Frequent PIK3CA mutations in eutopic endometrium of patients with ovarian clear cell carcinoma

Abstract

Recent studies have reported cancer-associated mutations in normal endometrium. Mutations in eutopic endometrium may lead to endometriosis and endometriosis-associated ovarian cancer. We investigated PIK3CA mutations (PIK3CAm) for three hotspots (E542K, E545K, H1047R) in eutopic endometrium in patients with ovarian cancer and endometriosis from formalin-fixed paraffin-embedded specimens by laser-capture microdissection and droplet digital PCR. The presence of PIK3CAm in eutopic endometrial glands with mutant allele frequency ≥ 15% were as follows: ovarian clear cell carcinoma (OCCC) with PIK3CAm in tumors, 20/300 hotspots in 11/14 cases; OCCC without PIK3CAm, 42/78 hotspots in 11/12 cases; high-grade serous ovarian carcinoma, 8/45 hotspots in 3/5 cases; and endometriotic cysts, 5/63 hotspots in 5/6 cases. These rates were more frequent than in noncancer nonendometriosis controls (7/309 hotspots in 5/17 cases). In OCCC without PIK3CAm, 7/12 (58%) cases showed multiple hotspot mutations in the same eutopic endometrial glands. In 3/54 (5.6%) cases, PIK3CAm was found in eutopic endometrial stroma. Multisampling of the OCCC tumors with PIK3CAm showed intratumor heterogeneity in three of eight cases. In two cases, PIK3CAm was detected in the stromal component of the tumor. Homogenous PIK3CAm in the epithelial component of the tumor matched the mutation in eutopic endometrial glands in only one case. Eutopic endometrial glands in ovarian cancer and endometriosis show high frequency of PIK3CAm that is not consistent with tumors, and multiple hotspot mutations are often found in the same glands. While the mutations identified in eutopic endometrium may not be driver mutations in the patient's cancer, these are still driver mutations but this specific clone has not undergone the requisite steps for the development of cancer.

Fig. 1. Examples of laser-capture microdissection.

A, B Endometrial glands of secretory phase (magnification, 63×). C Collected endometrial glands. D, E Endometrial stroma of secretory phase (63×). Scale bars: 100 µm.

Fig. 2. PIK3CA mutations in OCCC, HGSOC, EC, endometrial glands, and endometrial stroma.

A PIK3CA mutation in OCCC, HGSOC, EC, endometrial glands, and endometrial stroma in all cases. Tumor, endometriosis, endometrial glands, and endometrial stroma of each case were divided into three hotspots and arranged by sample. The spots with mutant allele frequency ≥ 15% are shown in red. For the OCCC cases, the cases on the left side of the dotted line have PIK3CA mutation in the tumor, while the cases on the right side are cases without PIK3CA mutation. a–k show cases which tumors were macro- or microdissected and multisampled (details are shown in Fig. 3C). B Comparison of the patient age of each group. The box and whisker plot shows the age of each group. OCCC ovarian clear cell carcinoma, HGSOC high-grade serous ovarian carcinoma, EC endometriotic cyst, T tumor, E endometriosis, G endometrial gland, S endometrial stroma, EM endometrium, NS not significant.

Fig. 3. PIK3CA mutations in tumors by macro- and microdissection.

A Association between PIK3CA mutations in tumor cells of origin and intratumor heterogeneity. If cells with driver PIK3CA mutations arise outside of the ovary, such as in the endometrium, and are transported to the ovary to form a tumor (left) or if the tumor cells have driver PIK3CA mutations at the time they arise in the ovary (middle), then PIK3CA mutations are likely to be present in all tumor cells. However, if PIK3CA mutations are not present at the time the tumor cells arise and emerge later, intratumor heterogeneity of PIK3CA mutations would occur (right). B Example of laser-capture microdissection. Clear cell carcinoma (case g from Fig. 2, hematoxylin and eosin staining; magnification 50×, toluidine blue staining; magnification 63×). “Te” represents the epithelial component of the tumor and “Ts” represents the stromal component of the tumor. Scale bar: hematoxylin and eosin staining; 200µm, toluidine blue staining; 100µm. C MAF of the epithelial component and stromal component of the tumor and eutopic endometrial glandular epithelium and stroma. MAFs of PIK3CA by macro- and microdissection are shown in the heat map. For the endometrial glandular epithelium, the sample with the highest MAF was extracted. The cases a–k in C are the same as those shown in Fig. 2A. The tumor of cases a, b, d, e, i, and j were multiple macrodissected. The epithelial and stromal components of the tumor of cases b, c, f–i, and k were microdissected. Macro macrodissection, micro laser-capture microdissection, EM endometrium, MAF mutant allele frequency.

Fig. 4. Comparison of PIK3CA MAF in OCCC, endometriosis, and eutopic endometrium.

Samples in which mutant alleles were detected are shown as dot plots for ten groups: OCCC (epithelial and stromal components of tumors sampled by laser-capture microdissection), endometriosis (comorbid endometriosis in OCCC and endometriotic cysts), and eutopic endometrium (endometrial glands of OCCC with PIK3CA mutation in the tumor, OCCC without PIK3CA mutation in the tumor, HGSOC, endometriotic cyst, healthy control, and endometrial stroma). The horizontal bar indicates the average. The dotted line indicates MAF = 15%. MAF mutant allele frequency, OCCC ovarian clear cell carcinoma, PIK3CAm PIK3CA mutation, HGSOC high-grade serous ovarian carcinoma.