Loss of GATA2 promotes invasion and predicts cancer recurrence and survival in uterine serous carcinoma

Abstract

BACKGROUNDA priori knowledge of recurrence risk in patients with nonmetastatic (International Federation of Gynecology and Obstetrics [FIGO] stage I) uterine serous carcinoma (USC) would enable a risk-stratified approach to the use of adjuvant chemotherapy. This would greatly reduce treatment-related morbidity and be predicted to improve survival.METHODSGATA2 expression was scored by IHC across a retrospective multiinstitutional cohort of 195 primary USCs. Associations between GATA2 levels and clinicopathologic metrics were evaluated using Student's t test, Fisher's exact test, Kaplan-Meier method, and Cox proportional hazard ratio. Invasion in patient-derived USC cells was assessed by Student's t test. RNA-Seq, anti-GATA2 ChIP-Seq, and confirmatory Western blotting enabled identification of GATA2 targets.RESULTSPatients with FIGO stage I GATA2hi USCs had 100% recurrence-free and 100% cancer-related survival, which was significantly better than patients with GATA2lo USCs. In patients for whom adjuvant chemotherapy was omitted, patients with GATA2hi USC had 100% recurrence-free 5-year survival compared with 60% recurrence-free survival in patients with GATA2lo USC. Depletion of GATA2 in patient-derived USC cells increased invasion in vitro.CONCLUSIONRoutine GATA2 IHC identifies 33% of patients with FIGO stage I USC who have a greatly reduced risk of posthysterectomy USC recurrence. Our results suggest that a GATA2-guided personalized medicine approach could be rapidly implemented in most hospital settings, would reduce treatment-related morbidity, and would likely improve outcomes in patients with USC.FUNDINGNIH grants R01 DK068634, P30 CA014520, S10 OD023526, K08 DK127244, T32 HL007899, the UW-Madison Department of Pathology and Laboratory Medicine, the UW-Madison Centennial Scholars Program, the Diane Lindstrom Foundation, the American Cancer Society, the V Foundation, The Hartwell Foundation, and the UMN Department of Obstetrics, Gynecology, and Women's Health.

Keywords: Cancer; Clinical Research; Obstetrics/gynecology; Oncology; Tumor suppressors.

Figure 1. GATA2 IHC correlates with low FIGO stage and with cancer-related and overall survival in USC.

(A) H&E sections and GATA2 IHC from representative USCs showing spectrum of GATA2 IHC staining. Tumor TMA 210 is an example of a GATA2^lo USC, while tumors TMA 180 and TMA 188 are both GATA2^hi. H&E and DAB, 600×. Scale bar: 50 μm. (B) Histogram depicting percent GATA2⁺ tumor nuclei in all 70 cases from UW1 USC cohort. Vertical dotted line indicates 15% cutoff between GATA2^lo and GATA2^hi USCs. (C–F) Distribution of patient age (C), tumor size (D), FIGO stage (E), and myometrial invasion (F) between GATA2^lo and GATA2^hi USCs (n = 70). (G) Kaplan-Meier curve depicting cancer-related survival in USCs from UW1 cohort based on GATA2^lo or GATA2^hi status (n = 64). Analysis in C, D, and F by Student’s t test and analysis in E by Fisher’s exact test. **P < 0.005, ***P < 0.0005.

Figure 2. GATA2 IHC predicts recurrence and survival in FIGO stage I USC.

(A) Histogram depicting percent GATA2⁺ tumor nuclei in combined FIGO stage I USC cohort (n = 127). These patients were pooled from UW1, UW2, Yale, and UMN cohorts. Vertical dotted line indicates 15% cutoff between GATA2^lo and GATA2^hi USCs. (B–D) Spectrum of patient age (n = 127) (B), tumor size at staging hysterectomy (n = 124) (C), and myometrial invasion (n = 121) (D) in FIGO stage I GATA2^lo and GATA2^hi USCs as analyzed by Student’s t test. (E–G) Kaplan-Meier curves depicting recurrence free (n = 123) (E), cancer-related (n = 78) (F), and overall survival (n = 121) (G) in FIGO stage I–II USCs based on GATA2^lo or GATA2^hi status. *P < 0.05.

Figure 3. GATA2 depletion promotes invasion in patient-derived USC cell lines.

(A) Level of chromosomal instability in GATA2^lo or GATA2^hi USCs (n = 60). (B and C) GATA2 is expressed in Ark1 and Ark2 patient-derived USC cells and can be depleted by anti-GATA2 siRNA (B) and doxycline-inducible shRNA (C). (D) Schematic of Matrigel-coated membrane invasion assay. (E) Ability of Ark1 and Ark2 USC cells to invade through Matrigel-coated membranes after GATA2 depletion (n = 11 for Ark1 and n = 7–8 for Ark2). (F) Schematic of hydrogel invasion assay. (G) Ability of Ark1 spheroids to invade into hydrogel after GATA2 depletion (n = 3–5). Analysis in E and G by Student’s t test, with Bonferroni corrections performed for both groups in E. *P < 0.05, ***P < 0.005, ****P < 0.0005. CIN, chromosomal instability; DOX, doxycycline.

Figure 4. GATA2 multi-omics identifies SIN3B as a GATA2 target that regulates USC invasion.

(A) Volcano plot of RNA-Seq data showing RNA expression changes after GATA2 depletion in Ark1 USC cells. (B) Number of genome wide GATA2 ChIP-Seq peaks that fall within different gene regulatory regions. (C) Transcription factor motifs enriched within regions of GATA2 ChIP-Seq occupancy. (D) Approach to identifying candidate GATA2-regulated genes in USC. (E) SIN3B locus with superimposed GATA2 ChIP-Seq occupancy. (F) Western blot depicting SIN3B levels after GATA2 depletion in Ark1 USC cells. (G) Western blot showing SIN3B levels after induction of shSIN3B or shScramble control in Ark1 USC cells. (H) USC invasion through Matrigel-coated membranes following depletion of SIN3B or scramble control (n = 11) as analyzed by Student’s t test with Bonferroni correction. *P < 0.05, **P < 0.005.

Figure 5. GATA2 IHC predicts recurrence and overall survival in FIGO stage I USC in the absence of adjuvant chemotherapy.

(A and B) Kaplan-Meier curves depicting recurrence-free (n = 24) (A) and overall survival (n = 24) (B) in GATA2^lo and GATA2^hi patients with FIGO stage I USC who did not receive adjuvant chemotherapy. These patients were pooled from UW1, UW2, Yale, and UMN cohorts. (C) Current NCCN guidelines and proposed GATA2-guided treatment model for FIGO stage I USC. Per the NCCN, patients with FIGO stage IA noninvasive USCs may receive either close observation or adjuvant therapy, whereas patients with invasive FIGO stage I USCs should receive adjuvant therapy. In a proposed GATA2-guided model, patients with GATA2^hi FIGO stage I USCs would be offered close observation, while patients with GATA2^lo FIGO stage I USC would be offered adjuvant therapy. *P < 0.05. NCCN, National Comprehensive Cancer Network.