A Rare Case of Scalp Metastasis from Encapsulated Angioinvasive Follicular Thyroid Carcinoma.

Cutaneous metastases from thyroid carcinomas are extremely rare; however, the scalp is a common site for cutaneous metastases from follicular thyroid carcinomas (FTCs). We report the case of a 77-year-old male patient with a blood-rich scalp lesion. Histopathological tests of punch biopsy specimens revealed subcutaneous well-formed follicular structures that were similar to those found in the thyroid gland. Immunohistochemistry using thyroid transcription factor-1 (TTF-1) and paired-box gene family 8 (PAX8) revealed an FTC metastasis. We performed total thyroidectomy and resection of the scalp lesion at the same time and administered postoperative radioactive iodine treatment. The primary thyroid lesion was diagnosed as an FTC based on extracapsular extension and vessel invasion. The patient has not experienced disease recurrence since the treatment. When scalp metastasis of thyroid carcinoma is suspected, we recommend total extirpation, including the primary tumor and scalp metastasis, for an improved prognosis.

Serum Levels of N- and C-ERC/Mesothelin and Clinicopathological Factors in Mesothelioma Patients and Those without Mesothelioma.

Objectives: ERC/mesothelin is a glycosylphosphatidylinositol (GPI)-anchor protein expressed in mesothelioma. A precursor protein is cleaved by proteases and an N-terminal fragment (N-ERC) is extracellularly secreted. A remaining C-terminal fragment (C-ERC) is tethered on cellular membranes by the GPI-anchor, but C-ERC is also released after cleavage by proteases. We and other groups reported that serum N-/C-ERC levels are associated with stages of mesothelioma and suggested the possibility of their usefulness as diagnostic markers. However, the N-ERC level is also influenced by renal functions that are not directly associated with conditions of mesothelioma. It is not known whether other clinical factors influence serum N-/C-ERC values. Furthermore, their relationship to the amount of ERC/Mesothelin in mesothelioma is not yet validated. The objective of this study is to clarify the relationship of serum N-/C-ERC levels and the status of mesothelioma and several clinical factors. Materials and Methods: We analyzed relations of serum N-/C-ERC levels and ages, gender and other clinical factors in 522 patients without mesothelioma and examined their relation to the amount of ERC/Mesothelin in mesothelioma tissues in 13 mesothelioma cases. Results: Serum N-ERC levels were influenced by renal functions. On the contrary, those of C-ERC were not influenced by any clinical factors examined in this study and were significantly correlated with the amount of ERC/Mesothelin in mesothelioma. Conclusion: Although both markers are good indicators of treatment-responses in individual patients with mesothelioma, only C-ERC reflected the amount of ERC/Mesothelin in mesothelioma among multiple patients, possibly because N-ERC was influenced by renal functions.

The Inhibitory Effects of Anti-ERC/Mesothelin Antibody 22A31 on Colorectal Adenocarcinoma Cells, within a Mouse Xenograft Model.

The expression of Renal Carcinoma (ERC)/mesothelin is enhanced in a variety of cancers. ERC/mesothelin contributes to cancer progression by modulating cell signals that regulate proliferation and apoptosis. Based on such biological insights, ERC/mesothelin has become a molecular target for the treatment of mesothelioma, pancreatic cancer, and ovarian cancer. Recent studies revealed about 50-60% of colorectal adenocarcinomas also express ERC/mesothelin. Therefore, colorectal cancer can also be a potential target of the treatment using an anti-ERC/mesothelin antibody. We previously demonstrated an anti-tumor effect of anti-ERC antibody 22A31 against mesothelioma. In this study, we investigated the effect of 22A31 on a colorectal adenocarcinoma cell line, HCT116. The cells were xenografted into BALB/c nu/nu mice. All mice were randomly allocated to either an antibody treatment group with 22A31 or isotype-matched control IgG1κ. We compared the volume of subsequent tumors, and tumors were pathologically assessed by immunohistochemistry. Tumors treated with 22A31 were significantly smaller than those treated with IgG1κ and contained significantly fewer mitotic cells with Ki67 staining. We demonstrated that 22A31 exhibited a growth inhibitory property on HCT116. Our results implied that ERC/mesothelin-targeted therapy might be a promising treatment for colorectal cancer.

Basal cell adenocarcinoma of the maxillary sinus: A case report.

Basal cell adenocarcinoma is a low-grade malignancy of the salivary glands. Basal cell adenocarcinoma of the minor salivary gland is an extremely rare disease that originates from the maxillary sinus. The histopathological characteristics of basal cell adenocarcinomas are similar to those of basal cell adenomas. However, basal cell adenocarcinomas can be differentiated from basal cell adenomas based on their tendency to invade surrounding tissues. Surgical resection is the first-line treatment for basal cell adenocarcinomas. An 86-year-old man underwent operations for a maxillary sinus tumor twice in our department. The pathological results of the tumor at both times revealed basal cell adenoma. After 4 and 5 years since the last operation, the tumor recurred, and the patient was treated with partial maxillectomy using Weber-Ferguson incision. We observed invasions to the surrounding tissue, and based on immunohistochemical findings, the patient was diagnosed with basal cell adenocarcinoma. Herein, we present an extremely rare case of basal cell adenocarcinoma arising from the maxillary sinus, in detail.

Expression status of PD-L1 and B7-H3 in mesothelioma.

Mesothelioma is a rare, aggressive malignancy with poor outcome, and has limited treatment options. The aim of this study was to perform a comprehensive analysis of programmed death ligand 1 (PD-L1) and B7 homolog 3 (B7-H3) expression in mesothelioma. We investigated the protein expression of PD-L1 and B7-H3 and their potential correlation with histological subtype, which might help to develop new therapies targeting these immune checkpoint molecules. Expression analysis of PD-L1 and B7-H3 was performed by immunohistochemistry using serial tissue sections of specimens obtained from 31 patients with mesothelioma. Tumors were classified into 22 epithelioid, 6 sarcomatoid, and 3 biphasic types. Of the 31 patients, 13 (41.9%) were positive for PD-L1 and 28 (90.3%) were B7-H3 positive. Twelve of the 13 PD-L1 positive patients were positive for B7-H3. PD-L1 and B7-H3 were widely co-expressed in biphasic and sarcomatoid type tumor cells. These findings might provide a rationale for the use of combination therapy for mesothelioma by targeting PD-L1 and B7-H3, as well as the development of anti-B7-H3 or anti-PD-L1 single agents.

Metastatic seeding of human colon cancer cell clusters expressing the hybrid epithelial/mesenchymal state.

Emerging evidence supports the theory that tumor cell clusters efficiently metastasize to distant organs. However, the roles of epithelial-to-mesenchymal transition (EMT) in metastasizing tumor cell clusters have not yet been fully elucidated. To investigate this issue, tumor fragments were dissected from 40 colorectal cancer (CRC) patients and implanted subcutaneously into immunodeficient mice. We observed that tumors developed from the tumor fragments obtained from 28 of the 40 CRC patients. The tumors were then dissociated into cell suspensions to be orthotopically injected into secondary mice. The tumors from 13 of the 28 patients progressed. Furthermore, metastases formed spontaneously in the liver and lungs from the tumor fragments obtained from 8 of these 13 patients. Moreover, employing a mathematical analysis, we showed that tumor cell clusters seeded these metastases significantly more often than did single tumor cells. Membrane E-cadherin- and nuclear ZEB1-positive tumor cells indicating the hybrid epithelial/mesenchymal state were also detected in primary tumors of various CRC patients, and in the corresponding patient-derived xenografts (PDXs) and circulating tumor cell clusters in the bloodstreams of mice. In contrast, ZEB1 staining was barely detectable in the patient-matched liver metastases presumably developing through mesenchymal-to-epithelial transition. Inhibition of E-cadherin or ZEB1 expression by shRNA notably prevented the PDX-derived tumor organoids from colonizing the liver, when injected intrasplenically into mice, indicating E-cadherin and ZEB1 expressions to be required for their metastatic colonization. Taken together, these findings suggest that the epithelial/mesenchymal state mediates metastatic seeding of human CRC cell clusters into distant organs.

Usefulness of immunohistochemistry for mismatch repair protein and microsatellite instability examination in adenocarcinoma and background endometrium of sporadic endometrial cancer cases.

AIM: Microsatellite instability (MSI), which reflects loss of DNA mismatch repair (MMR) activity, and immunohistochemistry (IHC) for MMR proteins are employed as screening examinations for Lynch syndrome (LS). Recent studies revealed that there is a population of MSI-high tumors in sporadic endometrial cancer (EC). However, MSI data for Japanese EC patients are scarce. Furthermore, sporadic estrogen-dependent EC (type I) is generally considered to arise from hyperplasia. Because LS is usually associated with type I EC, we hypothesized that MSI might be involved in the oncogenic process in some sporadic EC. We conducted MSI testing to reveal MSI status in sporadic Japanese EC. IHC for MMR proteins was also performed. METHODS: Ninety-eight tissue samples of sporadic ECs from Japanese patients were used for IHC and MSI examinations. We also evaluated MMR protein expressions in the background normal endometrium. RESULTS: Microsatellite instability-high was observed in 10.2% of 98 cases with sporadic EC, a lower percentage than that in Western studies. Loss of some MMR proteins was observed in 23 cases (23.5%) and there was a significant correlation with MSI-high status (P < 0.001). Concerning the background endometrium, two cases showed partial loss of MLH1 and PMS2, corresponding to adjacent EC lesions, suggesting that MMR deficiency may already be present in the background endometrium. CONCLUSION: The MSI-high rate was low in our Japanese cohort. Our data confirmed the usefulness of MMR protein assessment for MSI screening in Japanese EC patients. Furthermore, IHC of the background endometrium might reveal the mechanism of MSI-high tumorigenesis.

Establishment of anti-mesothelioma monoclonal antibodies.

BACKGROUND: Mesotheliomas are aggressive, therapy-resistant tumors that are predicted to increase in incidence at least until 2020. The prognosis of patients with mesothelioma is generally poor because they are typically diagnosed at a late stage and their tumors are resistant to current conventional therapies. For these reasons, improved diagnosis and therapy are urgently required. To address these issues, the aim of our research was to develop novel mesothelioma-specific monoclonal antibodies (mAbs) as diagnostic and therapeutic agents. METHODS: To develop anti-mesothelioma mAbs useful for diagnosis and therapy, we repeatedly immunized a BALB/c mouse with viable mesothelioma cells, alternating between those from three mesothelioma cell lines. We hybridized the spleen cells from this immunized mouse with P3U1 myeloma cells. We then screened supernatants harvested from the hybridoma clones by assessing whether they bound to a mesothelioma cell line not used for immunization and altered its morphology. We designed this developmental strategy to reduce the risk of obtaining clonotypic mAbs against a single mesothelioma cell line. RESULTS: Our newly generated mouse anti-human mAbs immunostained clinical samples of mesotheliomas. One of the newly generated mAbs did not react with any other tumor cell line tested. Two other mAbs significantly inhibited the proliferation of mesothelioma cells. CONCLUSION: These newly generated anti-mesothelioma mAbs are potentially useful as diagnostic and therapeutic agents for mesothelioma. Moreover, our novel strategy for establishing antitumor mAbs may facilitate the development of new diagnostic and therapeutic techniques for mesotheliomas and other malignancies.

Establishment of a Therapeutic Anti-Pan HLA-Class II Monoclonal Antibody That Directly Induces Lymphoma Cell Death via Large Pore Formation.

To develop a new therapeutic monoclonal Antibody (mAb) for Hodgkin lymphoma (HL), we immunized a BALB/c mouse with live HL cell lines, alternating between two HL cell lines. After hybridization, we screened the hybridoma clones by assessing direct cytotoxicity against a HL cell line not used for immunization. We developed this strategy for establishing mAb to reduce the risk of obtaining clonotypic mAb specific for single HL cell line. A newly established mouse anti-human mAb (4713) triggered cytoskeleton-dependent, but complement- and caspase-independent, cell death in HL cell lines, Burkitt lymphoma cell lines, and advanced adult T-cell leukemia cell lines. Intravenous injection of mAb 4713 in tumor-bearing SCID mice improved survival significantly. mAb 4713 was revealed to be a mouse anti-human pan-HLA class II mAb. Treatment with this mAb induced the formation of large pores on the surface of target lymphoma cells within 30 min. This finding suggests that the cell death process induced by this anti-pan HLA-class II mAb may involve the same death signals stimulated by a cytolytic anti-pan MHC class I mAb that also induces large pore formation. This multifaceted study supports the therapeutic potential of mAb 4713 for various forms of lymphoma.

IL-6 signal blockade ameliorates the enhanced osteoclastogenesis and the associated joint destruction in a novel FcγRIIB-deficient rheumatoid arthritis mouse model.

OBJECTIVE: We earlier found that TNFα but not interleukin (IL)-17 is indispensable in the pathogenesis of spontaneously occurring rheumatoid arthritis (RA)-like disease in our newly established FcγRIIB-deficient C57BL/6 (B6) mouse model, designated KO1. Here, we examined the role of IL-6 in the pathogenesis of RA features in KO1, with particular reference to cartilage and bone destruction in arthritic joints. METHODS: To evaluate the preventive effect of MR16-1, a rat anti-mouse IL-6 receptor (IL-6R) mAb, 4-month-old preclinical KO1 mice were divided into three groups: the first treated with MR16-1 for 6 months, the second treated with normal rat IgG, as a control, and the third left untreated. The incidence and severity of arthritis, immunological abnormalities, and transcription levels of receptor activator of NF-κB ligand (RANKL), osteoprotegerin (OPG), and inflammatory cytokines/chemokines in ankle joint tissues were compared among the three groups. The therapeutic effect of MR16-1 was examined by treating 7-month-old KO1 mice in the early stages of arthritis for 2 months. RESULTS: Compared with the findings in the KO1 mice left untreated or treated with normal rat IgG, the development of arthritis was markedly suppressed in mice with MR16-1 treatment started from preclinical stages. The suppression was associated with the decrease in production of autoantibodies, rheumatoid factors (RF), and anti-cyclic citrullinated peptide (CCP). Histologically, marked synovitis, pannus formation, and cartilage and bone destruction associated with the increase in tartrate-resistant acid phosphatase (TRAP)-positive osteoclast generation were evident in the two control groups; however, these findings were virtually absent in MR16-1-treated mice. Real-time PCR analysis revealed that the up-regulated expression levels of MCP-1, IL-6, and TNFα, and the aberrantly high RANKL/OPG expression ratio in synovial joint tissues from the two control groups of mice with overt arthritis were significantly suppressed in MR16-1-treated mice. In mice with therapeutic MR16-1 treatment, there was no progression in arthritis score and the RANKL/OPG ratio in joint tissues was significantly suppressed. CONCLUSIONS: Administration of an anti-IL-6R mAb ameliorated spontaneously occurring RA-like disease features, indicating that IL-6, as well as TNFα, plays a pivotal role in the pathogenesis of RA in KO1 mice. Current studies showed that, in addition to the role in enhancing autoantibody production, IL-6 promotes synovial tissue inflammation and osteoclastogenesis, leading to the severe synovitis with pannus formation and the progressive cartilage and bone destruction in multiple joints.

TNFα but not IL-17 is critical in the pathogenesis of rheumatoid arthritis spontaneously occurring in a unique FcγRIIB-deficient mouse model.

OBJECTIVE: TNFα and IL-17 have been shown to be the major inflammatory cytokines involved in the pathogenesis of rheumatoid arthritis (RA). Here, we examined the effect of these cytokines on spontaneously occurring RA in our newly established arthritis-prone FcγRIIB- deficient C57BL/6 (B6) mice, designated KO1, by introducing genetic deficiency of TNFα and IL-17 into KO1 mice. METHODS: KO1.TNFα(-/-) and KO1.IL-17(-/-) mice were established by crossing KO1 with TNFα-deficient and IL-17-deficient B6 mice, respectively. The incidence and severity of RA, cartilage and bone destruction, immunological abnormalities, and transcription levels of receptor activator of NF-κB ligand (RANKL)/osteoprotegerin (OPG) and inflammatory cytokines/chemokines in ankle joints were compared among KO1, KO1.TNFα(-/-), and KO1.IL-17(-/-) mice. RESULTS: The development of RA was completely inhibited in KO1.TNFα(-/-) mice. In contrast, KO1.IL-17(-/-) mice unexpectedly developed severe RA comparable to KO1. Compared with those in KO1 and KO1.IL-17(-/-) mice, frequencies of peripheral monocytes, known to be containing osteoclast precursors, were significantly decreased in KO1.TNFα(-/-) mice. Intriguingly, while RANKL expression levels in ankle joints did not differ among the three strains, OPG expression levels were drastically decreased in arthritis-prone, but not arthritis-free, mice. The expression levels of inflammatory cytokines/chemokines, such as MCP-1, IL-6, and TNFα, were up-regulated in arthritis-prone mice. CONCLUSION: TNFα is indispensable while IL-17 is dispensable in the pathogenesis of RA in KO1 mice. In this model, TNFα may contribute to the development of arthritis, through mediating the increase in frequencies of osteoclast precursors in circulation and their migration into the joints, and the decrease in OPG expression, leading to the up-regulated osteoclastogenesis associated with severe cartilage and bone destruction.

Nephrotoxic effect of subchronic exposure to S-(1,2-dichlorovinyl)-L-cysteine in mice.

The effect of subchronic exposure of S-(1,2-dichlorovinyl)-L-cysteine (DCVC), an active metabolite of trichloroethylene (TCE), was investigated in mice, as a part of mechanistic assessment of renal toxicity of TCE. To examine the subchronic effects of DCVC on kidney function, Balb/c male mice were administered DCVC orally and intraperitoneally once a week for 13 weeks at 1, 10 and 30 mg/kg (Main Study) and for 8 weeks at 30 mg/kg (PCR Study). At the terminal sacrifice, mice orally and intraperitoneally administered with 10 and 30 mg/kg showed significantly lower kidney weight and significantly higher blood urea nitrogen levels than the control group. Pathological examination revealed that a dose of 30 mg/kg delivered by both routes resulted in renal tubular degeneration characterized by tubular necrosis and interstitial fibrosis, and in degradation of the cortex. Degenerative changes were accompanied by the increased expression of tumor necrosis factor-α, interleukin-6 and cyclooxygenase-2 mRNAs in the kidney of mice treated with 30 mg/kg for 8 weeks. These pathohistological observations mostly corresponded to those in short-term toxicity studies on DCVC. DCVC might be a direct cause of renal toxicity, which is suggested from the aggravation in these symptoms with the dose increase.

Mesothelin (MSLN) promoter is hypomethylated in malignant mesothelioma, but its expression is not associated with methylation status of the promoter.

Gene methylation leads to malignant progression in some tumors. The mechanism by which mesothelin is expressed in malignant mesothelioma (MM) is not well understood. MM is histologically divided into 3 subtypes, that is, the epithelioid, sarcomatoid, and biphasic types, and it was shown that mesothelin expression was restricted to the epithelioid type and the epithelioid component of the biphasic type of MM. However, its regulatory mechanism of expression has not been clarified. Here, we studied the expression of mesothelin by immunohistochemistry along with the methylation status of 20 CpG sites in the promoter of the mesothelin gene (MSLN) in 118 lung specimens, including 39 MM, 41 lung carcinoma, 26 nonneoplastic pulmonary lesions, and 12 normal lung tissue samples by the methylation-sensitive single nucleotide primer extension technique. We confirmed that mesothelin was expressed in the epithelioid type and epithelioid component of the biphasic type of MM but neither in the sarcomatoid type nor sarcomatous component of the biphasic type. Surprisingly, the MSLN promoter was significantly hypomethylated in the MM cases regardless of its subtype, compared with the other pulmonary lesions and normal lung tissue samples. These findings suggested that hypomethylation of the MSLN promoter may be specifically associated with the formation of MM, regardless of its expression status, and that the expression of mesothelin protein was lost in the sarcomatoid type by some unknown posttranscriptional regulatory mechanism. We also identified 4 CpG sites, among the 20 sites studied, to be more specifically hypomethylated in MM cases.

Birt-Hogg-Dubé (BHD) gene mutations in human gastric cancer with high frequency microsatellite instability.

Birt-Hogg-Dubé (BHD) syndrome is a rare inherited genodermatosis characterized by benign hamartomatous skin lesions and an increased risk of pneumothorax and renal tumors. Many of the patients harbor insertion/deletion mutations in the hypermutable poly(C)(8) tract in exon 11 of the BHD gene. This mutational hot spot is also reported to be a target of mutation in microsatellite instability (MSI) sporadic colorectal tumors. To test if the BHD gene is a potential mutational target in gastric cancer, we screened for mutations in all of the coding exons of the BHD gene in 30 cases of MSI gastric cancer as well as 50 cases of microsatellite stable (MSS) gastric cancer. Mutations in the poly(C)(8) tract of BHD were detected in 3 of 19 MSI-high cases (15.8%), and none of 11 MSI-low cases. All BHD mutated cases also showed mutations of both BAX and TGFbetaRII. No mutations were detected in the other exons of the BHD gene. No BHD mutations were found in MSS gastric cancer cases. Taken together, these findings show that the BHD gene is a rare target in MSI-high gastric cancer, and BHD mutation tends to occur downstream in the mutational events of other major MSI-high target genes.

Genetic dissection of the effects of stimulatory and inhibitory IgG Fc receptors on murine lupus.

Immune complex (IC)-mediated tissue inflammation is controlled by stimulatory and inhibitory IgG Fc receptors (FcgammaRs). Systemic lupus erythematosus is a prototype of IC-mediated autoimmune disease; thus, imbalance of these two types of FcgammaRs is probably involved in pathogenesis. However, how and to what extent each FcgammaR contributes to the disease remains unclear. In lupus-prone BXSB mice, while stimulatory FcgammaRs are intact, inhibitory FcgammaRIIB expression is impaired because of promoter region polymorphism. To dissect roles of stimulatory and inhibitory FcgammaRs, we established two gene-manipulated BXSB strains: one deficient in stimulatory FcgammaRs (BXSB.gamma(-/-)) and the other carrying wild-type Fcgr2b (BXSB.IIB(B6/B6)). The disease features were markedly suppressed in both mutant strains. Despite intact renal function, however, BXSB.gamma(-/-) had IC deposition in glomeruli associated with high-serum IgG anti-DNA Ab levels, in contrast to BXSB.IIB(B6/B6), which showed intact renal pathology and anti-DNA levels. Lymphocytes in BXSB.gamma(-/-) were activated, as in wild-type BXSB, but not in BXSB.IIB(B6/B6). Our results strongly suggest that both types of FcgammaRs in BXSB mice are differently involved in the process of disease progression, in which, while stimulatory FcgammaRs play roles in effecter phase of IC-mediated tissue inflammation, the BXSB-type impaired FcgammaRIIB promotes spontaneous activation of self-reactive lymphocytes and associated production of large amounts of autoantibodies and ICs.

Frequent down-regulation of HIVEP2 in human breast cancer.

The HIVEP2 gene, located on 6q23-q24, belongs to a family of genes that encodes large zinc fingers containing transcription factor proteins. Although this gene has been implicated in the regulation of immune responses and cellular proliferation, its functions are largely unknown. In the present study, we investigated HIVEP2 gene abnormalities in microdissected breast cancer tissue. For real-time quantitational RT-PCR analysis of paired normal and tumor tissues, mRNA levels were down-regulated to a maximum of 96%. The overall median expression level in breast cancer (33 cases) was significantly lower than that in normal breast tissue (normalized median value of 4.49 versus 17.68; p < 0.0001). Through full-length 5'-RACE (rapid amplification of cDNA ends) analysis, we identified multiple exons in the 5'-untranslated regions with multiple transcriptional start sites, four of which were located in a large CpG island. No tissue- or cancer-specific usage patterns for the transcription start sites were identified by multiplex RT-PCR analysis. Only faint methylation was detected in the 5' region of the island in normal cells and breast cancer tissue, indicating physiological, aging and no tumor-specific methylation. Mutation screening showed only germline polymorphisms. Thus, down-regulation of the HIVEP2 genes frequently occurs and may be one of the genetic events responsible for breast cancer, and their transcription may be regulated by complex mechanisms involving interactions with other factors and/or by other genetic/epigenetic mechanisms.

Genetics of synchronous uterine and ovarian endometrioid carcinoma: combined analyses of loss of heterozygosity, PTEN mutation, and microsatellite instability.

Synchronous development of carcinomas in the endometrium and ovaries is a fairly common phenomenon, but distinction of a single clonal tumor with metastasis from 2 independent primary tumors may present diagnostic problems. To determine clonality and the occurrence of progression, we microdissected multiple foci from 17 cases of synchronous endometrioid carcinomas and studied loss of heterozygosity (LOH), microsatellite instability (MI), and PTEN mutations. In 14 of the 17 cases, genetic alterations were either homogeneous or found in only some of the foci. LOH was detected for 10q (4 cases), 17p (2 cases), and 2p, 5q, 6q, 9p, 11q, 13q, and 16q (1 case each). Four cases had the MI phenotype with discordant MI patterns between both tumor sites, thus indicating a biclonal or triple clonal process. In 3 of 6 cases with PTEN mutations, identical mutations in both tumor sites indicated a single clonal neoplasm. Altogether, 14 synchronous tumors were genetically diagnosed as follows: single clonal tumor, characterized by concordant genetic alterations in both tumor sites, including identical LOH, identical PTEN mutations, and/or identical sporadic allelic instability patterns (4 cases); single clonal tumor with genetic progression, homogeneous LOH or identical PTEN mutations in both tumor sites and progressive LOH in ovarian metastatic foci (2 cases); and double (7 cases) or triple clonal tumors (1 case), determined by discordant PTEN mutations, heterogeneous LOH, and/or discordant MI patterns. Thus, 35% of synchronous tumors were monoclonal, 47% were polyclonal, and 18% were undetermined. The favorable prognosis of synchronous endometrioid carcinomas may be due to the occurrence of PTEN mutations in both independent and metastatic tumors, the MI-positive independent primary tumors, and the low frequency of LOH.