Value of thyroid specific peroxidase and Ki-67 stains in preoperative cytology for thyroid follicular tumors.

BACKGROUND: The aim of this study was to elucidate immunocytochemically whether thyroid specific peroxidase (TPO) and Ki-67 can complement fine-needle aspiration (FNA) cytology as useful markers in order to distinguish between follicular adenoma (FA) and follicular carcinoma (FC). METHODS: We studied 40 FAs and 68 FCs obtained by surgical resection. FNA cytology smears were divided into two groups: Cytology-A (Cy-A) (94 cases) with typical benign cytology and Cytology-B (Cy-B) (14 cases) with atypical cytology. FCs were divided into two groups: FC-I (42 cases) without any poorly differentiated structures and FC-II (26 cases) with some poorly differentiated structures. Cytology smears and histology from FAs and FCs were studied immunocytochemically for thyroid specific peroxidase (TPO) and Ki-67. RESULTS: TPO expression was negative in 12.5% FAs, 21.4% FC-I, and 46.2% FC-II. In 68 FC cases, Cy-B were more frequently observed in TPO-negative cases (38.1%) than in TPO-positive cases (12.8%). The mean Ki-67 LI was 0.46 in FAs, 0.53 in FC-I, and 1.13 in FC-II. The high Ki-67 LI was correlated with Cy-B. Moreover, higher Ki-67 LI showed a close relationship with distant metastasis. In 94 Cy-A cases, 54 cases were FCs. When 38 cases with negative TPO or Ki-67 LI over 0.62 were extracted from them, as many as 28 cases were FCs, the rate of FCs were significantly higher than the rest. CONCLUSION: Therefore, addition of TPO stain and Ki-67 stain to routine Papanicolaou stain could improve the diagnostic reliability of FNA cytology for FC with high degree of malignancy.

Synovial sarcoma arising in the vulva cytogenetically confirmed by SYT break-apart rearrangement fluorescence in situ hybridization: A case report and discussion of diagnostic methods.

Synovial sarcoma (SS) is a soft tissue sarcoma of unknown histogenesis that rarely occurs in the female genital tract. We report a case of SS occurring in the right vulva of a young Japanese female. The tumor was composed of poorly differentiated rounded cell areas, surrounded by fibroblastic spindle-shaped cell areas. Immunohistochemically, the tumor cells were focally positive for cytokeratin, vimentin, CD99, Bcl-2 and neuron-specific enolase. The tumor was suspected, but was difficult to confirm as it was an SS based solely on light-microscopic and immunohistochemical findings. Although reverse transcription polymerase chain reaction (RT-PCR) failed to detect SS-specific SYT-SSX fusion gene transcripts using an RNA sample extracted from the formalin-fixed paraffin-embedded tumor tissue, SYT break-apart rearrangement fluorescence in situ hybridization (SYT bar-FISH) successfully confirmed our diagnosis of SS for the tumor. Thus, SYT bar-FISH may be more suitable for the purpose of the molecular diagnosis of SS than conventional RT-PCR when using archival formalin-fixed paraffin-embedded tissue specimens.

Immunohistochemical examination of Aκ amyloidosis with antibody against adjacent portion of the carboxy terminus of immunoglobulin kappa light chain.

For the purpose of investigating the carboxy terminus distribution of immunoglobulin κ light chain in Aκ amyloid deposits in tissue sections, we examined the immunostaining pattern of Aκ amyloidosis with conventional rabbit clonal antibody against peptide derived from the C-terminal sequence of human κ light chain. This antihuman kappa light chain clone II (clone H16-E) reacted with the adjacent region of the C terminus of the κ light chain constant region in SPOT analysis. Immunohistochemically, this antibody reacted with amyloid deposits in all 18 cases of Aκ amyloidosis. In 15 cases, this antibody reacted with amyloid deposits almost uniformly. In this study, we demonstrated for the fi rst time that the peptides adjacent to the carboxy terminus of immunoglobulin κ light chain or full-length κ light chain were constituents of Aκ amyloidosis, and these molecules were distributed uniformly in almost all cases of Aκ amyloidosis in tissue sections.

Combined evaluation of CK5/6, ER, p63, and MUC3 for distinguishing breast intraductal papilloma from ductal carcinoma in situ.

The differentiation of intraductal papilloma (IDP) in the breast from ductal carcinoma in situ (DCIS) is sometimes difficult. Fifty papillary lesions (25 DCIS and 25 IDP) were immunohistochemically examined using a panel of antibodies, including CK5/6, ER, p63, Ki-67, chromogranin A, synaptophysin, neuron specific enolase, CD56, MUC1, MUC3, CD44, p21, p27, and p53. The immunohistochemical staining pattern of each antibody was evaluated using the Allred scoring system. Then, the area under curve (AUC) for each antibody was computed by receiver operating characteristic (ROC) analysis. DCIS typically showed high scores for ER and MUC3 reactivity compared with IDP, and the AUC for ER and MUC3 were 0.941 and 0.908, respectively. In contrast, IDP showed high scores for CK5/6 and p63 reactivity compared with DCIS, and the AUC for CK5/6 and p63 were 1.00 and 0.954, respectively. We devised a 'Differential Index' (DI) using the following formula: [S(ER) + S(MUC3)]/[S(CK5/6) + S(p63) + 1]. The distributions of the DI for IDP and DCIS did not overlap when the cutoff value was placed arbitrarily at DI = 1.0. From these results, it is concluded that a panel of four CK5/6, ER, p63, and MUC3 antibodies provide valuable information for differentiating IDP from DCIS.

Long-term follow-up after lamellar keratoplasty in a patient with bilateral idiopathic corneal keloid.

PURPOSE: To report a case of bilateral idiopathic corneal keloid. METHODS: Retrospective review of clinical features, histopathological findings, clinical management, and outcome. RESULTS: A 2-year-old boy with bilateral corneal keloid was treated with lamellar keratoplasty and tranilast eye drops. Peripheral localized white corneal nodules had been present bilaterally since the age of approximately 6 months in the absence of any history of trauma, inflammatory disease, or relevant family history. Pathological examination of the excised corneal buttons revealed myofibroblast proliferation (positive staining for α-smooth muscle actins), a haphazard arrangement of collagen bundles, and the absence of inflammatory cells. On the basis of these findings, a diagnosis of corneal keloid was assigned. The size of the corneal lesion in the right eye decreased in response to therapy with tranilast eye drops. Lamellar keratoplasty resulted in improved bilateral visual acuity, which was maintained at the 12-year follow-up. CONCLUSIONS: This report describes a very rare case of bilateral corneal keloid in the absence of trauma or inflammation that was diagnosed by histological and immunohistochemical examination and electron microscopy. Good visual acuity was maintained over an extended period of postsurgery follow-up. Tranilast may represent a novel adjuvant therapy for corneal keloid.

9p21 index as estimated by dual-color fluorescence in situ hybridization is useful to predict urothelial carcinoma recurrence in bladder washing cytology.

Recent studies have shown that chromosome 9p21 locus is frequently deleted in the early stages of urothelial carcinogenesis. To study the predictive value of the 9p21 aberrations in recurrence of urothelial carcinoma of the urinary bladder, we applied dual-color fluorescence in situ hybridization for 9p21 and chromosome 9 centromere to the bladder washing cytology samples that were obtained from the patients with urothelial carcinoma of the urinary bladder treated by transurethral resection. For the evaluation, the 9p21 index was defined as the ratio of the mean number of 9p21 signals per nucleus for that of the chromosome 9 centromere signals per nucleus in each of the bladder washing cytology samples. The 9p21 index values of the bladder washing cytology samples with no (G0) cytologic atypia were significantly higher than those of the bladder washing cytology samples with moderate (G2) (P < .01) and severe (G3) (P < .001) cytologic atypia, but the index values did not statistically differ from those of the bladder washing cytology samples with mild (G1) cytologic atypia. Recurrence-free survival in the patients with a low 9p21 index value (<0.9) was significantly poorer in comparison with the patients with a high 9p21 index value (>0.9). Furthermore, 2 patients of bladder washing cytology G1 with a low 9p21 index value recurred much sooner than the other patients of the bladder washing cytology G1 category. These findings indicate that a decreased 9p21 index value is associated with recurrence of urothelial carcinoma of the urinary bladder, and the 9p21 index may be useful as a marker to identify patients with elevated risk of recurrence of urothelial carcinoma of the urinary bladder.

Tranilast, an anti-allergic drug, down-regulates the growth of cultured neurofibroma cells derived from neurofibromatosis type 1.

Neurofibromas are benign tumors that comprise primarily of Schwann cells and fibroblasts. Mast cells have been found scattered in the tumor tissue, and their role in promoting the proliferation of neurofibroma has been suggested. Tranilast (N-[3,4-dimethoxycinnamolyl]anthranilic acid) is an anti-allergic drug that inhibits release of the chemical mediators from mast cells and it used for the treatment of keloids and hypertrophic scars by its inhibition of growth-promoting transforming growth factor (TGF)-beta(1) from fibroblasts. We assumed that tranilast would suppress neurofibroma cell growth. In order to prove this hypothesis, we investigated the effectiveness of tranilast in inhibiting the tumor growth using a new cell culture system obtained from patients with neurofibromas. We called this culture system with the mixture of Schwann cells and fibroblasts "NF1 cells culture". Mast cells were differentiated from CD34(+) peripheral blood mononuclear cells of normal healthy subjects, and were co-cultured with NF1 cells. Three days after tranilast (10 approximately 100 microM) added to the culture dishes, we counted viable cell numbers and measured the concentrations of TGF-beta(1), stem cell factor (SCF) and tryptase, which exists in the histamine granule, in the culture medium. Tranilast significantly suppressed proliferation of the NF1 cells and lowered the levels of TGF-beta(1), SCF and tryptase. These results suggest that tranilast retards tumor proliferation through not only suppression of cell growth factor, but also the inhibition of a chemical mediator released from mast cells. Thus, tranilast can be a potent therapeutic agent to inhibit the growth of neurofibromas.

Risk of concomitant carcinoma in situ determining biopsy candidates among primary non-muscle-invasive bladder cancer patients: retrospective analysis of 173 Japanese cases.

OBJECTIVES: To determine candidates for bladder biopsies among Japanese primary non-muscle-invasive bladder cancer patients according to the risk of concomitant carcinoma in situ (CIS). METHODS: Between January 1992 and August 2006, 173 primary non-muscle-invasive bladder cancer cases underwent transurethral resection of the bladder tumor with bladder biopsies for the detection of CIS. Correlations between biopsy results and preoperative/pathological features were retrospectively analyzed. RESULTS: Positive cytology was statistically associated with the presence of concomitant CIS in multivariate analysis (P < 0.01). Abnormal cystoscopic appearance outside the tumor almost achieved statistical significance in multivariate analysis among preoperative factors (P = 0.06). In our series, one (12.5%) of eight low-risk, 18 (24.7%) of 73 intermediate-risk and 41 (59.4%) of 69 high-risk cases had CIS in normal-looking sites, respectively. In cases with a single papillary tumor and negative cytology, one of 16 (6.3%) had concomitant CIS in their biopsy specimens at the normal-looking sites. CONCLUSIONS: All non-muscle-invasive bladder cancer patients with positive cytology are candidates for additional random biopsies. Targeted biopsies should be performed for all suspicious areas in the bladder mucosa. Random biopsies should be considered in cases with the macroscopic types of cancer for predicting intermediate- and high-risk cancer.

Discrepancies between cytology, cystoscopy and biopsy in bladder cancer detection after Bacille Calmette-Guerin intravesical therapy.

OBJECTIVES: To evaluate discrepancies in the detection of Bacille Calmette-Guerin (BCG)-resistant bladder cancer by cystoscopy, bladder biopsy and urinary cytology. METHODS: Between January 1992 and August 2006, 127 bladder cancer patients underwent a cycle of eight weekly BCG instillations. Four weeks after the last BCG instillation, urinary cytological analysis and cystoscopy with targeted biopsy in addition to eight-nine selected-site biopsies were performed. RESULTS: Biopsy-proven cancer was found in 11/27 (40.7%), 5/42 (11.9%), and 11/58 (19.0%) of positive, suspicious, and negative cytology cases, respectively. Abnormal and normal cystoscopic findings correlated with a biopsy-proven cancer in 13/53 (24.5%) and 14/74 (18.9%) cases, respectively. The combination of a macroscopic cystoscopic suspicion and a positive cytology missed malignant cases in 15.9% of the cases. In 100 cases without biopsy-proven cancer, the rates of denuded urothelium at biopsy in the cases with positive and non-positive cytology were 7/16 (43.8%) and 16/84 (19.0%), respectively. CONCLUSIONS: According to our study, routine biopsy is recommended in the evaluation of BCG treatment, even if the timing, limitations and disadvantages of the procedure should be taken into account.

Influence of cytological stains on comparative genomic hybridization analysis for DNA extracted from cytological smears.

In the present study, we investigated the influence of cytological stains in analyzing DNA extracted from cytological slides by comparative genomic hybridization (CGH). Multiple imprint cytological slides were prepared for fresh-frozen breast cancer tissue samples and the slides were stained by three staining methods for each sample. Under microscopic observation, cancer cells were selectively microdissected from the slides and forwarded to DNA extraction, whole genome amplification, and CGH analysis. CGH was successfully performed for all methylgreen-stained and May-Grunwald-Giemsa (MGG)-stained cytological smear slides, but for two Papanicolaou (PAP)-stained slides. The number of chromosomal imbalances detected were 5-10 in methylgreen-stained slides and 5-9 in MGG-stained slides. The chromosomal imbalances resemble each other between methylgreen-stained and MGG-stained slides. The present study indicates that the MGG stain is preferred to the PAP stain for the purpose of cytogenetical analysis by CGH for DNA extracted from cytological smear slides.

Comparison of three cytologic preparation methods and immunocytochemistries to distinguish adenocarcinoma cells from reactive mesothelial cells in serous effusion.

We assessed whether a panel of seven antibodies is useful in the differentiation of adenocarcinoma cells (ACCs) from reactive mesothelial cells (RMCs) in effusion samples and to determine optimal specimen preparation conditions for immunocytochemical analysis of effusion samples. Immunocytochemistry (ICC) was performed on three types of effusion preparations from the same effusion specimens: ethanol-fixed smears, ethanol-fixed cell-blocks, and formalin-fixed cell-blocks. Commercially available antibodies MOC-31, Ber-EP4, CA19-9, CEA, EMA, CA125, and HBME-1 were tested on RMCs from four samples of various etiology and 15 samples of adenocarcinoma from various primary sites. Ethanol-fixed smears showed strong immunoreactivity to all antibodies tested. The immunoreactivity of ethanol-fixed and formalin-fixed cell-blocks was significantly lower with all antibodies except CA19-9. Smear preparations are more sensitive than cell-blocks for immunocytochemical study. A panel of antibodies MOC-31, Ber-EP4, CA19-9, and CEA appears to be suitable to distinguish between ACCs and RMCs.

Acceleration of murine amyloidosis by implantation of amyloid-containing grafts.

We examined the transmissibility of amyloidosis by the implantation of amyloid-containing tissue. If the transmissibility similar to prion diseases is applicable, using amyloid-containing tissue for transplantation in humans might be a risk factor. In this study, AA amyloidosis occurred in mice that underwent implantation of AA amyloid-containing grafts to the liver and subsequent inflammatory stimulation. AApoAII amyloidosis occurred after implantation of AApoAII amyloid-containing grafts to the liver or to the subcutaneous space without inflammatory stimulation. Both types of amyloidoses occurred in the recipient mice sooner than expected. Moreover, AA and AApoAII amyloid deposits were found at 12 weeks after implantation in mice given AApoAII amyloid-containing grafts and inflammatory stimulation. These results suggest that implanted amyloid deposits have an AEF effect and that implanted amyloid-containing tissue can promote and accelerate a different type of amyloidosis. In another experiment, mice received amyloid-containing or normal tissue grafts. The degree of amyloid deposition was compared after 6 days and 5 weeks of inflammatory stimulation and when the mice were killed. There was no obvious difference in the degree of amyloid deposition between each group, indicating that the lag-time is shortened by implantation of amyloid-containing tissue, resulting in severe amyloidosis in the short term.

A case of Epstein-Barr Virus (EBV)-associated thymic carcinoid and investigation of existence of EBV-infected cells in thymus and thymic tumors.

We describe the first case of Epstein-Barr virus (EBV)-associated thymic carcinoid tumor found by in situ hybridization (ISH) on paraffin-embedded sections. ISH revealed that both tumor cells and infiltrated lymphocytes were EBV positive, while a few EBV-infected lymphocytes were detected in 2 of 11 thymuses and 1 of 11 thymomas.

Immunohistochemical study of cytokeratins in amyloid deposits associated with squamous cell carcinoma and dysplasia in the oral cavity, pharynx and larynx.

The frequency of amyloid deposits associated with squamous cell carcinoma (SCC) and dysplasia in the oral cavity, pharynx and larynx was examined. In addition, the origin of amyloid proteins by immunohistochemical staining with a panel of anticytokeratin monoclonal antibodies was investigated. Amyloid deposits were found in eight of 73 (11.0%) SCC and one of seven (14.3%) dysplasias in the oral cavity, in eight of 22 (36.4%) SCC and zero of two (0%) dysplasias in the pharynx, and in 22 of 37 (59.5%) SCC and four of 10 (40.0%) dysplasias in the larynx. Eight of 12 different cytokeratin (CK) antibodies reacted with these deposits: 34 beta E12 (CK1, -5, -10, -14) reacted with amyloid deposits in 19 of 19 cases (100%), LL002 (CK14) in eight of 18 cases (44.4%), MNF116 (CK5, -6, -8, -17) in eight of 19 cases (42.1%), D5/16B4 (CK5, -6) in five of 18 cases (27.8%), DE-K10 (CK10) in four of 17 cases (23.5%), RCK108 (CK19) in three of 18 cases (16.7%), 34 beta B4 (CK1) in three of 19 cases (15.8%) and AE8 (CK13) in two of 17 cases (11.8%). These antibodies always reacted with the cytoplasm of squamous cell lesions. Amyloid deposits in two cases contained a CK5 and CK14 pair, and in another two cases they contained both a CK5 and CK14 pair, and a CK1 and CK10 pair. Anti-CK antibodies, including OV-TL12/30 (CK7), c-51 (CK8), DC10 (CK18) and IT-Ks20.8 (CK20) did not react with the amyloid deposits. We conclude that the amyloid deposits associated with SCC or dysplasia in the oral cavity, pharynx or larynx were derived from CK of cancer cells and that some amyloid deposits might be assembled by two or more different CK.

Enhanced tumor necrosis factor- alpha expression in small sized abdominal aortic aneurysms.

Circulating levels of tumor necrosis factor alpha (TNF- alpha) are elevated in the patients with abdominal aortic aneurysm (AAA). We investigated TNF- alpha expression and cellular infiltration in the walls of AAAs of different sizes. Twenty-seven surgical specimens of AAAs were categorized according to the maximum aneurysm diameter into a small size group (less than 50 mm in diameter, n = 8; S group), a medium-sized group (50 to 59 mm in diameter, n = 11; M group), and a large size group (larger than 59 mm in diameter, n = 8; L group). The level of TNF- alpha and interleukin-1 beta(IL-1 beta) in the aneurysm wall was measured by ELISA. Immunohistochemical staining was performed to observe the TNF- alpha expression and the infiltration of macrophages and lymphocytes in aneurysm walls. Enzyme-linked immunosorbent assay showed that the level of TNF- alpha in the S group (5.47 +/- 3.48 pg/mg protein) was significantly higher ( p < 0.05) than that in the M group (2.70 +/- 1.33 pg/mg protein) or the L group (1.82 +/- 1.21 pg/mg protein). No significant difference in IL-1 beta was observed between the S, M, and L groups. Immunohistochemical analysis also showed that TNF- alpha was expressed strongly in the S group but was negative or weakly positive in the M and L groups. Furthermore, the expression of TNF- alpha was seen mainly where the aneurysm wall showed atheromatous change and macrophage infiltration. These results indicated that the expression of TNF- alpha in the aneurysm wall was enhanced in small AAAs, and this enhancement might be related to the infiltration of macrophages.

Spindle-cell ameloblastic carcinoma: A case report with immunohistochemical, ultrastructural, and comparative genomic hybridization analyses.

Spindle-cell ameloblastic carcinoma is a classification proposed for a group of rare odontogenic carcinomas with sarcomatoid components and is distinguished from odontogenic carcinosarcoma. We report a case of spindle-cell ameloblastic carcinoma of the right mandible that occurred in a 67-year-old Japanese man. Growth of the tumor was destructive, there was extensive lung metastasis, and the outcome was unfavorable. Ultrastructural and immunohistochemical examination showed the spindle-cell component of the tumor to be epithelial in character. A gain of 5q with amplification of 5q13 was detected in the tumor by comparative genomic hybridization.

[Idiopathic adrenal hematoma: a case report].

We report a rare case of idiopathic adrenal hematoma. Including our case, 13 such cases have been described in Japan. A 63-year-old [correction of 65] woman was admitted to our hospital for further examination of a right adrenal mass on ultrasonography. Laboratory tests including hormonal assay were within the normal ranges. Computed tomography showed a tumor with calcification measuring 3.0 x 2.0 cm in the right adrenal gland. Magnetic resonance imaging (MRI) revealed a mass with heterogeneous low to iso signal intensity on T1-weighted images and high signal intensity on T2-weighted images. A peripheral rim of the mass was slightly enhanced on dynamic MRI. The patient underwent laparoscopic adrenalectomy. Histopathological examination revealed an old hematoma without neoplastic cells or vascular lesions and these findings were evidence of idiopathic adrenal hematoma.

Numerical aberrations of chromosome 9 in bladder cancer. A possible prognostic marker for early tumor recurrence.

To investigate whether nonrandom aberrations of chromosomal numbers could predict tumor recurrence in patients with bladder cancer, archival urine cytology specimens (Giemsa-stained) from patients previously treated for transitional cell carcinoma of the urinary bladder were studied retrospectively by fluorescence in situ hybridization. A total of 48 patients (pTis, 6; pTa, 2: pT1, 32; and pT2-4, 8) were consecutively enrolled in this study, and numerical aberrations of chromosomes 9 and 17 were investigated. Cytology was diagnosed as negative for malignancy in 18 patients and positive in 30 patients. Twenty-seven of the 48 patients (56%) had one or more chromosomal aberrations. The frequency of numerical aberrations of chromosome 17 was correlated with increasing stage and grade, whereas loss of copies of chromosome 9 (monosomy) was frequently observed at a lower stage and grade. Chromosomal aberrations were detected in 9 (50%) of 18 patients with negative or equivocal cytology (class I, II, or III) by the Papanicolaou classification. Of eight patients with negative or equivocal cytology who developed tumor recurrence, four (50%) showed monosomy 9 and one (14%) showed a numerical aberration of chromosome 17. All six patients who showed monosomy of chromosome 9 developed tumor recurrence within 12 months, whereas four of the nine patients who did not show monosomy of this chromosome developed recurrence within 12 months (P<0.05, Fisher test). These results suggest that monosomy of chromosome 9 might be a prognostic marker for early tumor recurrence in patients with negative or equivocal cytology specimens.

Acceleration of murine AA amyloidosis by oral administration of amyloid fibrils extracted from different species.

We herein report that experimental murine amyloid A (AA) deposition is accelerated by oral administration of semipurified amyloid fibrils extracted from different species. Three groups of mice were treated with semipurified murine AA amyloid fibrils, semipurified bovine AA amyloid fibrils or semipurified human light chain-derived (A(lambda)) amyloid fibrils for 10 days. After 3 weeks, each mouse was subjected to inflammatory stimulation by subcutaneous injection with a mixture of complete Freund's adjuvant supplemented with Mycobacterium butyricum. The mice were killed on the third day after the inflammatory stimulation, and the spleen, liver, kidney and gastrointestinal tract were examined for amyloid deposits. Amyloid deposits were detected in 14 out of 15 mice treated with murine AA amyloid fibrils, 12 out of 15 mice treated with bovine AA amyloid fibrils and 11 out of 15 mice treated with human A(lambda) amyloid fibrils. No amyloid deposits were detected in control mice receiving the inflammatory stimulant alone or in amyloid fibril-treated mice without inflammatory stimulation. Our results suggest that AA amyloid deposition is accelerated by oral administration of semipurified amyloid fibrils when there is a concurrent inflammatory stimulation.