Cytomorphological characteristics of low-grade papillary urothelial carcinoma for differential diagnosis from benign papillary urothelial lesions: logistic regression analysis in SurePath(™) liquid-based voided urine cytology.

OBJECTIVE: The diagnosis of low-grade papillary urothelial carcinoma (LGPUC) in urine cytology specimens is challenging because of its subtle, minimally atypical findings. Furthermore, as SurePath(™) liquid-based cytology (LBC) is becoming a widely used method in urine cytology, the inevitable cytomorphological alterations resulting from this technique call for new morphological diagnostic criteria in LGPUC. METHODS: Logistic regression analysis was carried out on SurePath slides from surgically proven voided urine specimens. The study was designed to include a test set (n = 141) and a validation set (n = 61), and evaluated significant discriminative parameters between LGPUC and benign papillary urothelial neoplasm (BPUN). RESULTS: Of the seven cytological findings that were found to have statistical significance in univariate analysis, five were found to be independent variables: loss of polarity of papillaroid clusters, irregular contours, absence of columnar cells, hobnail features and hyperchromasia. These independent variables had an area under the curve (AUC) of 0.781. CONCLUSIONS: The distinctive cytological criteria identified above may prove to be helpful in cases in which other conventional criteria for LGPUC are insufficient for diagnosis.

Prognostic and predictive values of EGFR overexpression and EGFR copy number alteration in HER2-positive breast cancer.

BACKGROUND: Epidermal growth factor receptor (EGFR) is overexpressed in a subset of human epidermal growth factor receptor 2 (HER2)-positive breast cancers, and coexpression of HER2 and EGFR has been reported to be associated with poor clinical outcome. Moreover, interaction between HER2 and EGFR has been suggested to be a possible basis for trastuzumab resistance. METHODS: We analysed the clinical significance of EGFR overexpression and EGFR gene copy number alterations in 242 HER2-positive primary breast cancers. In addition, we examined the correlations between EGFR overexpression, trastuzumab response and clinical outcome in 447 primary, and 112 metastatic HER2-positive breast cancer patients treated by trastuzumab. RESULTS: Of the 242 primary cases, the level of EGFR overexpression was 2+ in 12.7% and 3+ in 11.8%. High EGFR gene copy number was detected in 10.3%. Epidermal growth factor receptor overexpression was associated with hormone receptor negativity and high Ki-67 proliferation index. In survival analyses, EGFR overexpression, but not high EGFR copy number, was associated with poor disease-free survival in all patients, and in the subgroup not receiving adjuvant trastuzumab. In 447 HER2-positive primary breast cancer patients treated with adjuvant trastuzumab, EGFR overexpression was also an independent poor prognostic factor. However, EGFR overexpression was not associated with trastuzumab response, progression-free survival or overall survival in the metastatic setting. CONCLUSIONS: Epidermal growth factor receptor overexpression, but not high EGFR copy number, is a poor prognostic factor in HER2-positive primary breast cancer. Epidermal growth factor receptor overexpression is a predictive factor for trastuzumab response in HER2-positive primary breast cancer, but not in metastatic breast cancer.

Mutation spectrum and inhibitor risk in 100 Korean patients with severe haemophilia A.

Haemophilia A (HA) is an X-linked recessive bleeding disorder caused by defects in the F8 gene encoding the coagulation factor VIII. Mutation analysis in HA is important to confirm the diagnosis, genotype-phenotype correlations and for genetic counselling and family study. The aim of this study was to detect causative mutations of F8 in severe HA patients in Korea and to correlate the mutation type with the risk of inhibitor development. A total of 100 unrelated Korean patients with severe HA were enrolled for this study. The Nijeman modification of the Bethesda assay was used to determine the presence of inhibitor. Molecular analysis of F8 was performed using a combination of molecular techniques, including long-distance polymerase chain reaction, direct sequencing and multiplex ligation-dependent probe amplification (MLPA). We identified causative mutations in 98% of severe HA patients (98/100). Inv22 and Inv1 mutations were detected in 30 patients and one patient, respectively. A total of 59 unique mutations were identified in 69 non-inversion patients, including 24 novel mutations. The overall prevalence of inhibitor was 26%. Inhibitor risk was highest in patients with large deletion mutations identified using MLPA (100%). Among those with point mutations, the prevalence of inhibitor was highest when the mutation occurred in the A3 and C2 domains (60% and 50%, respectively). The molecular diagnostic strategy involving multiplex PCR, sequencing and dosage analyses identified causative mutations in most cases of severe HA. The high inhibitor risk was associated with large deletion mutations and point mutations in A3 and C2 domains.

A prospective longitudinal study evaluating the usefulness of a T-cell-based assay for latent tuberculosis infection in kidney transplant recipients.

We evaluated whether ELISPOT assay can predict tuberculosis (TB) development in kidney-transplantation (KT) recipients with a negative tuberculin skin test (TST). All adult patients admitted to a KT institute between June 2008 and December 2009 were enrolled; TB development after KT was observed between June 2008 and December 2010. Isoniazid (INH) was given to those patients with positive TST or clinical risk factors for latent TB infection (LTBI). ELISPOT assay was performed on all patients, and TB development after KT was observed by a researcher blinded to the results of ELISPOT. A total of 312 KT recipients including 242 (78%) living-donor KT were enrolled. Of the 312 patients, 40 (13%) had positive TST or clinical risk factors for LTBI and received INH; none developed TB after KT. Of the remaining 272 patients, 4 (6%) of 71 with positive ELISPOT assay developed TB after KT, whereas none of the 201 patients with negative (n = 171) or indeterminate ELISPOTs (n = 30) developed TB after KT (rate difference between positive and negative/indeterminate ELISPOT, 3.3 per 100 person-years [95% CI 1.4-5.1, p<0.001]). Positive ELISPOT results predict subsequent development of TB in KT recipients in whom LTBI cannot be detected by TST or who lack clinical risk factors for LTBI.

Diagnostic usefulness of a T cell-based assay for latent tuberculosis infection in kidney transplant candidates before transplantation.

BACKGROUND: The presence of latent tuberculosis (TB) infection (LTBI) should be evaluated before kidney transplantation. Although a new T cell-based assay for diagnosing LTBI gave promising results, this assay has not yet been compared with the tuberculin skin test (TST) for diagnosing LTBI in renal transplant candidates before transplantation. PATIENTS AND METHODS: All adult patients admitted to a single institute for renal transplantation over a 1-year period were prospectively enrolled. A clinically predictive risk of LTBI was defined as: (i) recent close contact with a person with pulmonary TB; (ii) abnormal chest radiography; (iii) a history of untreated or inadequately treated TB; or (iv) a new infection (i.e., a recent conversion of TST). RESULTS: Of 209 renal recipients, 47 (22%) had a positive TST> or =5 mm, 21 (10%) had a positive TST> or =10 mm, 65 (30%) had a positive T-SPOT.TB test, and 25 (12%) had an indeterminate T-SPOT.TB test. The induration size of TST was significantly associated with a high positivity rate on T-SPOT.TB (P<0.001). Agreement between T-SPOT.TB test and TST> or =10 mm was fair (k=0.24, 95% confidence interval 0.11-0.36). However, neither univariate nor multivariate analysis showed any association between the clinical risk for LTBI and positivity on T-SPOT.TB or TST. CONCLUSION: T-SPOT.TB test was more frequently positive than TST in renal transplant candidates. However, further longitudinal studies are awaited to determine whether the ability of T-SPOT.TB assay to detect LTBI in renal transplant recipients can better predict the development of TB than can TST after transplantation.

Age and HER2 expression status affect MRI accuracy in predicting residual tumor extent after neo-adjuvant systemic treatment.

BACKGROUND: Although recent studies suggest high accuracy of breast magnetic resonance imaging (MRI) in predicting residual tumor extent after neo-adjuvant systemic treatment (NST), its use is still controversial. In this study, we aimed to identify predictive factors of MRI accuracy after NST to determine a subgroup of patients in whom the use of MRI provides best additional benefit. MATERIALS AND METHODS: Clinicopathologic and molecular profiles of breast cancer patients were investigated and their relationships with MRI accuracy were analyzed. RESULTS: From January 2006 to February 2008, 195 patients received NST and preoperative MRI. In overall, MRI predicted residual tumor extent with higher accuracy than ultrasonography. Triple-negative (TN) tumors showed highest correlation between MRI-measured and pathologic tumor size (r = 0.781) when compared with other subtypes. Multivariate analysis showed age and HER2 expression status as independent factors predicting MRI accuracy. When patients were classified based on their age and HER2 status, relatively older patients (>45) with HER2-negative tumors showed highest MRI accuracy. This finding was further validated using an independent cohort of 63 consecutive patients. CONCLUSION: Age and HER2 status independently affected MRI accuracy after NST. This observation may guide more tailored approach in using MRI in breast cancer patients undergoing NST.

Overexpression of phospholipase C-gamma1 in rat 3Y1 fibroblast cells leads to malignant transformation.

Phospholipase C-gamma1 (PLC-gamma1) mediates signals from various extracellular origins to evoke cellular events such as mitogenesis. Previously, we reported that PLC-gamma1 was highly expressed in colorectal cancer and familial adenomatous polyposis, suggesting that PLC-gamma1 might be oncogenic. In this study, we have established rat 3Y1 fibroblasts that overexpress whole PLC-gamma1 and src homology 2 (SH2)-SH2-SH3 domain of PLC-gamma1. These cells showed a transformed phenotype and were tumorigenic when transplanted into nude mice. These results indicate that overexpression of PLC-gamma1 could transform rat fibroblasts, and the transformation is mediated by SH2-SH2-SH3 domain of PLC-gamma1.

Clinical benefit of nomogram for predicting positive resection margins in breast conserving surgery.

PURPOSE: Previously, we reported a nomogram for the prediction of positive resection margin (RM) after breast conserving surgery (BCS). This study was conducted to evaluate the clinical usefulness of the nomogram. METHODS: Prospective patients who underwent operations using the nomogram between July 2012 and August 2013 (nomogram group; N = 260) were compared with past control patients who underwent operations between July 2010 and October 2011 and underwent frozen section biopsy (FSB) without use of the nomogram (N = 266). In the nomogram group, an intraoperative assessment of RM using FSB was only performed when the nomogram score was higher than predefined cut-off (>80). In addition, we conducted retrospective analysis of additional 181 patients who received BCS in another institute (Kyoto University Hospital). These patients did not undergo FSBs for RMs. RESULTS: Of 260 patients, 161 (61.9%) presented low nomogram scores and avoided FSB. The surgical decision to use the nomogram did not significantly increase reoperation rate due to positive RM compared with the control FSB group (4.6% vs. 3.8%, p = 0.47). The surgery time was significantly reduced by 18.1% (mean 14.7 min) in nomogram group (p < 0.001). Of 99 nomogram high-score patients, 14 presented with positive RM on FSB and 11 of them avoided reoperation. In the Kyoto cohort, the reoperation rate was significantly lower in low-score patients than in high-score patients (2.7% vs. 11.4%, p < 0.001). CONCLUSIONS: We showed that our nomogram is useful to reduce FSBs without increasing reoperation rate for surgeons who perform routine FSBs. For most surgeons, it can give useful information about the possibility of tumor-positive RMs.

Reactivity of antibodies against 10-amino acid residue and pro-domain of stromelysin-3.

Stromelysin-3 (ST3) has two highly conserved domains in the pro-domain. In particular, an unusual 10-amino acid residue sandwiched between the pro-domain and the catalytic domain of ST3 exists in ST3 but not in other matrix metalloproteinases (MMPs). To specifically detect ST3 expression in human tumors, we have made two kinds of ST3-specific polyclonal antibodies. One was raised against the synthetic 10-amino acid residue (88GLSARNRQKR97) specific to ST3, and the other against recombinant ST3 pro-domain (62APATQEAPRPASSLRPPRCGVPDPSDGLSARNRQKR97) containing the decapeptide and PRCGVPD sequence obtained by expression in Escherichia coli. Two protein species, 59 kDa and 45 kDa which were consistent with those expected for pro-ST3 and the mature form of ST3, were specifically detected in 100-fold concentrated conditioned media of fetal lung fibroblast by Western blot analysis. Immunohistochemical staining indicated that in infiltrating ductal breast carcinoma and squamous cell carcinoma of the uterine cervix, reactivity of those antibodies was found not only in fibroblastic cells surrounding cancer cells but also in neoplastic cells. However, reactivity of two ST3 antibodies was inhibited by excess of the synthetic peptide (10-amino acid residue) not only in fibroblastic cells but also in neoplastic cells. These findings suggest that antibodies against the ST3 specific region may cross react with the recently known membrane type-metalloproteinase (MT-MMP), which have RXKR sequences between the pro- and catalytic domain.

Overexpression of phospholipase D1 in human breast cancer tissues.

Phospholipase D (PLD) catalyzes the hydrolysis of phosphatidylcholine (PC) to produce phosphatidic acid (PA) and choline. PLD is a major enzyme implicated in important cellular processes, such as cell proliferation. We designed this study to investigate the expression of PLD in human breast carcinomas and non-malignant tissues using RT-PCR, Western blot analysis, immunohistochemistry and an Arf-dependent PLD activity assay. We examined about 550 bp of PCR product and 120 kDa of PLD protein. Our results showed that PLD protein and mRNA levels were overexpressed in 14 of 17 breast cancer tissues. We also observed increased expression by immunohistochemistry and Arf-dependent PLD activity in microsomes of human breast tumors, which correlated well with PLD expression. PLD expression was elevated in human breast tumors compared with normal breast tissues. These results implicate a possible role of PLD in human breast tumorigenesis and suggest that PLD may be useful as a marker for malignant disease in the breast.

Cyclin E overexpression as an independent risk factor of visceral relapse in breast cancer.

AIM: Prognostic value of the cyclin E overexpression in breast cancer has not been clearly established, especially in relation to the pattern of recurrence. We investigated the implication of cyclin E overexpression for the pattern of recurrence in Korean breast cancer patients. METHODS: Using immunohistochemical methods, we retrospectively examined the cyclin E expression level in breast cancer specimens from 128 women who underwent curative breast surgery, and correlated the levels of expression with the pattern of relapse in patients. RESULTS: Cox model-based multivariate analysis indicated that distant relapse could be predicted by the number of positive axillary lymph nodes, high cyclin E expression, and the younger age (<35 years) of the patient. We tested further the association of cyclin E overexpression with the specific types of recurrence; multivariate analyses indicated that adjusted relative risks of bone and visceral relapse as the first events among high cyclin E group were 2.46 (95% confidence interval (CI), 0.86-7.02) (P=0.092), and 3.98 (95% CI, 1.23-12.94) (P=0.022), respectively. On the other hand, cyclin E overexpression was not associated with the risk of locoregional relapse. CONCLUSION: Our data suggest that cyclin E overexpression in primary breast carcinoma tissue could independently predict the risk of distant relapse, especially of visceral relapse, as the first failure after curative breast surgery.

Overexpression of peroxiredoxin in human breast cancer.

The peroxiredoxins (Prx) are a family of 25 kDa peroxidases that can reduce H2O2 using an electron from thioredoxin (Trx) or other substances. The mammalian Prx family is divided into six groups (Prx I-VI) on the basis of homology of amino acid sequences. They are located in the cytosol and play a role in the cell signaling system. Previous reports have shown that Prx II has proliferative and anti-apoptotic properties and thus may induce carcinogenic changes. We conducted this study to reveal the change in expression of Prx in human breast cancer in comparison to normal tissues. Western immunoblotting using Prx type I, II and III antibodies was undertaken on 24 human breast cancer tissues and normal counterparts. We used antibodies against purified recombinant NKEF-A/PAG, NKEF-B and MER 5 which are the Prx isoforms. Type I Prx was overexpressed in the cancer tissues of 21 patients (87.5%), type II in 18 patients (75%) and type III in 19 patients (79.2%) in relation to normal tissue. However, no significant relationship was found between Prx overexpression and clinicopathological parameters of breast cancer such as tumor size, lymphatic invasiveness, hormone receptor status or nuclear and histologic grade. In conclusion, Prx is overexpressed in breast cancer tissues to a great extent suggesting that Prx has a proliferative effect and may be related to cancer development or progression.

Expression of phospholipase C-gamma 1 and its transcriptional regulators in breast cancer tissues.

BACKGROUND: PLC-gamma 1 is activated through direct interaction with growth factor receptor tyrosine kinase but little is known about the mechanisms controlling PLC-gamma 1 expression and its biological significance. MATERIALS AND METHODS: Using immunoblotting, we evaluated PLC-gamma 1 protein overexpression in twenty breast cancer tissues. The expression of binding protein to GES1, GES2 and GES3, located in transcriptional regulator (GPE1) was found by electrophoretic mobility shift assay (EMSA). We also determined whether there was any correlation between prognostic factors (numbers of metastatic axillary nodes, histologic grade, c-erbB2, p53, and E-cadherin) and the overexpression of PLC-gamma 1 protein. RESULT: On immunoblotting, 17 of 20 breast cancer tissues showed overexpression of PLC-gamma 1, a result of which was corresponded to that of immunohistochemistry. The binding proteins to GES1, GES2 and GES3 were overexpressed only when PLC-gamma 1 protein overexpression was apparent. Positive expression of E-cadherin only was significantly associated with PLC-gamma 1 protein overexpression (x = 0.607, p = 0.045). CONCLUSION: GPE1 binding proteins might be the transcriptional regulator in PLC-gamma 1 overexpression and the relationship between expression of PLC-gamma 1 and E-cadherin would require further elucidation.

Fine needle aspiration cytology of palpable breast lesions. Histologic subtype in false negative cases.

OBJECTIVE: To evaluate the use of fine needle aspiration cytology (FNAC) in the management of benign and malignant breast disease in an outpatient breast clinic and to determine the guidelines for reporting on FNAC of breast lesions, we evaluated the test results in correlation with the histologic diagnosis. STUDY DESIGN: We evaluate the test results of 669 cases of FNAC of the breast in 17 months in our outpatient breast clinic in correlation with the histologic diagnosis, histologic subtype according to the Japan Mammary Cancer Society classification and mammography results in false negative cases. RESULTS: Among 669 cases, 25.3% were inadequate for cytologic diagnosis. An analysis of the results using the two-by-four contingency table, including cases with inadequate cytologic diagnosis, showed that 10.6% and 1.0% had false negative and false positive cytologic diagnoses, respectively; sensitivity and specificity were 76.9% and 91.6%, respectively. The false negative rate of 10.6% was higher than expected, and those belonged mostly to the "inadequate" cytologic diagnosis category even though they were discrete, obviously malignant masses clinically. To try to explain this, we compared the cytologic results and histologic subtype according to the Japan Mammary Cancer Society classification and the mammography results. Of 73 cases of infiltrating ductal carcinoma in which retrospective reexamination of the histology slide was possible, 27 (37.0%) were papillo-tubular type, 11 (15.1%) were solid-tubular type and 35 cases (47.9%) were scirrhous type. Although the scirrhous subtype was only 37.0% in the group in which a cytologic diagnosis of carcinoma was possible, more than two-thirds of the cases of false negative cytology were scirrhous type and had malignant mammographic findings. This finding was statistically significant. CONCLUSION: In cases of ductal carcinoma of scirrhous subtype, the FNAC tended to be inadequate and false negative, but mammography showed better discrimination in such cases.

FNAC of malignant lymphoma in an area with a high incidence of T-cell lymphoma. Correlation of accuracy of cytologic diagnosis with histologic subtype and immunophenotype.

OBJECTIVE: To analyze the usefulness of fine needle aspiration cytology on malignant lymphoma in an area with a high incidence of T-cell lymphoma and to correlate the accuracy of cytologic diagnosis with histologic subtype and immunophenotype. STUDY DESIGN: We retrospectively studied the usefulness of fine needle aspiration cytology in the diagnosis of 49 cases of nodal and extranodal non-Hodgkin's lymphoma (NHL) and seven cases of Hodgkin's disease in a total of 56 patients in whom subsequent excisional biopsy revealed lymphoid malignancy. Slides showing the results of cytologic investigation were reviewed together with the information on which histologic diagnosis was based. On the basis of pathologic variables, such as prognostic groups based on the Working Formulation, so-called grade, cell size based on the modified Rappaport classification, and--in cases of NHL--immunophenotype, the accuracy of original and reviewed cytologic diagnoses was compared. RESULTS: Of the 49 cases of NHL, 8 (16.3%) were inadequate for cytologic diagnosis, and malignant lymphoma was diagnosed or suspected in 36 (73.5%), excluding inadequate specimens; the diagnostic accuracy for NHL was 87.8%. In high grade cases, malignant lymphoma was more easily diagnosed or suspected than in those that were low or intermediate grade. The rate of inadequate cases was highest in the "mixed small and large cell" category, and cases that were "false negative" were either composed entirely of small cells or contained a small cell component. Cytologic diagnosis or suspicion of malignant lymphoma was easily obtained in the "large cell" category, followed by mixed small and large cell and "small cell." Aspirates from non-B-cell type were more frequently acellular than those of B-cell type; with regard to diagnostic accuracy, however, there was no noticeable difference between the two immunophenotypes. CONCLUSION: In many cases in the mixed small and large cell category or where the immunophenotype was non-B, the aspirate was inadequate, and no definitive diagnosis was possible. Many of our cases of T-cell lymphoma were mixed small and large cell, and in Korea, where the incidence of extranodal and T-cell lymphoma is high, the usefulness of FNAC for the initial diagnosis of malignant lymphoma is limited. For a definitive diagnosis, biopsy is required.

Fine needle aspiration cytology of gastric epithelioid leiomyosarcoma metastasized to the liver. A case report.

BACKGROUND: There have been only a few reports on fine needle aspiration (FNA) cytology of epithelioid leiomyosarcoma, especially of the stomach, and a summary of the cytologic findings in this tumor is needed. CASE: A case of epithelioid leiomyosarcoma of the stomach metastasized to the liver and was composed cytologically of peculiar binucleated cells. CONCLUSION: Similar findings in most cases are that the cells are round or polygonal, with eccentrically located nuclei. The most variable findings relate to the texture of the cytoplasm, which varies from granular to dense to vacuolar. FNA cytology of epithelioid leiomyosarcoma can show cells that are mononuclear, binucleated or multinucleated, with eccentric nuclei and dense to vacuolar cytoplasm, with the variations probably depending on fixation status.

Comparing the accuracy of ThinPrep Pap tests and conventional Papanicolaou smears on the basis of the histologic diagnosis: a clinical study of women with cervical abnormalities.

OBJECTIVE: To confirm that the ThinPrep Pap test (TP) is as effective as or more effective than the conventional Papanicolaou smear (CS) in detecting epithelial cell abnormalities in a population with cervical abnormalities. STUDY DESIGN: In a blinded, split-sample, matched-pair study, a CS was prepared using a cytobrush, and then TP slides were prepared from the remainder of the sample. All slides were evaluated as defined and classified by the Bethesda System. The results of the two cytologic tests were compared in 483 women relative to the histologic diagnoses of subsequent colposcopically directed cervical biopsies in 158 cases. RESULTS: The cytologic diagnoses from the two methods agreed exactly in 91.4% of cases. The comparison between the two cytologic diagnoses with reference to the histologic diagnosis of subsequent colposcopically directed cervical biopsies showed that TP was significantly more specific for diagnosing lesions than was CS. The sensitivity of the two methods was equivalent. CONCLUSION: In a population with cervical abnormalities, TP is more specific than and as effective as CS in detecting cervical epithelial cell abnormalities. TP improved the specificity of disease detection by reducing the atypical squamous cells of undetermined significance category and/or false positive cases.

Case Report. CT and US features of renal matrix stones with calcified center.

We report imaging findings of renal matrix stones in a patient with congenital ureteropelvic junction obstruction associated with urinary tract infection. The stones were composed of a calcified center and a non-calcified peripheral matrix that was slightly high attenuated on CT and nonshadowing on US.

The clear cell variant of epithelioid intravenous leiomyomatosis of the uterus: report of a case.

A case of very rare uterine clear cell epithelioid leiomyomatosis is reported. The patient presented with a recurrent pelvic mass after hysterectomy 2 years earlier. The recurrent tumor was located mainly in the vessels of the broad ligament as worm-like plugs. The reviewed hysterectomy specimen showed a typical multi-lobulated intramural mass together with worm-like plugs within the myometrial vessels. Computer tomography exhibited metastatic nodules in both lungs. According to an histological examination, all the tumor cells were of epithelioid type and contained abundant clear cytoplasm. Characteristic large, thick-walled blood vessels were observed. Immunohistochemical staining and ultrastructural examination supported smooth-muscle origin in this case. Electron microscopic study revealed that the clear cytoplasm was attributed to the presence of numerous dilated mitochondria that had lost their cristae. The patient is still alive with the disease 28 months after surgery.