Figure 1.
Nuclear HNF1B expression is associated with cytoplasmic clearing across multiple tumor types.
Representative H&E (10×objective, and magnified inset) and positive HNF1B immunostains are shown for (A) ovarian CCC, (B) renal CCC, and (C) mixed endometrioid/clear cell ovarian carcinoma. In the ovarian carcinoma with mixed histology (C), the upper row of panels depicts a region of the tumor with clear cell histology (HNF1B-positive); the middle row of panels (insets) depicts a different region of the same tumor with endometrioid histology and clear cell features (note papillary pattern, highlighted by green box) (also HNF1B-positive); and the bottom row (insets) depicts yet another region of the same tumor with endometrioid histology but without clear cell features (and with correspondingly weaker HNF1B-immunostaining). Note that this patient experienced a tumor-associated thromboembolic event (see main text). (D) Graphical display of proportion of HNF1B positive cases (shaded red) among different carcinoma types with or without cytoplasmic clearing. Gynecologic carcinomas include those from the endometrium, cervix and ovary. Other neoplasms represent diverse anatomic sites (see Table S1). P-values (two-sided Fisher’s exact test) for pairwise comparisons are indicated.
Figure 2.
HNF1B CpG island is hypomethylated in carcinomas with cytoplasmic clearing.
(A) Representative bisulfite-modified DNA sequences indicative of CpG island methylation (left) and hypomethylation (right). CpG methylation (at numbered sites) is indicated with an asterisk. (B) Schematic summary of CpG methylation within a 148bp region (PCR primers excluded) of the CpG island spanning HNF1B exon 1. The numbered boxes represent each of the 16 CpG dinucleotides ordered across the region; black fill denotes presence of methylation. Data are shown for individual specimens, including normal blood leukocytes, ovarian carcinomas of serous (n = 4) and clear cell (n = 4) histotype, and renal CCC (n = 5). The proportion of methylated CpGs is significantly higher among serous vs. clear cell ovarian cancers (P<0.001; Student’s t-test).
Figure 3.
HNF1B transcriptional targets are enriched for starch metabolism and clotting cascade pathways.
(A) GSEA analysis shows enrichment of computationally predicted (by binding sites) HNF1 transcriptional targets (n = 259) among genes upregulated following tetracycline-induced HNF1B expression in HEK293 kidney cells [24]. (B) The “leading edge” enriched genes (n = 64) are significantly over-represented among select functional gene sets (nominal P values<0.01); the top 10 gene sets are shown. Columns on the left depict relative log2 gene expression levels (red/blue scale indicated) among replicate cell lines with or without HNF1B induction. Columns on the right indicate membership (gray fill) among the top 10 most significant gene sets, which include liver-specific genes, clotting cascade (represented by five different gene sets), and starch/sucrose metabolism.
Figure 4.
HNF1B transcriptional targets and clotting cascade are enriched in ovarian CCC.
(A) GSEA analysis shows enrichment of putative HNF1 targets (“leading edge” from Fig. 3) among genes selectively expressed in ovarian carcinoma with clear cell vs. other histotype [25]. (B) The leading edge enriched genes determined here (n = 25) are significantly over-represented among select functional gene sets (nominal P values<0.01); the top 10 gene sets are shown. Columns on the left depict relative log2 gene expression levels (red/blue scale indicated) among normal ovary and ovarian carcinomas of different histotype. Columns on the right indicate membership (gray fill) among the top 10 most significant gene sets, which include liver-specific genes, and the clotting cascade (represented by five different gene sets). (C) Clotting factor genes (FGA, FGB, F2, and F13B) are more highly expressed in laser-microdissected ovarian CCC vs. normal ovarian surface epithelium (dataset from [26]). Heatmap depicts all microarray probes for the respective genes; corresponding P-values (two-sided Student’s t-test) are indicated.
Figure 5.
HNF1B-positive gynecologic malignancies are associated with cytoplasmic prothrombin expression, and increased clotting factor transcript levels.
(A) Representative prothrombin immunostains (10×objective, and magnified inset) are shown for HNF1B-negative, prothrombin-negative mixed-histology endometrial carcinoma (left); and HNF1B-positive, prothrombin-positive (moderate cytoplasmic staining) mixed endometrioid/clear cell ovarian carcinoma (right; same case as depicted in Fig. 1C). (B) Q-RT-PCR analysis demonstrates increased clotting factor transcript levels in ovarian CCC (n = 6) compared to ovarian serous carcinoma (n = 7). Transcript levels are normalized to GAPDH, then set relative to a single serous carcinoma “reference” sample, and reported as log2 values. For graphs shown, undetected transcript levels (green diamonds) are arbitrarily set to smallest detectable levels. Red bars indicate average levels; P-values (non-parametric Mann-Whitney U-test) are shown.
Table 1.
Tumor HNF1B expression is associated with venous thrombosis.