Application of Immunohistochemistry and Molecular Diagnostics to Clinically Relevant Problems in Endometrial Cancer Bojana Djordjevic, Shannon Westin, Russell R. Broaddus

Abstract

A number of different clinical scenarios are presented in which lab-based analyses beyond the usual diagnosis based on light microscopic examination of H&E stained slides - immunohistochemistry and PCR-based assays such as sequencing, mutation testing, microsatellite instability analysis, and determination of MLH1 methylation - are most helpful for guiding diagnosis and treatment of endometrial cancer. The central goal of this information is to provide a practical guide of key current and emerging issues in diagnostic endometrial cancer pathology that require the use of ancillary laboratory techniques, such as immunohistochemistry and molecular testing. The authors present the common diagnostic problems in endometrial carcinoma pathology, types of endometrial carcinoma, description of tissue testing and markers, pathological features, and targeted therapy.

Keywords: Lynch Syndrome; endometrial cancer; targeted therapy.

Figure 1. Typical immunohistochemical expression pattern for an endometrial endometrioid adenocarcinoma

Endometrioid-type endometrial adenocarcinoma (A, representative H&E stain) is usually strongly and diffusely positive for vimentin (B), negative for monoclonal CEA (C), positive for ER (D), and has patchy positive expression of p16 (E). Higher grade endometrioid tumors may lose some nuclear ER expression, but even the grade 3 endometrioid tumors will have at least some ER expression.

Figure 2. Typical immunohistochemical expression pattern for an endocervical adenocarcinoma

In biopsies, it can frequently be difficult to distinguish an endocervical adenocarcinoma from an endometrial adenocarcinoma. This distinction is important, as the surgical approaches can differ between the two tumor types. Endocervical adenocarcinoma (A, representative H&E) is usually negative for vimentin (B; strong staining in left portion of figure is stroma), strongly positive for monoclonal CEA (C), negative for ER (D), and diffusely positive for p16 (E).

Figure 3. Hysterectomy specimen with mucinous adenocarcinoma involving the endocervix and lower uterine segment

Unfortunately, immunohistochemistry cannot always resolve difficult pathology diagnostic problems. Based on the gross examination and examination of H&E stained slides (A), the differential diagnosis of this tumor includes endocervical adenocarcinoma, endometrial endometrioid adenocarcinoma with mucinous differentiation, and metastatic colorectal adenocarcinoma. The tumor was negative for cytokeratin 20 (B) and strongly positive for cytokeratin 7(C). While this profile favors a gynecological origin for this tumor, it must be remembered that a subset of MSI-High colorectal adenocarcinomas can express CK 7(109). The tumor was negative for vimentin and ER and had weak and patchy nuclear expression of CDX2, patchy expression of p16, and strong expression for CEA (data not shown). The immunohistochemistry profile therefore could not definitively establish a primary site for this malignancy. A subsequent colonoscopy was negative, so this tumor most likely represents a primary endocervical or endometrial adenocarcinoma. An endocervical primary may be favored because of the lack of ER and vimentin expression.

Figure 4. Uterine serous carcinoma

This serous carcinoma (representative H&E, A) shows a typical immunohistochemical expression pattern. The tumor has strong and diffuse nuclear expression of p53 (B) and p16 (C). WT-1 is positive (nuclear) in most ovarian high grade serous carcinomas. However, uterine serous carcinoma is usually negative for WT-1 (D). This tumor is ER positive (E).

Figure 5. Uterine serous carcinoma with atypical immunohistochemical expression pattern

By routine H&E staining (A), this tumor has features typical of uterine serous carcinoma, specifically high nuclear grade and loss of nuclear intra-epithelial polarity. However, the tumor is negative for p53 (B). The tumor did show strong and diffuse expression of p16 (C) and was negative for WT-1 (D). Unlike the serous carcinoma presented in Figure 4, this example is negative for ER (E).

Figure 6. MSI analysis chromatogram

In this example, the tumor has allelic shift for the microsatellites BAT26 and D17S250, as demonstrated by the extra peaks in the tumor tracings compared to that of normal tissue. The five other microsatellites in the panel had similar allelic shift, so this is an MSI-High endometrial carcinoma.

Figure 7. MLH1 methylation analysis

Extracted DNA is treated with bisulfite, which converts cytosine to uracil; methylated cytosines are resistant to such conversion, and this forms the basis of this assay. Concurrent analyses of control cell lines (top tracing, negative control K562; middle tracing, positive control RKO) are important. The endometrial tumor (bottom tracing) has both unmethylated and methylated MLH1. The unmethylated peak is likely due to the presence of contaminating normal stromal cells.

Figure 8. Immunohistochemical expression of DNA mismatch repair proteins in endometrial carcinoma

This tumor is negative for MSH2 (A) and has positive nuclear expression for MLH1 (B). In (A) note the presence of positive staining stromal cells. Such cells are a useful internal positive control.

Figure 9. Patterns of PTEN immunohistochemical expression seen in endometrial carcinoma

Positive (A), heterogeneous (B), and negative (C) examples are provided. In cases with PTEN negative staining, the presence of the positive staining stromal cells serves as a useful internal positive control.