Immunohistochemical detection of hepatocyte nuclear factor-4α in vertebrates.

Hepatocyte nuclear factor-4α (HNF4α) presents in multiple isoforms generated using alternative promoter (P1 and P2) and splicing. Neither conservation of tissue distribution of HNF4α isoforms, nor presence of alternative promoter usage is known. In this study, to detect the expression of HNF4α in some species of animals, we have applied monoclonal antibodies against P1 (K9218) and P2 (H6939) promoter-driven and P1/P2 promoter-driven H1415 HNF4α for immunohistochemistry and western blot analysis. Antibody K9218 was observed in the hepatocytes, proximal tubules of the kidney, and epithelial cells in the mucosa of the small intestine and colon of rats, chicken, and tortoise, whereas antibody H6939 signal were detected in the stomach, pancreas, bile duct, and pancreatic duct of human and rats. The signal for antibody K9218 was recognized in tissues of a wide range of mammals, bird, reptile, amphibian, and fish as well. Antibody H1415 displayed a positive reaction in hepatocytes and intestinal epithelial cells in chicken and tortoise, whereas the bile duct, mucosal epithelial cells in the stomach, or pancreas in these animals were negative. Western blotting showed the binding of the antibody with HNF4α protein from each animal. The sequence of human HNF4α was 100% identical to murine and rat HNF4α, 88.9% to chicken, 77.8% to Xenopus HNF4α, and 81.5% to medaka. However, the specific part of human and invertebrate Drosophila HNF4 shares only 14.8% sequence identity. This antibody is useful for detecting HNF4α isoforms in a wide range of vertebrates, and suggests many insights into animal evolution.

Adenoid Cystic Carcinoma With Sialolithiasis of the Left Submandibular Gland: A Case Report and Literature Review.

Adenoid cystic carcinoma is one of the most common salivary gland malignancies with poor long-term prognosis, but the coexistence of sialoliths is extraordinarily rare. In this article, we report a case of 30-year-old woman with a history of submandibular area swelling with intermittent pain increasing during mealtimes that had led her attending physician to diagnose a sialolith in the left submandibular gland on a radiograph 10 years before. However, the surgical specimen proved to be an adenoid cystic carcinoma accompanied with a sialolith. Histopathologically, the submandibular gland was displaced with a fibrous granulation tissue containing a small cribriform carcinoma invading the extracapsular region of the gland. We performed fluorescence in situ hybridization examination with an MYB-NFIB fusion probe of the lesion, with positive results. The patient underwent a supraomohyoid neck dissection as additional procedure because of the possibility of the extracapsular cancer nest remaining around the submandibular gland, but she remains well and disease free 11 years after the first operation.

Roundabout1 distribution in neoplastic and non-neoplastic diseases with a focus on neoangiogenesis.

Slit and its receptor Roundabout (Robo) are important for neuronal development and neo-angiogenesis in various neoplastic and non-neoplastic diseases. Angiogenesis is a key factor for tumor growth and other angiogenesis-dependent diseases including rheumatoid arthritis, and chronic inflammation Recently, over-expression of Slit/Robo1 family proteins has been reported in several types of malignancy. We explored the expression of Robo1 in neoplastic and non-neoplastic diseases with a focus on newly formed blood vessels. Three hundred and thirty four cases of malignancy and forty five cases of angiogenic diseases were recruited. Using the A7241A Robo1 monoclonal antibody, Robo1 expression was validated by immunohistochemistry. Among malignant cases, endothelial cells of newly formed blood vessels in 283 tumors (84.7%) exhibited positive staining with above antibody. In non-neoplastic diseases, newly formed blood vessels were positive in 70.6% (12/17) cases of chronic inflammation, 100% (18/18) cases of pyogenic granuloma and 83.3% (5/6) cases of rheumatoid arthritis. Recently, newly anti-angiogenesis therapy is drawing attention as effective therapy for angiogenesis-dependent diseases without regard to their neoplastic or non-neoplastic nature. Our results showed a large number of neoplastic and non-neoplastic diseases showed positive staining for ROBO1 by immunohistochemistry. Thus, Robo1 targeted therapy may create new strategies for the treatment of angiogenic-dependent diseases through the suppression of angiogenesis. Further, besides the majority of liver cell carcinomas (23/28, 82.1%), Robo1 was positive in 100% of the squamous cell carcinoma of the esophagus, uterine cervix, lung and skin. Thus, immunohistochemical evaluation of Robo1 may be useful as an additional diagnostic tool for liver cell carcinomas and squamous cell carcinomas.

Mammary analogue secretory carcinoma of salivary glands: a clinicopathologic and molecular study including 2 cases harboring ETV6-X fusion.

Mammary analogue secretory carcinoma (MASC) is a recently described low-grade carcinoma with morphologic and genetic similarity, including ETV6-NTRK3 fusion, to secretory carcinoma of the breast. ETV6 is frequently involved in other epithelial and nonepithelial tumors, and many fusion partners of ETV6 have been reported. In the present study, 14 Japanese MASC cases were clinicopathologically and molecularly analyzed. The median age of the patients was 39 years, and the male:female ratio was 6:8. All cases showed histopathologic findings compatible with those previously described for MASC and harbored an ETV6 split as visualized by fluorescence in situ hybridization. Two cases showed thick fibrous septa and invasive features including vascular or perineural tumor involvement, findings that are rare in MASC. In addition, in these 2 cases, non-NTRK3 genes appeared to fuse with ETV6 (ETV6-X fusion). NTRK1 and NTRK2, both members of the NTRK family, were not involved. Of the 14 MASC cases, the ETV6-NTRK3 fusion transcript was positive in 6 cases, and the relative expression level of the ETV6-NTRK3 fusion transcript was variable, ranging from 1 to 5.8. Results of the present study of MASC suggest that (1) ETV6 occasionally fuses with unknown non-NTRK3 genes, (2) ETV6-X cases might have an invasive histology, (3) for molecular diagnosis of MASC, fluorescence in situ hybridization to detect ETV6 splits is the method of choice, and (4) the expression level of the ETV6-NTRK3 fusion transcript is considerably variable. These findings provide a novel insight into the oncogenesis, histopathology, diagnosis, treatment, and prognosis of this newly recognized carcinoma.

MRI for differentiation of renal cell carcinoma with sarcomatoid component from other renal tumor types.

PURPOSE: To investigate the usefulness of MRI for detection of sarcomatoid renal cell carcinoma (SRCC) components within RCC and differentiation from other renal tumors. METHODS: Two observers independently interpreted T2-weighted images of 10 patients with pathologically confirmed RCCs with SRCC and 131 with non-SRCC renal tumors, with special reference to conspicuously low signal intensity (SI) areas (T2LIA) compared to the renal cortex. SRCC probability was classified as (1) definitely non-SRCC, no T2LIA; (2) probably non-SRCC, <1 cm T2LIA; (3) low probability of SRCC, homogeneous tumor with 1-3 cm T2LIA; (4) probably SRCC, heterogeneous tumor with 1-3 cm T2LIA; and (5) definitely SRCC, >3 cm T2LIA, multiple >1 cm T2LIAs, or showing disruption of the pseudocapsule. The observers used chemical shift imaging to exclude the area representing hemorrhage or hemosiderin deposition from T2LIA. Scores of 4/5 were regarded as positive for evaluating the accuracy and area under the receiver operating characteristic curve. The SI ratio of the lowest SI in the tumor to that of the renal cortex in the 1 and ≥2 score groups was compared using Mann-Whitney's U test. RESULTS: Sensitivity, specificity, accuracy, and positive and negative predictive values were 90%, 95%, 94%, 56%, and 99%, respectively, and area under the receiver operating characteristic curve was 0.93. The mean SI ratio of the lowest SI in the tumor to that of the renal cortex was significantly lower in the ≥2 score group (0.58) than in the 1 score group (1.36). CONCLUSIONS: MRI predicted RCC with SRCC with a moderate positive predictive value and a high negative predictive value.

Characteristic MRI findings of sarcomatoid renal cell carcinoma dedifferentiated from clear cell renal carcinoma: radiological-pathological correlation.

PURPOSE: To evaluate MRI findings of sarcomatoid renal cell carcinoma (SRCC). MATERIAL AND METHODS: Eleven patients with pathologically proven SRCC dedifferentiated from clear cell renal carcinoma (CCRC) underwent preoperative renal MRI. The MRI findings were compared with histological findings. On MRI, the following findings were evaluated: the presence and distribution of areas showing heterogeneous iso to high signal intensity (SI) on T2-weighted images (T2HIA) and conspicuously low SI areas (T2LIA) compared to normal renal cortex, areas showing high SI on T1-weighted images and unenhanced areas on dynamic contrast-enhanced images, disruption of pseudocapsule, and the SIs of T2HIA and T2LIA on diffusion-weighted imaging (DWI). The apparent diffusion coefficient (ADC) values and SI ratios to muscle on dynamic contrast-enhanced imaging (DCE) were compared between T2HIA and T2LIA using the t test. RESULTS: The distribution of T2HIA and T2LIA was as follows: a mixed pattern alone in five, nodular T2LIA pattern alone in one, both mixed and nodular T2LIA patterns in four, and a separated pattern in one. Disruption of the pseudocapsule was seen in all cases. The imaging findings suggesting intratumoral hemorrhage and necrosis were seen in 18% and 63%, respectively. The SIs of T2HIA and T2LIA were low intermediate and high on DWI, respectively. T2LIA and T2HIA corresponded to the components of SRCC with abundant fibrosis and CCRC, respectively. T2LIA showed significantly lower enhancement at all DCE phases and a lower ADC value than T2HIA. CONCLUSION: The presence of T2LIA corresponding to the area showing a hypovascular nature and markedly restricted diffusion might be characteristic findings of SRCC. Intratumoral hemorrhage and necrosis were seen, but they were not specific findings.

Thymic extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue: a gene methylation study.

Although rare, thymic mucosa-associated lymphoid tissue (MALT) lymphoma is considered to be a distinct clinicopathological entity. Using a methylation-specific polymerase chain reaction, we analyzed thymic MALT lymphomas (n = 18) for their methylation of the following seven tumor suppressor genes: DAPK1, p16(INK4A), p14(ARF), CDH1, RARB, TIMP3 and MGMT. Reactive lymph nodes (n = 16) were used as a control. Of the seven genes examined, thymic MALT lymphomas had an increased number of genes that were methylated (2.9 genes) as compared with reactive lymph nodes (0.63, p = 0.0003). In particular, thymic MALT lymphomas showed a frequent methylation of DAPK1, CDH1, TIMP3 and p14(ARF). In addition, gene methylation of p14(ARF) was associated with a larger tumor size, while that of the other three genes was not associated with any clinicopathological features examined. This study suggests that methylation of tumor suppressor genes may play an important role in thymic MALT lymphoma.

Clinicopathological significance of MAML2 gene split in mucoepidermoid carcinoma.

CRTC1-MAML2 and CRTC3-MAML2 fusions have been associated with favorable clinicopathological features of mucoepidermoid carcinomas. However, the significance of the MAML2 gene split has not been fully clarified. In the present study, 95 mucoepidermoid carcinomas (paraffin-embedded materials) were analyzed for CRTC1-MAML2 and CRTC3-MAML2 fusions by RT-PCR and for the MAML2 gene split by FISH. Quantitative RT-PCR for the CRTC1-MAML2 transcript was performed in selected cases. MLL gene involvement, which has been reported in some leukemia cases, was examined by FISH in fusion partner-unknown cases. CRTC1-MAML2 and CRTC3-MAML2 fusions were detected in 37 and 6 cases, respectively. The MAML2 gene split was detected in 62 cases, which included all CRTC1/3-MAML2 fusion-positive cases. The level of CRTC1-MAML2 transcript expression was highly variable, and its clinicopathological impact was unclear. The MLL gene split was not detected. Mucoepidermoid carcinomas negative for CRTC1/3-MAML2 and positive for the MAML2 gene split (n = 19) showed favorable clinicopathological tumor features similar to those positive for CRTC1/3-MAML2 fusions. Compared with negative cases (n = 33), mucoepidermoid carcinomas positive for the MAML2 split (n = 62) were associated with lower patient age, a mild female predilection, a smaller tumor size, less frequent nodal metastasis, a lower clinical stage, a lower histological grade, and longer overall and disease-free survival. The MAML2 gene split emerged as an independent prognostic factor for both overall and disease-free survival in multivariate prognostic analysis. The presence of the MAML2 gene split defines a distinct mucoepidermoid carcinoma subset that is associated clinicopathologically with favorable tumor features.

A case of central nervous system lymphoma manifesting as multiple patchy white matter lesions with a past history of tonsil lymphoma.

Lymphoma of the central nervous system (CNS) parenchyma is known to present as a well-demarcated mass with strong and homogenous gadolinium enhancement in the periventricular and/or superficial region. We report a case of central nervous system lymphoma (CNSL) manifesting as multiple white matter lesions with non-tumorous patchy or ring-like enhancement and partial spontaneous resolution on magnetic resonance imaging (MRI). Such findings are unusual and could lead to misdiagnosis without pathological evaluation.

Intramucosal stomach adenocarcinoma metastasizing as a large intraabdominal mass with focal choriocarcinomatous differentiation.

We report a case of gastric intramucosal adenocarcinoma giving rise to a large metastatic intraabdominal mass with focal choriocarcinomatous differentiation. The main histological picture of the surgically resected abdominal tumor was well differentiated adenocarcinoma with focal choriocarcinomatous differentiation. Serum hCGß level showed 710mIU/dl postoperatively, and the gastric lesion featured small foci of well differentiated adenocarcinoma in adenoma with no choriocarcinomatous differentiation. The origin of the abdominal tumor was the main point of question. HNF4α is a transcription factor of embryonic liver differentiation, whose distribution is restricted to hepatocytes and certain neoplastic tissue including gastric adenocarcinoma. Hep Par 1 is originally developed for the discrimination of hepatocellular carcinoma, but a part of gastric adenocarcinoma also shows positive staining. Immunostaining with panel of antibodies for CK7, CK20, HNF4α, and Hep-Par1 showed pattern of gastric adenocarcinoma, and a diagnosis of a very rare intramucosal gastric adenocarcinoma metastatic to the abdominal cavity was established. Diagnostic utility of the panels of above antibodies for discrimination of the tumor origin was confirmed, and the relation between the metastatic ability of the gastric adenocarcinoma and its choriocarcinomatous differentiation is discussed.

Characterization of chromosomal aberrations in thymic MALT lymphoma.

Mucosa-associated lymphoid tissue (MALT) lymphoma arising in the thymus is a rare disorder that shows a strong association with autoimmune disease. Several MALT-lymphoma-specific and -associated chromosomal abnormalities, including t(11;18), t(14;18), t(1;14), trisomy 3 and trisomy 18, are known to occur. The former translocation results in apoptosis inhibitor 2 gene (API2)-MALT lymphoma-associated translocation 1 (MALT1) fusion. In this study, we examined 14 cases of thymic MALT lymphomas for API2-MALT1 fusion using multiplex reverse transcription polymerase chain reaction and looked for trisomy 3, trisomy 18 and abnormalities of MALT1 and IGH genes using fluorescence in situ hybridization. Thymic MALT lymphoma cases had a high frequency of trisomy 3 (7/14 cases), a very low incidence of trisomy 18 (1/14) and no detectable MALT1-associated (0/13) or IGH-associated (0/13) gene abnormalities including t(11;18). A review of the literature showed that the pattern of chromosomal aberrations in thymic MALT lymphoma was similar to those of thyroid and salivary gland MALT lymphomas. Although frequently detected, trisomy 3 was not associated with any of the clinicopathological factors analyzed, suggesting that trisomy 3 may play a role in lymphoma development. In conclusion, the present study showed that thymic MALT lymphoma has a characteristic pattern of chromosomal aberrations that may be similar to those of other autoimmune-associated MALT lymphomas.

Impact of CRTC1/3-MAML2 fusions on histological classification and prognosis of mucoepidermoid carcinoma.

AIMS: The aim of study was to evaluate the impact of CRTC1-MAML2 and CRTC3-MAML2 fusions on the histological classification of mucoepidermoid carcinoma (MEC) of the salivary glands and on the prognosis of patients. METHODS AND RESULTS: MEC cases (n = 111) were screened for CRTC1-MAML2 and CRTC3-MAML2 fusions by reverse transcription polymerase chain reaction. We developed a system of 'molecular Armed Forces Institute of Pathology (AFIP) classification' that combined the AFIP histological classification proposed by Goode et al. and the presence of CRTC1-MAML2 or CRTC3-MAML2 fusions. MEC cases positive for CRTC1-MAML2 or CRTC3-MAML2 fusion formed a favourable tumour subset that was distinct from fusion-negative cases. When positive for the fusions, 'high-risk' patients, including those with a higher histological grade or an advanced clinical stage, showed an excellent prognosis. For overall survival, 'molecular AFIP classification' was selected as a powerful independent prognostic factor (P=0.0038), as was the clinical stage (P =0.0032). For disease-free survival, 'molecular AFIP classification' was also selected as an independent prognostic factor (P = 0.0006). CONCLUSIONS: Molecular AFIP classification may be useful in predicting the prognosis of patients with MEC.

Analysis of API2-MALT1 fusion, trisomies, and immunoglobulin VH genes in pulmonary mucosa-associated lymphoid tissue lymphoma.

Pulmonary mucosa-associated lymphoid tissue lymphoma is unique in that chronic inflammation is rare and that API2-MALT1 fusion, resulting from t(11;18)(q21;q21), occurs frequently. In this study, we examined 20 cases for API2-MALT1 fusion using the multiplex reverse-transcription polymerase chain reaction and looked for trisomy 3, trisomy 18, and abnormalities of MALT1 and IGH genes using fluorescence in situ hybridization. In addition, we analyzed VH genes by subcloning of the monoclonal polymerase chain reaction products. Of 20 cases studied, we detected gene abnormalities in 16: API2-MALT1 fusion in 9, trisomy 3 in 5, trisomy 18 in 4, MALT1 abnormality in 13, and IGH abnormality in 1. MALT1 gene abnormalities were concordant with API2-MALT1 fusion or trisomy 18. One case showed API2-MALT1 fusion and trisomy 3. On detection of API2-MALT1 fusion and trisomies, we were able to divide our cases into 3 groups, API2-MALT1 positive, trisomy positive, and no detectable gene abnormality, suggesting that tumor development had processed along different genetic pathways. All 20 cases were analyzed for VH genes. Most of the VH genes selected by the lymphomas belonged to the VH3 family, but there was no restriction to any particular VH fragment. Of interest, VH genes were unmutated in 7 cases, suggesting that T-cell-independent extrafollicular B-cell maturation may be important in the development of this lymphoma. In addition, both mutated and unmutated tumor cases were found to carry the API2-MALT1 fusion and trisomy 3. This observation suggests that these gene abnormalities may occur in microenvironments found before or outside of follicular germinal centers.

A case of oncocytic papillary renal cell carcinoma.

Papillary renal cell carcinomas (RCC) are the second most frequently identified pathological subtypes of RCC. Occasionally, papillary RCC demonstrate pathological characteristics of renal oncocytomas (RO), benign renal tumors. We report the case of an 81-year-old woman with an oncocytic papillary RCC, which was difficult to differentiate from a hybrid of RO and papillary RCC, who underwent left radical nephrectomy. Morphological examination showed oncocytic tumor region and partially scattered regions with papillary structure. Immunohistochemical examinations demonstrated strongly positive staining of alpha-methylacyl-CoA racemase in the papillary region and negative staining of progesterone receptor and CD117 in both regions. Fluorescence in situ hybridization confirmed the increased number of copies of chromosome 7 in the papillary region. Comprehensively, this case could be diagnosed as oncocytic papillary RCC. No evidence of disease recurrence was found at 12 months' follow up.

Composite glandular-endocrine cell carcinoma of the stomach. Report of two cases with goblet cell carcinoid component.

Composite glandular-endocrine cell carcinoma (CGECC) is recognized as a special type of gastric tumor composed of ordinary adenocarcinoma and neuroendocrine tumors. Goblet cell carcinoid (GCC) is a well-established type of appendiceal carcinoid, but the GCC component has not been well delineated in CGECC of the stomach. We report on two gastric CGECCs with a GCC component, analyzing the histologic components by immunohistochemistry. On initial biopsy, both cases were diagnosed as signet-ring cell carcinoma. However, the resected tumors consisted of three components: signet-ring cell carcinoma, GCC, and glandular adenocarcinoma. Although some signet-ring carcinoma cells and goblet carcinoid cells were indistinguishable by hematoxylin and eosin staining, E-cadherin immunostaining disclosed a definitive difference regarding the staining pattern in these cells. Both patients are well, with no recurrent tumor for about 10 years of follow-up. CGECC with a GCC component may have been confused with conventional adenocarcinoma with signet-ring cells. In cases of advanced signet-ring cell carcinoma with good prognosis, the possibility of such CGECC has to be considered.