Nuclear grade and comedo necrosis of ductal carcinoma in situ as histopathological eligible criteria for the Japan Clinical Oncology Group 1505 trial: an interobserver agreement study.

OBJECTIVE: The Japan Clinical Oncology Group 1505 trial is a single-arm multicentre prospective study that examined the possibility of non-surgical follow-up with endocrine therapy for patients with low-grade ductal carcinoma in situ. In that study, the eligible criteria included histopathological findings comprising low to intermediate nuclear grade and absence of comedo necrosis, and cases were entered according to the local histopathological diagnosis. Nuclear grade is largely based on the Consensus Conference criteria (1997), whereas comedo necrosis is judged according to the Rosen's criteria (2017). The purpose of this study was to standardize and examine the interobserver agreement levels of these histopathological criteria amongst the participating pathologists. METHODS: We held slide conferences, where photomicrographs of haematoxylin-eosin-stained slides from 68 patients with ductal carcinoma in situ were presented using PowerPoint. The nuclear grade and comedo necrosis statuses individually judged by the pathologists were analysed using κ statistics. RESULTS: In the first and second sessions, where 22 cases each were presented, the interobserver agreement levels of nuclear grade whether low/intermediate grade or high grade were moderate amongst 29 and 24 participating pathologists, respectively (κ = 0.595 and 0.519, respectively). In the third session where 24 cases were presented, interobserver agreement levels of comedo necrosis or non-comedo necrosis were substantial amongst 25 participating pathologists (κ = 0.753). CONCLUSION: Although the concordance rates in nuclear grade or comedo necrosis were not high in a few of the cases, we believe that these results could provide a rationale for employing the present criteria of nuclear grade and comedo necrosis in the clinical study of ductal carcinoma in situ.

Promoter DNA Hypermethylation of the Cysteine Dioxygenase 1 (CDO1) Gene in Intraductal Papillary Mucinous Neoplasm (IPMN).

BACKGROUND: Intraductal papillary mucinous neoplasm (IPMN) involves adenoma (IPMA), a precancerous lesion, cancer (IPMC) including high-grade dysplasia (HGD), and invasive carcinoma (IC). DNA markers of IPMN are required for detection of invasive disease, and cysteine dioxygenase 1 (CDO1) gene promoter hypermethylation is a potential candidate. However, it has never been investigated in the context of IPMN. PATIENTS AND METHODS: A total of 107 IPMN tumor tissues, including 41 IPMC and 66 IPMA, were studied. CDO1 promoter methylation was quantified using TaqMan quantitative methylation-specific polymerase chain reaction (qMSP) in patients with IPMN and other pancreatic cystic disorders after pancreatectomy. RESULTS: The methylation values (TaqMeth Vs) of CDO1 increased when noncancerous pancreas tissues were compared with IPMA and HGD (p < 0.0001). Among IPMC, the TaqMeth Vs in IC were not significantly higher than in HGD. The TaqMeth Vs of the solid tumors were higher than those of the cystic tumors (p = 0.0016), which were in turn higher than the corresponding noncancerous tissues (p < 0.0001). Prognostic analysis revealed that high TaqMeth Vs (≥ 14.1) resulted in a poorer prognosis than low TaqMeth Vs (< 14.1) (p < 0.0001). In other pancreatic cystic diseases, only malignant mucinous cystic neoplasm showed DNA hypermethylation of its promoter. A pilot study in pancreatic juice confirmed methylation in all IPMN samples but not in benign pancreatic diseases (p = 0.0277). CONCLUSIONS: CDO1 promoter hypermethylation is extremely specific to IPMN and may accumulate with IPMN tumor progression during the adenoma-carcinoma sequence. It might be a promising candidate as a diagnostic marker of pancreatic cystic diseases.

Nodal induces apoptosis and inhibits proliferation in ovarian endometriosis-clear cell carcinoma lesions.

BACKGROUND: Expression of Nodal, a member of the TGF-ß superfamily, is commonly absent in differentiated tissues, while its re-expression occurs in a variety of human malignancy. However, little is known about its involvement in ovarian tumorigenesis. Herein, we focused on the functional roles of Nodal in ovarian endometriosis-carcinoma lesions. METHODS: Regulation and function of Nodal and its associated molecules, including Smad2, GSK-3ß, and several cell kinetics-related molecules, were assessed using clinical samples consisting of 108 ovarian carcinomas and 33 endometriotic lesions, as well as ES-2 (ovarian clear cell carcinoma; OCCCa) and Ishikawa (endometrial carcinoma) cell lines. RESULTS: Nodal expression was significantly higher in endometriosis and OCCCa lesions as compared to that of non-OCCCas, with positive correlations to phosphorylated forms of both Smad2 (pSmad2) and GSK-3ß. When compared to endometriotic lesions, the expression of Nodal and pSmad2 was significantly decreased in OCCCa. Treatment of Ishikawa cells with TGF-ß1 resulted in transcriptional upregulation of Nodal, along with increased pSmad2 expression, while inhibition of GSK-3ß also induced an increase in Nodal expression at the posttranslational level. Both ES-2 and Ishikawa cells stably overexpressing Nodal had increased susceptibility to apoptosis in response to treatment with cisplatin and doxorubicin, respectively, together with higher cleaved caspase-3 expression and decreased Bcl2/Bax ratio. Moreover, the stable Nodal-overexpressing cells showed reduced cell proliferation, along with increased expression of p27kip1 and p21waf1. In clinical samples, a significantly higher number of apoptotic cells and lower Ki-67 labeling indices were observed in Nodal-positive as compared to Nodal-negative OCCCa. CONCLUSIONS: These findings suggest that Nodal is a multifunctional cytokine involved in the modulation of cell kinetics in ovarian endometriosis-OCCCa lesions.

A functional role of LEFTY during progesterone therapy for endometrial carcinoma.

BACKGROUND: The left-right determination factor (LEFTY) is a novel member of the TGF-ß/Smad2 pathway and belongs to the premenstrual/menstrual repertoire in human endometrium, but little is known about its functional role in endometrial carcinomas (Em Cas). Herein, we focused on LEFTY expression and its association with progesterone therapy in Em Cas. METHODS: Regulation and function of LEFTY, as well as its associated molecules including Smad2, ovarian hormone receptors, GSK-3ß, and cell cycle-related factors, were assessed using clinical samples and cell lines of Em Cas. RESULTS: In clinical samples, LEFTY expression was positively correlated with estrogen receptor-α, but not progesterone receptor (PR), status, and was inversely related to phosphorylated (p) Smad2, cyclin A2, and Ki-67 levels. During progesterone therapy, expression of LEFTY, pSmad2, and pGSK-3ß showed stepwise increases, with significant correlations to morphological changes toward secretory features and decreased Ki-67 values. In Ishikawa cells, an Em Ca cell line that expresses PR, progesterone treatment reduced proliferation and induced increased expression of LEFTY and pGSK-3ß, although LEFTY promoter regions were inhibited by transfection of PR. Moreover, inhibition of GSK-3ß resulted in increased LEFTY expression through a decrease in its ubiquitinated form, suggesting posttranslational regulation of LEFTY protein via GSK-3ß suppression in response to progesterone. In addition, overexpression or knockdown of LEFTY led to suppression or enhancement of Smad2-dependent cyclin A2 expression, respectively. CONCLUSION: Upregulation of LEFTY may serve as a useful clinical marker for the therapeutic effects of progesterone for Em Cas, leading to inhibition of tumor cell proliferation through alteration in Smad2-dependent transcription of cyclin A2.

Identification of LEFTY as a molecular marker for ovarian clear cell carcinoma.

To identify proteins involved in ovarian clear cell carcinoma (OCCCa), shotgun proteomics analysis was applied using formalin-fixed and paraffin-embedded samples of ovarian carcinoma. Analysis of 1521 proteins revealed that 52 were differentially expressed between four OCCCa and 12 non-OCCCa samples. Of the highly expressed proteins in OCCCa, we focused on left-right determination factor (LEFTY), a novel member of the transforming growth factor-ß superfamily. In 143 cases of ovarian epithelial carcinoma including 99 OCCCas and 44 non-OCCCas, LEFTY expression at both mRNA and protein levels was significantly higher in OCCCas compared with non-OCCCas, with the mRNA expression of LEFTY1 being predominant compared to that of LEFTY2. OCCCa cells stably overexpressing LEFTY1 showed reduced cell proliferation, along with decreased pSmad2 expression, and also either displayed an activated p53/p21waf1 pathway or increased p27kip1 expression, directly or indirectly. Moreover, the treatment of stable cell lines with cisplatin led to increased apoptotic cells, together with the inhibition of protein expression of a pSmad2-mediated X-linked inhibitor of apoptosis and a decreased bcl2/bax ratio. Blocking LEFTY1 expression with a specific short hairpin RNA inhibited cisplatin-induced apoptosis, probably through the increased expression of both XIAP and bcl2, but not bax. In clinical samples, a significantly higher number of apoptotic cells and lower Ki-67 labeling indices were observed in OCCCas with a high LEFTY score relative to those with a low score. These findings suggest that LEFTY may be an excellent OCCCa-specific molecular marker, which has anti-tumor effects in altering cell proliferation and cellular susceptibility to apoptosis.

BRCAness and Prognosis in Triple-Negative Breast Cancer Patients Treated with Neoadjuvant Chemotherapy.

BRCAness is defined as the set of traits in which BRCA1 dysfunction, arising from gene mutation, methylation or deletion, results in DNA repair deficiency. In the present study, we addressed BRCAness, therapeutic efficacy, recurrence, and survival in patients with triple negative breast cancer (TNBC) who were treated with neoadjuvant chemotherapy at Kitasato University Hospital, Japan, between April 2006 and October 2012. BRCAness was determined by preoperative core needle biopsy (CNB) specimens and surgical specimens. Assay was performed using Multiplex Ligation-dependent Probe Amplification (MLPA) with P376-B2 BRCA1ness probemix (MRC-Holland, Amsterdam, The Netherlands). The relative copy number ratio of each sample was compared to Human Genomic DNA (Promega, Madison, WI, USA) as reference samples was calculated with Coffalyser.NET default settings. The BRCAness score was calculated with the relative copy number ratio of various DNA sequences. Values of 0.5 or more were determined as the BRCA1-like Type (BRCAness) and those of less than 0.5 as the Sporadic Type to analyze pathological complete response (pCR) rate, recurrence, and survival. pCR (ypT0/Tis/N0) was observed in 15 patients (pCR rate: 37.5%). These patients had no recurrence. Twelve patients recurred, 8 died from breast cancer. The BRCA1-like Type were 22 and Sporadic Type were 18 in CNB specimens. No major differences were observed between the BRCA1-like Type and Sporadic Type with pCR rate, recurrence rate and survival. Twenty four surgical specimens of non-pCR patients were available and 9 were BRCA1-like Type, who had more recurrences (7/9 vs. 5/15), and their relapse-free survival was also lower (p<0.05) than that of Sporadic Type. Seven BRCA1-like Type patients remained BRCA1-like Type in surgical specimens, were worse in recurrence (p<0.01) and survival (p<0.05) compared with 6 patients whose BRCA status in surgical specimens turned to Sporadic Type. New clinical trials assessing the true recurrence (TR) rate of BRCA-type patients are expected since neither platinum-containing drugs nor poly (ADP-ribose) polymerase (PARP) inhibitors are effective against tumors with nonfunctional BRCA genes.

Distinct ß-catenin and PIK3CA mutation profiles in endometriosis-associated ovarian endometrioid and clear cell carcinomas.

OBJECTIVES: We focused on the differences in molecular mechanisms in the early stages of endometriosis-associated ovarian endometrioid carcinoma (OEMCa) and ovarian clear cell carcinoma (OCCCa). METHODS: Alterations in the ß-catenin and PIK3CA genes, as well as expression of their associated markers, were investigated. RESULTS: Mutations in exon 3 of the ß-catenin gene were identified in 21 (60%) of 35 OEMCas. The mutations were also detected in the coexisting nonatypical (52.4%) and atypical (73.3%) endometriosis, and the single-nucleotide substitutions were identical in most cases. In contrast, the mutations were not identified in any of the OCCCas and their coexisting endometriosis. PIK3CA mutations were observed in 11 (31.4%) of 35 OEMCas and 10 (35.7%) of 28 OCCCas. Ten of 11 OEMCas had PIK3CA mutations in exon 9, and eight of 10 OCCCas had them in exon 20. The same mutations were also detected in the coexisting nonatypical and/or atypical endometriosis in three OEMCas and four OCCCas. In addition, significant differences in the expression of pAkt, hepatocyte nuclear factor 1ß, hypoxia-inducible factor 1α, p65, and inducible nitric oxide synthase were evident between the two types of tumors and their coexisting endometriosis. CONCLUSIONS: Distinct molecular events may occur in relatively early stages of tumorigenesis of endometriosis-associated OEMCas and OCCCas.

Transcriptional upregulation of HNF-1ß by NF-κB in ovarian clear cell carcinoma modulates susceptibility to apoptosis through alteration in bcl-2 expression.

Hepatocyte nuclear factor-1ß (HNF-1ß) is a transcriptional factor that has an important role in endometriosis-ovarian clear cell carcinoma (OCCC) sequence by modulating cell kinetics and glucose metabolism. However, little is known about the detailed molecular mechanisms that govern its regulation and function. Herein, we focus on upstream and downstream regulatory factors of HNF-1ß in OCCCs. In clinical samples, HNF-1ß expression was positively correlated with the active form of NF-κB/p65 in OCCCs, and closely linked with a low nuclear grade and non-solid architecture. In cell lines, transfection of p65 resulted in increased HNF-1ß mRNA and protein expression in TOV-21G cells (OCCC cell line with endogenous HNF-1ß expression), in line with activation of the promoter, probably through interacting with the basic transcriptional machinery. Suppression of endogenous HNF-1ß expression by siRNA increased apoptosis in TOV-21G cells, while treatment of Hec251 cells (endometrial carcinoma cell line with extremely low endogenous HNF-1ß expression) stably overexpressing exogenous HNF-1ß with doxorubicin abrogated apoptosis of the cells, along with increased ratio of bcl-2 relative to bax. Moreover, overexpression of HNF-1ß led to upregulation of bcl-2 expression at the transcriptional level in TOV-21G cells, which provided evidence for a positive correlation between HNF-1ß and bcl-2 expression in OCCCs. These data, therefore, suggest that association between HNF-1ß and NF-κB signaling may participate in cell survival by alteration of apoptotic events, particularly in mitochondria-mediated pathways, through upregulation of bcl-2 expression in OCCCs.

Usefulness of MRI of microcalcification lesions to determine the indication for stereotactic mammotome biopsy.

BACKGROUND: With the recent rise in mammography (MMG) screenings there has been an increase in the identification of microcalcifications without lump. Therefore, a vacuum-assisted needle biopsy under stereotactic guidance (ST-MTB) is frequently performed for diagnosis. However, ST-MTB is a highly invasive examination. In this study, we investigated the effectiveness of utilizing contrast-enhanced magnetic resonance imaging (MRI) to differentiate between benign and malignant category 3 (C3) calcifications. MATERIALS AND METHODS: One hundred and sixty-eight patients with microcalcifications underwent contrast-enhanced MRI prior to ST-MTB in our hospital. Their MRI scans were reviewed to determine whether the contrast-enhanced MRI findings were consistent. We calculated the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of contrast-enhanced MRI. RESULTS: No malignancy was not found in the 51 of the 168 cases analyzed by MRI. The calculated sensitivity, specificity, PPV and NPV of contrast-enhanced MRI were 84%, 82%, 58% and 95%, respectively. CONCLUSION: Contrast-enhanced MRI for Category 3 calcified lesions would be a useful diagnostic tool for identifying ST-MTB-indicated patients.

[Improvised surgical technique for elderly women with advanced breast cancer accompanied by extensive skin invasion].

Patient 1 was a 63-year-old woman whose chief complaint was a mass in the left breast. Physical examination revealed an inverted left nipple, a very large mass on the anterior aspect of the sternum, and erythema. Because the tumor had directly invaded the sternum, T4cN3M0, stage IIIC breast cancer was diagnosed. The patient preoperatively received chemotherapy with 6 courses of FEC100 (5-fluorouracil, epirubicin, and cyclophosphamide) and 5 courses of nanoparticle albumin -bound paclitaxel (260 mg/m2), which enabled a partial response. Patient 2 was an 83-year-old woman whose chief complaint was a mass in the upper internal and external quadrants of the right breast measuring 20×15 cm and erythema. The mass was accompanied by enlarged right axillary lymph nodes(T4bN1M0, stage IIIB breast cancer). Both patients underwent core needle biopsy of the skin and breast masses. They were both diagnosed with invasive, lobular, triple-negative breast cancer (estrogen receptor negative, progesterone receptor negative, human epidermal growth factor receptor 2 negative). The surgical resection line was drawn to include the extensive skin invasion, and mastectomy and axillary dissection were performed. Skin grafting was scheduled but the retromammary space on the healthy side was dissected to the anterior border of the latissimus dorsi muscle, and the skin of the healthy side was used to cover the defect on the affected side. Consequently, the pendulous breast on the healthy side was elevated. This surgical technique provided an excellent aesthetic outcome without any skin problems, because autologous skin was used to fill the defect. Radiotherapy could subsequently be administered as scheduled. This procedure may be useful for elderly patients.

Mixed angiosarcoma, clear cell adenocarcinoma and mature teratoma elements in an ovarian tumor: a case report and literature review.

Malignant transformation of a mature teratoma in the ovary is a rare event, with an approximate rate of only 1-2%. Here, we report an ovarian tumor with a unique combination of epithelial and non-epithelial malignant components, including mature teratoma elements. A 59 year-old postmenopusal woman underwent total hysterectomy and bilateral salpingo-oophorectomy to remove a huge solid mass of the right ovary. The ovarian tumor was 16 × 12 × 4.5 cm in dimensions, composed of red-brown and greyish-white tissue with several cystic areas. Microscopically, atypical cells immunopositive for both CD31 and CD34 formed irregular ectatic vascular patterns with a high MIB-1 labeling index in red-brown areas. In contrast, tubule-cystic and papillary structures were lined by HNF-1ß-immunopositive atypical cuboidal and hobnail cells with clear cytoplasm in greyish-white areas. In addition, normal-looking epithelial and stromal components, including mature squamous, cuboidal and ciliated epithelial cells, and adipose tissues, were observed in red-brown areas, suggesting an ovarian tumor combining angiosarcoma, clear cell adenocarcinoma, and mature teratoma features. We could demonstrate identical X-chromosome inactivation patterns among all three components by human androgen receptor gene (HUMARA) assays, pointing to complex inter-relationships regarding their pathogenesis. These observations suggest that a malignant tumor composed of two characteristic phenotypes arose in mature teratoma.

Nuclear survivin is associated with cell proliferative advantage in uterine cervical carcinomas during radiation therapy.

BACKGROUND: Although the anticancer effects of radiation therapy for patients with uterine cervical squamous cell carcinoma (U-SCC) are widely acknowledged, little is known about the resultant morphological alterations in tumour tissue kinetics. AIMS: To make a detailed assessment of possible roles of survivin expression in apoptosis and cell proliferation in U-SCC during radiation therapy. METHODS: 181 biopsy specimens from 55 consecutive U-SCCs of patients receiving radiation therapy were studied using a combined morphological (apoptosis) and immunohistochemical (MIB-1 and survivin) approach. The intracellular distribution of various splice variants of the survivin gene was also examined. RESULTS: Tumour cell proliferation, determined as MIB-1 labelling indices (LIs), as well as nuclear survivin (N-Surv) LIs, were inversely correlated with irradiation dosage, in contrast to relatively minor changes in apoptotic indices, suggesting a shift in tumour tissue kinetics towards a relative predominance of cell deletion. In addition, the low N-Sur LI category showed significant stepwise decrease in MIB-1 LIs during therapy, in contrast to no changes in the high category. Exogenous overexpression of three variants of the survivin gene resulted in different expression patterns, showing cytoplasmic staining with or without dot formation for survivin and survivin-2B and distinct nuclear accumulation for survivin-deled exon 3 (Ex3). CONCLUSIONS: Results showed that nuclear survivin, including survivin itself and the survivin-Ex3 splice variants, may participate in modulation of altered cell kinetics of U-SCC during radiation therapy.

[A rare case of huge triple-negative of accessory breast cancer enlarged during the course of chemotherapy].

A 38-year-old premenopausal woman with a right axillary mass was told that she had an atheroma. The mass enlarged. An infectious atheroma was suspected, and incision and drainage were performed. A mass 4 cm in diameter was palpated in the right axillary region. Ultrasonography showed a mass 6 cm in diameter, and magnetic resonance imaging revealed a hypervascular mass at the same site. Stage IIIB triple-negative of accessory breast cancer (T4bN1M0) was diagnosed. The patient received four courses of FEC 100 (5-fluorouracil, epirubicin, and cyclophosphamide) as preoperative chemotherapy, but progressive disease was diagnosed, and the regimen was switched to weekly paclitaxel. The tumor became ulcerated and friable, and the hemoglobin level fell to 6 g/dL. Emergency surgery was thus performed. Postoperatively, the patient received six courses of chemotherapy with paclitaxel plus gemcitabine. Accessory breast cancer is extremely rare, but should be borne in mind when an axillary mass is encountered. In our patient, progressive disease had developed during the preoperative chemotherapy for accessory breast cancer. Although treatment was switched to a different regimen, bleeding was detected, and the patient underwent a semi-emergency surgery.

CD3- and CD4-positive plasmablastic lymphoma: a literature review of Japanese plasmablastic lymphoma cases.

Plasmablastic lymphoma (PBL) is a very rare and recently-described subtype of diffuse large B-cell lymphoma. A maxillary tumor in an 84-year-old HIV-negative Japanese-man was referred. The biopsied specimen showed a diffuse proliferation of mature plasma cells, expressing CD3 (+), CD4 (+), CD20 (-), CD138 (+) and EBER (+) by immunohistochemistry. He was diagnosed as a plasmablastic lymphoma; radiation therapy (RT) was started, but the response to the RT was only a partial response. To our knowledge, this is the first report of a patient with PBL expressing CD3 and CD4.

[A long-term survival case of progressive breast cancer detected in gastric metastasis].

A 51-year-old postmenopausal woman was diagnosed as having adenocarcinoma (gastric cancer type 4) from gastric biopsy by upper endoscopy. Her chief complaint was abdominal dilatation. Meanwhile, a breast CT suggested tumor in her left breast and was diagnosed as an invasive lobular carcinoma based on a core needle biopsy. After gastric biopsy, tissues are stained by ER and PgR in immunohistochemistry. The diagnosis was modified from gastric cancer to T2N1M1, stage IV left breast cancer, accompanied by a treatment. Chemotherapy with EC 6 course consisted of a weekly PTX 4 course (epirubicin, cyclophosphamide-weekly paclitaxel) was performed. After the chemotherapy, breast mass, ascites and tumor marker were dramatically improved. Then hormonal therapy was administered. She passed away 2 and 1/2 years after her first visit to the hospital. Metastatic gastric tumors simulating type 4 advanced gastric cancer (MGTS type 4) and invasive lobular carcinoma are known to have an unfavorable prognosis. There is no doubt, however, that the multidisciplinary treatments have brought a satisfaction to her and family. We should keep in mind a possibility of gastric metastasis of breast cancer, when consulting a female patient with gastric cancer type 4.

Significant increase of colonic mutated crypts correlates with age in sporadic cancer and diverticulosis cases, with higher frequency in the left- than right-side colorectum.

Mild periodic acid-Schiff (mPAS) staining can discriminate non-O-acetylated (mPAS-positive) from O-acetylated (mPAS-negative) epithelial sialoglycoproteins in human colonic mucosa, allowing the three haplotypes expressed from a single polymorphic autosomal gene (oat) to be distinguished. In heterozygotes, we previously demonstrated wholly mPAS-positive (stem cell mutated) crypts and clusters of two or more mPAS-positive crypts to be significantly increased with duration of ulcerative colitis. To establish whether such an increase in the number of mutated crypts with age also occurs in normal individuals or in cases with diverticulosis, the O-acetylation phenotype in the non-cancerous colonic mucosa of 47 sporadic colorectal cancer patients who were heterozygotes for oat was tested with mild-PAS staining. PAS-positive crypts were assessed histologically in relation to age and compared between the left (sigmoid colon and rectum) and right (cecum and ascending colon) sides of the colorectum. Wholly mPAS-positive (stem cell mutated) crypts and foci in heterozygotes were found to be increased significantly (P < 0.0001) in the left side with aging (r = 0.598 and 0.643, respectively). Such a positive correlation with aging was also confirmed in 19 diverticulosis cases without cancer (r = 0.797 and 0.793, respectively). The frequency of mutated crypts and foci on the right side was significantly lower than on the left side in both spontaneous colorectal cancer and diverticulosis cases. The results provide support for an intimate relationship between accumulation of mutated crypts with aging, possibly with significance for colorectal cancer development. Furthermore, the environment in the right side of the colon may be different from that in the left side in this regard.

Immunohistochemical separation of follicular variant of papillary thyroid carcinoma from follicular adenoma.

The accurate diagnosis of differentiated thyroid tumors is very important for clinical management of patients. The histopathological distinction between some types of differentiated thyroid tumors can be very difficult even for experienced pathologists. We used immunohistochemical markers from published data obtained from DNA expression profiling, tissue microarray analysis, and immunohistochemistry to analyze a series of 157 thyroid tumors and 5 normal thyroids. These analyses showed that several antibodies were useful in distinguishing follicular adenomas from follicular variant of papillary thyroid carcinomas including HBME-1, CITED1, galectin-3, cytokeratin 19, and S100A4 (p < 0.0001). A combination of markers consisting of a panel of HBME-1, galectin-3, and CK19 or a panel of HBME-1, CITED1, and galectin-3 was usually most effective in distinguishing follicular adenoma from follicular variant of papillary thyroid carcinoma. Because individual tumors may not express some of these markers, the use of a panel of antibodies is recommended. These results indicate that some individual antibodies or a panel of antibodies combined with histopathological analysis can be useful in separating follicular adenoma (FA) from follicular variant of papillary thyroid carcinoma (FVPTC).

Galectin-3 Expression in Functioning and Silent ACTH-Producing Adenomas.

Galectin-3 (Gal-3), a beta galactoside-binding protein, has been implicated in a variety of biological functions including cell growth, differentiation, tumor cell adhesion, angiogenesis, tumor progression, and metastasis. We recently reported that Gal-3 was expressed in a subset of normal pituitary cells and tumors including PRL, ACTH, and in folliculo-stellate (FS) cells and tumors and that Gal-3 had an important regulatory role in pituitary cell proliferation. We further investigated the expression of Gal-3 protein in ACTH- and PRL-producing tumors and the expression of various galectin mRNAs by RT-PCR in pituitary adenomas and normal pituitary. Most silent ACTH subtypes 1 and 2 adenomas were negative or only focally positive for Gal-3 expression compared to functioning ACTH tumors from patients with Cushing's disease and Nelson's syndrome. In the normal pituitary, Gal-3 was expressed in less than 1% of the basophil-invading cells (ACTH cells present in the posterior pituitary) and in a subset of the anterior lobe ACTH-positive cells. RT-PCR analyses showed that many members of the galectin family including galectins 1, 2, 3, 4, 5, 6, 7, 8, and 9 were expressed in normal pituitary and in functioning ACTH- and PRL-producing tumors. These results indicate that Gal-3 is associated with functioning ACTH and PRL tumors and is expressed infrequently in silent ACTH adenomas, suggesting that Gal-3 protein and/or gene is altered in non-functioning ACTH tumors. The use of ACTH and Gal-3 immunostaining should help in the diagnosis of silent ACTH adenomas.

RASSF1A and NORE1A methylation and BRAFV600E mutations in thyroid tumors.

We analyzed RASSF1A and NORE1A methylation and BRAF mutation in 89 thyroid tumors, 42 non-neoplastic thyroid tissues and three thyroid tumor cell lines using polymerase chain reaction (PCR), methylation-specific PCR, Western blotting and DNA sequencing in order to study thyroid tumor pathogenesis and progression. RASSF1A promoter methylation was present in all three thyroid cell lines and in 27/78 (35%) of benign and malignant thyroid tumors. We showed for the first time that there was generally good agreement between RASSF1A methylation status and RASSF1A protein expression. We also examined for the first time NORE1A promoter region methylation in thyroid cell lines and primary tumors and showed that two of three thyroid cell lines were methylated in the NORE1A promoter region, while all primary thyroid tumors analyzed (n=51) were unmethylated. BRAF mutation was present in 38% of papillary thyroid carcinomas (PTC), including 20% of PTC with a follicular variant pattern and 67% of the tall cell variant of PTC. Hyalinizing trabecular tumors (n=23), which had nuclear features similar to PTC, did not have BRAF mutations, indicating that the presence of BRAF mutations can help to separate these two tumor types. Phospho-MEK expression was increased in the NPA cell line, which had a BRAF mutation, supporting the importance of the BRAF pathway alterations in PTC pathogenesis. These results indicate that RASSF1A epigenetic changes are an early event in thyroid tumor pathogenesis and progression and that NORE1A methylation is uncommon in primary thyroid tumors. BRAF mutation occurs later in thyroid tumor progression and is restricted mainly to PTC and anaplastic thyroid carcinoma.

Cyclooxygenase-2 and thromboxane synthase in non-endocrine and endocrine tumors: a review.

Prostaglandins (PG) are members of a large group of hormonally active fatty acids derived from free fatty acids. They are formed from arachidonic acid-the major PG precursor. Cyclooxygenase (COX)-1 and -2 are the rate-limiting steps in PG synthesis. COX-2 is overexpressed in many human non-endocrine and endocrine tumors including colon, breast, prostate, brain, thyroid, and pituitary. COX-2 has an important role in angiogenesis and tumor growth. Thromboxane synthase (TS) catalyzes the synthesis of thromboxane A2 (TXA2), which is derived from arachidonic acid and prostaglandin H2 and is a vasoconstrictor and inducer of platelet aggregation. TXA2 stimulates tumor growth and spread of some tumors and TS appears to have a critical role in tumorigenesis in some organ systems. In this review, we examine the role of COX-2 and TS in various non-endocrine tumors, especially colon, breast, prostate, and brain as well as in endocrine tumors. The accumulating evidence points to an increasingly important role of COX-2 and TS in tumor progression and metastasis.