values were calculated with the log-rank test.

  • Table 1 Histopathological and clinical features of TNBC.

    CharacteristicN%
    Type of graft
      Primary BC2255%
      Primary nodes of BC25%
      Residual tumors after neoadjuvant1640%
    Mean age at diagnosis56 (29–89)
    TNM
      T1820%
      T21948%
      T3923%
      T425%
      N02358%
      N11538%
      N213%
      M03998%
      M113%
    Breast surgery
      Tumorectomy1640%
      Mastectomy2460%
    Lymph node surgery
      Sentinel node biopsy615%
      Lymphadenectomy3383%
    Histology
      Invasive carcinoma of no special type3793%
      Metaplastic BC38%
      Lymphovascular invasion (LVI)+1435%
      LVI2665%
    Rank SBR
      Grade SBR 100%
      Grade SBR 213%
      Grade SBR 33998%
    Recurrence
      No relapse1537.5%
      Local relapse820%
      Distant relapse1742.5%

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/12/531/eaax2625/DC1

    Fig. S1. Transcriptomic expression of TOP1 and MDR1 genes in PDX models.

    Fig. S2. Expression of SLFN11 in TGCA TNBC.

    Fig. S3. HR-mediated repair and DNA damage checkpoint activation in response to TOPI and TOPII inhibitors.

    Table S1. Comparison of cisplatin and irinotecan responses in 20 PDXs.

    Table S2. Characteristics of the 250 TNBCs.

    Table S3. Multivariate COX analysis of OS for SLFN11 mRNA expression in the series of 250 TNBCs.

    Data file S1. Clinical and pathological characteristics of the TNBCs corresponding to PDXs.

    Data file S2. Characteristics of TNBC PDXs (BRCAness, BRCA1/2 mutations, RB1, and SLFN11 status).

    Data file S3. List of genes differentially expressed between PDXs in the “response group (R)” as compared to “progressive disease (PD)” group.

    Data file S4. Individual data points.

  • The PDF file includes:

    • Fig. S1. Transcriptomic expression of TOP1 and MDR1 genes in PDX models.
    • Fig. S2. Expression of SLFN11 in TGCA TNBC.
    • Fig. S3. HR-mediated repair and DNA damage checkpoint activation in response to TOPI and TOPII inhibitors.
    • Table S1. Comparison of cisplatin and irinotecan responses in 20 PDXs.
    • Table S2. Characteristics of the 250 TNBCs.
    • Table S3. Multivariate COX analysis of OS for SLFN11 mRNA expression in the series of 250 TNBCs.

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Data file S1 (Microsoft Excel format). Clinical and pathological characteristics of the TNBCs corresponding to PDXs.
    • Data file S2 (Microsoft Excel format). Characteristics of TNBC PDXs (BRCAness, BRCA1/2 mutations, RB1, and SLFN11 status).
    • Data file S3 (Microsoft Excel format). List of genes differentially expressed between PDXs in the “response group (R)” as compared to “progressive disease (PD)” group.
    • Data file S4 (Microsoft Excel format). Individual data points.

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