Endometrial adenocarcinoma is the most common gynecological malignancy in the Western world. It is estimated that endometrial adenocarcinoma would be diagnosed in 47,130 women and that 8010 women would die of cancer of the uterine corpus, in 2012. Endometrial carcinoma is divided into different subtypes based on histology and biology. Molecular data analysis from several studies have shown frequent genetic alterations, which include PTEN inactivation, microsatellite instability, mutations of k-ras, beta-catenin,1,2 and p53. Whereas p53 mutations characterize tumors of serous differentiation and a subset of high-grade endometrioid carcinoma,3 the other alterations have not been systematically studied by histologic subtype.
K-ras mutations have proven to be drivers of oncogenesis and are present in approximately 25% of human cancers.4,5 In addition, k-ras mutations may cause resistance to epidermal growth factor receptor (EGFR) inhibitors, and testing is routinely performed in cases of colorectal carcinoma before targeted therapy.6–8 Most functional mutations occur in codons 12, 13, or 61; and diagnostic testing has focused on these regions.
Previously published studies have shown that k-ras gene mutation is found in 10% to 30% of endometrial carcinomas without specifying the subcategories or the presence or absence of significant mucinous differentiation.9–12 K-ras mutation has been reported in association with microcystic elongated and fragmented gland pattern of myometrial invasion in endometrioid carcinoma (EC).13 However, k-ras mutations in uterine mucinous adenocarcinoma and uterine endometrioid adenocarcinoma with a significant mucinous component have not yet been systematically investigated. Given the functional importance of k-ras and the presence of k-ras mutations in mucinous tumors of the pancreas and ovary.14–16 we examined whether the degree of mucinous differentiation in endometrial carcinomas predicted a k-ras mutation. The current study compares the frequency of k-ras mutations in endometrial mucinous carcinoma (MC), EC with significant mucinous differentiation (ECMD), pure forms of EC, and endometrial serous carcinoma (SC).
MATERIALS AND METHODS
After receiving Institutional Review Board approval, 55 endometrial carcinomas diagnosed between 2005 and 2010 were selected from the archival files of the surgical pathology database at Women and Infants Hospital (Providence, RI). Hematoxylin and eosin– stained slides were reviewed in all cases to confirm the diagnosis and International Federation of Gynecology and Obstetrics grade. All uterine SCs were classified as high grade. The cases were subcategorized as follows: MC (>50% tumor cells showing mucinous differentiation); ECMD (10%–50% of tumor cells with mucinous differentiation); EC (<10% of tumor cells with mucinous differentiation), and uterine SC. The criterion of 10% minimal mucinous component was selected in keeping with the conventional criteria for diagnosing a mixed-type endometrial carcinoma (requiring a minimum of 10% for the second component). Examples of MC, ECMD, and EC subtypes are illustrated in Figure 1.
Of the 55 cases, 10 cases were MC, 18 cases were ECMD, 16 cases were pure EC, and 11 cases were uterine SC. Periodic acid Schiff (PAS) stains with and without diastase were performed in selected cases to confirm the presence of diastase-resistant intracytoplasmic mucin. All patients were treated and followed at the Program of Women’s Oncology of Women and Infants Hospital, which is a member of the Gynecologic Oncology Group. All patients had undergone surgery performed by board-certified gynecologist oncologists and were staged according to the current International Federation of Gynecology and Obstetrics system. In addition, all endometrial carcinoma cases at our institution are consistently sampled for pathologic examination, with a minimum of 6 representative sections of tumor submitted for each case or tumor in toto in the case of smaller lesions.
Microdissection and DNA Extraction
For the current study, polymerase chain reaction (PCR) technique with direct genomic DNA sequencing was selected to minimize potential false-positive results. The technique is still considered as a criterion standard, although it is less sensitive and requires higher tumor cell contents from tissue samples. Microdissection technique was applied to ensure that study samples contain more than 80% tumor cells. Samples with positive or equivocal results by direct DNA sequencing were confirmed by resequencing and by peptide nucleic acid (PNA) mutant enrichment PCR.17 In the ECMD group, tumor cells with predominant mucinous differentiation were selected for DNA extraction. DNA extraction, purification, and PCR amplification of all samples were performed at the Molecular Laboratory of the Department of Pathology, Rhode Island Hospital (Providence, RI). Each sample was deidentified and assigned a sequential study number per institutional review board policy. All molecular analyses were performed without prior knowledge of morphologic subtypes or histological features.
DNA was purified using a standard phenol-chloroform procedure. Briefly, dissected tissue was deparaffinized in xylene and washed with ethanol. The sample was incubated overnight in lysis solution containing proteinase K. DNA was purified in a series of phenol-chloroform washes, eluted in 100% ethanol containing ammonium acetate, and washed with ice-cold 70% ethanol. The DNA pellet was dried and resuspended in TE buffer. DNA purity and concentration were evaluated with a Nanodrop 2000c spectrophotometer (Thermo Scientific, Waltham, MA).
A 224-base pair fragment encompassing codons 12 and 13 of exon 2 was PCR amplified using forward and reverse primers 5′-GTGTGACATGTTCTAATATAGTCA-3′ and 5′-CTGTATCAAAGAATGGTCCTGCAC-3′ (Integrate DNA Technologies, Coralville, IA), respectively. Each sample was amplified in triplicate 25-μL reactions containing 0.5 μmol/L of each primer, 1× PCR buffer, 1.5-mmol/L MgCl2, 0.2 mmol/L each deoxyribonucleotide triphosphate, 0.7-U AmpliTaq Gold DNA polymerase (Applied Biosystems, Carlsbad, CA), and 5 μL (100–500 ng per reaction) genomic DNA. After amplification, triplicate reactions were purified using Qiaquick PCR purification kit (Qiagen, Valencia, CA) and were confirmed by electrophoresis using 2% agarose gel.
DNA Sequencing and Analysis
Direct DNA sequencing was performed by Keck DNA Sequencing Laboratory at Yale University, New Haven, CT. Automated fluorescent sequencing was carried out on an ABI 3730xL DNA sequencer (Applied Biosystems) using the Big Dye Terminator version 3.1 sequencing kit, after a standardized variation of the kit insert protocol. Sequencing data were analyzed by Sequence Scanner version 1.0 software (Applied Biosystems). Electrographs were visually inspected for a mutant k-ras peak. Samples that were determined as positive or equivocal by direct DNA sequencing were confirmed by resequencing after a PNA mutant enrichment PCR. The PNA was designed to inhibit k-ras wild-type amplification, thereby enriching the mutant sequence.
Statistical analyses were performed using the Fisher exact test to compare the difference of k-ras mutation among the groups. Differences were considered statistically significant if P < 0.05.
Table 1 shows that k-ras mutations were identified in 19 (63%) of 30 grade 1 endometrial carcinomas and 5 (38%) of 13 grade 2 endometrial carcinomas. Six (86%) of 7 cases of grade 1 MCs and 2 (67%) of 3 grade 2 MCs revealed k-ras mutations. Of the 18 tumors classified as ECMDs, k-ras mutations were present in 9 (69%) of 13 grade 1 tumors and 3 (60%) of 5 grade 2 tumors. Cases classified as MC included tumors morphologically comparable to endocervical-type MC. Features include abundant intracytoplasmic mucin, predominantly low-grade nuclear features, and villoglandular and cribriform growth patterns. Of all type I endometrial carcinomas examined, 21 cases were accompanied by lymph node dissections, of which 5 cases showed lymph node metastasis, an example of which is shown in Figure 2. All 5 cases of metastasis arose from primary MCs or ECMDs. Two of the 5 metastases demonstrated both (1) mucinous morphology in the metastasis and (2) the same k-ras mutation as the primary tumor. Materials from 2 external consultation cases were not available for further analysis. A fifth case did not have enough tumor cells in the lymph node for further evaluation.
Whereas some endometrial carcinomas included in this study exhibited dilated glands with luminal and/or extracellular pools of mucin, intracytoplasmic mucin was required to classify a case as having mucinous differentiation. None of the endometrial carcinomas with mucinous differentiation in this study exhibited intestinal-type differentiation. In tumors designated as ECMD, foci of mucinous differentiation exhibited mucin-containing cytoplasm within cribriform, papillary, and cystically dilated endometrial glands. Such foci may be found admixed or separate from the endometrioid component. Squamous metaplasia, although present in some cases of EC, was not a focus of this particular study and was therefore not independently examined in the final analysis.
Periodic acid Schiff staining with and without diastase was used in select cases to distinguish between carcinomas with endocervical-like mucinous differentiation versus areas with possible secretory or clear cell–type changes. Mucinous differentiation in MC and ECMD cases was supported by the presence of PAS-positive diastase-resistant staining.
Point mutations were detected in 55% of type I endometrial carcinomas, with one point mutation identified per case. Six distinct k-ras mutations were identified: G12A, G12C, G12D, G12S, G12V, and G13D. Each morphologic subtype demonstrated the following frequency of mutations: EC (25%), MC (80%), ECMD (67%), and SC (9%). Figure 3 highlights the distribution of specific mutations in each group.
Most point mutations were found in codon 12 (17 cases), with the most prevalent mutation being G12D (codon 12, GGT > GAT, from glycine to aspartic acid, seen in 6/17 cases; Fig. 3). Seven cases exhibited point mutations at codon 13, all of which were G13D. Statistically significant difference in k-ras mutation is noted between the following groups: MC versus EC (P < 0.01), ECMD versus EC (P < 0.05), MC versus SC (P < 0.01), and ECMD versus SC (P < 0.01) by the Fisher exact test. No significant difference was seen between MC versus ECMD and EC versus SC (Fig. 4).
The results from this study show that 47% (16/34) EC and only 9% (1/11) SC demonstrated k-ras mutations. The frequency of k-ras mutation in EC in the current series is higher compared to other published reports, ranging from 15% to 30%.9–12,18 The reason for this higher frequency of K-ras mutation detection may be explained by the unique case selection criteria used to include cases in this study. The current study included MC and cases with significant mucinous component.
Among the 34 endometrial carcinoma cases, 18 cases showed a significant portion of tumor cells with mucinous differentiation. The term “mucinous differentiation” in this study specifically refers to tumor cells that are endocervical like, with abundant intracytoplasmic PAS-positive diastase-resistant mucin. When k-ras mutation of the ECMD subgroup was compare with that of the EC subgroup, a significantly higher frequency of k-ras mutation was detected (67% vs 25%). As expected, the frequency of k-ras mutation was less in the ECMD group compared to the pure MC group (80%), but the difference was not statistically significant. These findings showed that MC and ECMD are strongly associated with k-ras mutation in their tumorigenesis.
K-ras mutational analysis in the current study revealed that each affected case only contained a single-point mutation, either within codons 12 or 13. Our findings are comparable to what has been reported in the literature for endometrial carcinomas. A recent study identified k-ras mutations at codons 12 and 13, with G12D and G12V as the 2 most common mutations.12 In our study, the 2 most common mutations overall were G12D and G13D. It is interesting to note that limited data on mucinous ovarian tumors have also shown k-ras mutations at codon 12, with the most common mutations being G12D as well.15 Mutations in codon 61 have been previously reported to be clinically relevant in other tumors, for example, gastrointestinal tract. However, codon 61 mutations in endometrial carcinomas have only been reported rarely, with a recent study reporting no mutations at codon 61 in any of the 67 cases of endometrial carcinoma examined.19 As a result, k-ras mutations in codon 61 were not investigated in the current study.
Aberrant methylations of CpG islands in the gene promoter are reported to be linked to carcinogenesis in endometrial carcinomas and other human cancers.20,21 O 6-methylguanine DNA methyltransferase (MGMT) is a key enzyme in the DNA repair gene, which is frequently methylated in colorectal cancers. O 6-methylguanine DNA methyltransferase removes mutagenic and cytotoxic adducts from O6-guanine in DNA, the preferred point of attack of many carcinogens and alkylating chemotherapeutic agents.22 Researchers have found that MGMT methylation in colorectal cancers is associated with G to A mutations at k-ras.21,23 It is interesting that our data show that most (83% [20/24]) of k-ras mutations are at the second G of a GG pair of codon 12 and codon 13, particularly G-A transition (54% [13/24]). The association between MGMT methylation and G to A transition mutations have not, however, been consistently reproduced in colorectal carcinomas. Further investigation regarding the association between k-ras mutation and aberrant methylations of CpG islands in ECMD may be an interesting future study.
Standard treatment currently practiced for endometrial carcinomas is surgical removal of the tumor with total hysterectomy, bilateral salpingo-oophorectomy, and lymph node dissection. Some patients may receive systemic chemotherapy and/or radiation therapy, depending on tumor stage and the presence of poor prognostic indicators. Although most patients initially respond to these therapies, recurrence or metastasis with platinum-based drug-resistant disease is common. Some patients eventually succumb to their disease.
Targeted therapies are being administered in some human cancer with some success. Epidermal growth factor receptor inhibitors might be an option in patients with recurrent or metastatic endometrial carcinoma. Epidermal growth factor receptor family members have been shown to be highly expressed in endometrial cancers. Therefore, anti-EGFR–targeted therapies are currently being investigated.24 Of all types of endometrial cancers, 60% to 80% overexpress EGFR, and 20% to 30% overexpress Her-2/neu. These agents result in down-regulation of the mitogen-activated protein kinase and PI3K/AKT signal transduction pathways.25 More recent studies have proposed that k-ras mutation may result in clinical resistance to EGFR inhibitors and suggested that k-ras mutations are useful biomarker of resistance to EGFR-based therapies in colorectal carcinoma. Wild-type k-ras is required for panitumumab efficacy in patients with metastatic colorectal cancer.8 All these knowledge may be relevant in selecting targeted therapies for endometrial carcinomas as well.
In summary, we have found that MC and ECMD are associated with higher frequency of k-ras mutations. Our results point to an association between increased k-ras mutations in endometrial carcinomas and endocervical-like mucinous differentiation. This association suggests that routine identification of endocervical-like mucinous morphology in endometrial carcinomas may be useful for identifying cases that may benefit from additional k-ras testing in the future. The mutation status of k-ras gene in this subtype of endometrial carcinomas may have the potential to be used as a parameter in choosing specific targeted therapies and may even be useful in predicting treatment response to EGFR inhibitors.
Future directions from this study include the examination of k-ras mutational status between primary and metastatic tumors as well as within different areas of the same tumor. Subsequent studies examining a subset of recurrent ECMD may shed light on whether this histologic feature is associated with a more aggressive biological behavior. In addition, clinical follow-up data with a larger sample size may also provide an opportunity to determine if mucinous differentiation, in conjunction with k-ras and/or other oncogenic mutations, may be a histological identifier with prognostic value.
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Keywords:© 2013 by the International Gynecologic Cancer Society and the European Society of Gynaecological Oncology.
Endometrial carcinoma; Mucinous differentiation; K-ras mutation; DNA sequencing