Immunohistochemical differentiation between type B3 thymomas and thymic squamous cell carcinomas

Type B3 thymomas and thymic squamous cell carcinomas have some overlapping histological features, so it is difficult to make the differential diagnosis between these two entities, especially when the biopsy specimen is small. Only a few markers such as CD5 and CD 117 were applied to the differential diagnosis, the purpose of this study is to identify other diagnostic markers to help making the differential diagnosis more accurate. GLUT-1, MUC-1, CD117, CD5, CEA, P63, CK19, CK5/6, CD1a and TdT were evaluated using 16 cases of type B3 thymoma and 20 cases of thymic squamous cell carcinoma. Staining scores were obtained by calculating the percentage of positive cells. The sensitivity of GLUT-1 or MUC-1 for thymic squamous cell carcinomas was highest (100%), followed by CK5/6 (95%), CD117 (90%), P63 (85%), CD5 (80%) and CEA (75%). The specificities of CD5, CD117 and CEA for thymic squamous cell carcinomas all were 100%, next was MUC-1 (56.3%), followed by GLUT-1 (50%), P63 (25%), CK5/6 (12.5%). The sensitivities of CK19, TdT, and CD1a for type B3 thymomas were 100%, 93.8% and 87.5%, respectively. The specificity of CD1a for type B3 thymomas was highest (100%), followed by TdT (95%), CK19 (10%). The reactivity of GLUT-1, MUC-1, CD117, CD5, CEA, CD1a and TdT in thymic squamous cell carcinomas and type B3 thymomas had significant difference. Usually a panel of markers is needed, if we combine GLUT-1 or MUC-1 which sensitivity for thymic squamous cell carcinomas is highest with CD5, CD117, CEA, CD1a or TdT which have high specificity, we can make the differential diagnosis effectively.     

Tumour eosinophilia combined with an immunohistochemistry panel is useful in the differentiation of type B3 thymoma from thymic carcinoma

It is sometimes difficult to differentiate between type B3 thymoma from thymic carcinoma histologically. Given the rarity of these tumours, studies have been limited. A series of 66 thymic neoplasms were reviewed and classified according to the World Health Organization (WHO) scheme. We performed a tissue microarray analysis of surgically resected thymic tumour specimens including 12 thymic carcinomas, 17 type B3 thymomas and 37 thymomas of other types. Percentage and staining intensity of immunohistochemical markers were recorded. Tumour eosinophilia was recorded positive if at least one eosinophilic cell identified. Positive staining of the following markers significantly differentiated type B3 thymoma from thymic carcinoma: cytokeratin 5/6 (15 vs. 3), Mesothelin (0 vs. 5), cytoplasmic androgen receptor (10 vs. 0), CD57 (9 vs. 0), CD5 (0 vs. 7), TdT (lymphocytic) (14 vs. 1), CD1a (lymphocytic) (14 vs. 2), CD117 (1 vs. 9), MOC31 (2 vs. 6), p21 (2 vs. 8), cytoplasmic Survivin (0 vs. 4), and tumour eosinophilia (1 vs. 11). Combining two or three markers was able to differentiate these two tumours with area under the curve percentage of at least 92%. Tumour eosinophilia combined with a panel of immunohistochemistry could differentiate type B3 thymoma from thymic carcinoma.     

Gastrointestinal stromal tumours (GISTs) negative for KIT (CD117 antigen) immunoreactivity

Gastrointestinal stromal tumours (GISTs) are currently defined as mesenchymal tumours of the gastrointestinal tract that express KIT receptor tyrosine kinase. However, a small subgroup of tumours that fulfil the clinical and morphological criteria for GISTs lack KIT expression. So far, the biological features of these tumours have rarely been addressed. The present study describes seven gastrointestinal stromal neoplasms that presented clinicopathological features typical of GISTs but showed absence of CD117 expression as detected by immunohistochemistry. The tumours originated from the stomach (n = 5), duodenum (n = 1), and colon (n = 1), showing histologically either predominantly epithelioid (n = 3), mixed spindled and epithelioid (n = 2), or anaplastic/spindle cell (n = 2) type features. CD34 and alpha-smooth muscle actin (alpha-SMA) positivity was present in four and three tumours, respectively. Chromosomal analysis was performed in two cases, both showing losses of chromosomes 14, 22, and 1p, which is the characteristic feature of GISTs. Dual-colour interphase fluorescence in situ hybridization (FISH) analysis, utilizing chromosome 1p-, 14-, and 22-specific probes, revealed a similar cytogenetic profile in the remaining five tumour specimens. Mutational analysis of exons 9, 11, 13, and 17 of KIT, and exons 12 and 18 of PDGFRA was performed in all cases by denaturing high-pressure liquid chromatography (DHPLC) pre-screening, followed by direct sequencing. None of the tumours showed KIT mutant isoforms. Three tumours harboured PDGFRA exon 18 activating mutations; two were Asp --> Val(842) missense substitutions and one was a DIM842-844 amino acid deletion. KIT and PKC theta (protein activated in interstitial cells of Cajal and GISTs) expression was determined by western immunoblotting of the total cell lysates from three tumour biopsies. None of these three tumours expressed KIT, while all specimens showed expression of PKC theta protein. These findings indicate that there is a subgroup of KIT-negative GISTs that exhibit the same morphological, cytogenetic, and molecular features as KIT-positive tumours. While intragenic PDGFRA activating mutations are present in some of these tumours, the oncogenic events underlying the pathogenesis of the others remain unknown.     

Rare KIT (CD117) expression in multiple myeloma abrogates the usefulness of imatinib mesylate treatment

Background Imatinib mesylate blocks the tyrosine kinase activity of KIT (CD117) and is an effective treatment for gastrointestinal stromal tumors. In multiple myeloma, KIT expression has been detected by flow cytometry in about 33% of specimens, but no previous immunohistochemical assessment has yet been made of the expression pattern of KIT.Materials and methods We performed immunohistochemical analyses of 100 patients, including 72 with multiple myeloma (MM), 8 with lymphoplasmacytic lymphoma (LPL), 10 with monoclonal gammopathy of undetermined significance (MGUS) and 10 with reactive plasmocytosis. One KIT-positive MM was sequenced using polymerase chain reaction analysis.Results In MM, only 2 cases (2.8%) were KIT positive. The great majority of the cases (97, 2%) did not express the KIT receptor tyrosine kinase. No mutation of the c-kit gene was detected.Conclusions KIT expression is a rare event in MM and not detectable in MGUS and LPL. Therefore, treatment with imatinib is unlikely to be effective in these patients.     

High-level expression of CD109 is frequently detected in lung squamous cell carcinomas

CD109 is a glycosylphosphatidylinositol (GPI)-anchored cell surface protein, which is a member of the alpha2-macroglobulin/C3, C4, C5 family of thioester-containing proteins. It has been reported that CD109 is expressed in a subset of hematopoietic cells, endothelial cells and several kinds of human tumors. Herein it is reported that the CD109 protein is preferentially expressed in lung squamous cell carcinomas compared with other types of lung carcinoma including adenocarcinomas, large cell carcinomas and small cell carcinomas. Immunohistochemical staining of surgically resected lung specimens using an anti-CD109 antibody detected CD109 expression in basal cells of bronchial and bronchiolar epithelia and myoepithelial cells of bronchial secretary glands, but not in bronchial and bronchiolar apical epithelial cells and alveolar epithelial cells. Furthermore, the CD109 immunoreactivity was observed in squamous cell carcinomas at a high frequency compared with other types of lung carcinoma. Although the detailed function of CD109 protein is unclear, these results suggest that CD109 expression may play a role in the development of lung squamous cell carcinoma.     

Rare expression of KIT (CD117) in lymphomas: a tissue microarray study of 1166 cases

AIMS: Imatinib mesylate specifically inhibits KIT tyrosine kinase activity, and has been proven to be effective in the treatment of gastrointestinal stromal tumours. Because other KIT-expressing malignancies might benefit from Imatinib therapy, we evaluated the distribution and expression of KIT in 1166 cases of malignant lymphoma. MATERIALS AND RESULTS: Tissue microarrays (TMAs) containing 824 non-Hodgkin's lymphoma (NHL) and 342 Hodgkin's lymphoma (HL) cases were immunohistochemically analysed for the expression of the KIT protein. Two KIT-positive NHLs were sequenced using polymerase chain reaction analysis. One T-cell lymphoma and one follicular lymphoma of the 747 NHL cases (0.3%) were positive for KIT. All HLs were Kit-negative. None of the KIT-positive cases showed a kit gene mutation. CONCLUSIONS: KIT expression is a very rare event in NHL and virtually absent in HL. In the few positive cases, the aberrant expression is not caused by a mutation in the 'hot-spots' of the kit gene, indicating that treatment of these tumours with Imatinib may be ineffective.     

The role of CD5 expression in thymic carcinoma: possible mechanism for interaction with CD5+ lymphoid stroma (microenvironment)

Recently, an increasing number of TFE3 rearrangement‐associated tumours have been reported, such as TFE3 rearrangement‐associated perivascular epithelioid cell tumours (PEComas), melanotic Xp11 translocation renal cancers and melanotic Xp11 neoplasms. We have suggested that these tumours belong to a single clinicopathological spectrum. ‘Xp11 neoplasm with melanocytic differentiation’ or ‘melanotic Xp11 neoplasm’ have been proposed to designate this unique neoplasm. Herein, we describe the first case of an Xp11 neoplasm with melanocytic differentiation to be described in the prostate, bearing the novel NONO–TFE3 gene fusion. This study both adds to the spectrum regarding melanotic Xp11 neoplasms and expands its gene fusion spectrum. Moreover, we discuss the relationship of these rare tumours to neoplasms such as conventional PEComas, alveolar soft part sarcomas, malignant melanomas, clear cell sarcomas and Xp11 translocation renal cancers.     

Clonality analysis performed using human androgen receptor assay in a rare case of undifferentiated thymic carcinoma coexisting with type AB thymoma

We report a very rare case of combined thymic carcinomas: undifferentiated thymic carcinoma coexisting with type AB thymoma. The precise mechanism underlying the coexistence of these tumors remains unknown. Therefore, we used clonality analysis to ascertain whether the two tumors were clonally related. A 63‐year‐old woman with thyroid cancer visited our hospital. Chest computed tomography also revealed an anterior mediastinal tumor. The patient was treated with total thyroidectomy and surgery for mediastinal tumors together with left upper lobe partial resection. The mediastinal tumor was pathologically diagnosed as undifferentiated thymic carcinoma coexisting with type AB thymoma. Multiple pulmonary metastases were detected in the patient and stage IV disease was diagnosed. The tumor was treatment‐resistant, and the patient received fourth‐line chemotherapy. We conducted clonality analysis using an improved human androgen receptor gene‐amplification assay that involves random X‐chromosome inactivation through methylation, followed by methylated gene‐specific PCR amplification after sample DNA digestion with HpaII, a methylation‐sensitive restriction enzyme. Clonality analysis demonstrated identical X‐chromosome inactivation in cells present in both thymoma and thymic carcinoma areas, and thus revealed clonal proliferation. The two lesions in the patient might have arisen through the transformation of a preexisting thymoma into a more malignant lesion.     

Expression of cancer testis antigens in thymic epithelial tumors

Cancer testis antigens (CTAs) are detected in cancer cells but not in healthy normal tissues, with the exception of gametogenic tissues. However, to our knowledge, expression of the antigens in thymic epithelial tumors has not been examined yet. We examined the immunohistochemical expression of five CTAs (MAGE-A, NY-ESO-1, MAGE-C1, SAGE and GAGE7) in 192 cases of thymic epithelial tumor. The CTAs were variably expressed in the thymic epithelial tumors. Type B component of type AB thymomas, type B1/B2/B3 thymomas, and thymic carcinomas showed a generally positive correlation between the malignancy grades and positive expression rates in four CTAs other than MAGE-C1. In thymic squamous cell carcinomas (SqCCs), four antigens except for MAGE-C1 showed high expression rates ranging from 23.1% to 43.6%. In the prognostic analysis, a positive expression of SAGE (P = 0.0485) and GAGE7 (P = 0.0289) were associated with a shorter overall survival in type B2/B3 thymomas, respectively. In thymic SqCC, a positive MAGE-A expression was significantly associated with an increased level of programmed death ligand in tumor-infiltrating lymphocytes (P = 0.0181). We showed (i) a frequent CTA expression, (ii) a general correlation of CTA expression with tumor malignancy grades and (iii) a prognostic impact in some of the CTAs.     

Immunohistochemical localization of Mcl-1 and bcl-2 proteins in thymic epithelial tumours

Mcl-1 protein is a new member of the bcl-2 protein family. It is believed to be a blocker of apoptosis but might be different from bcl-2 in the control of apoptosis. Using immunostaining of formalin-fixed, paraffin-embedded sections, we investigated the expression of Mcl-1 in 42 thymic epithelial tumours: three medullary thymomas, five mixed thymomas, seven cortical thymomas, eight well-differentiated thymic carcinomas, 14 squamous cell carcinomas, four lymphoepithelioma-like carcinomas and one undifferentiated carcinoma. bcl-2 immunocytochemical localization was also performed for comparison. High-grade thymic carcinomas, especially squamous cell carcinomas, revealed more intense Mcl-1 immunoreactivity as compared to other subtypes ( P < 0.001). In cases that co-expressed Mcl-1 and bcl-2, the less differentiated cells had more intense expression of bcl-2, while the more differentiated cells displayed stronger Mcl-1 immunoreactivity. The differential expression of Mcl-1 and bcl-2 in neoplastic cells provides evidence that these proteins may play different roles in the processes of programmed cell death in thymic neoplasms. The finding that thymic carcinomas have stronger immunoreactivity for Mcl-1 indicates that this protein could be a useful marker to differentiate aggressive thymic epithelial tumours from indolent ones.     

Neoplastic thymic epithelial cells of human thymoma support T cell development from CD4−CD8− cells to CD4+CD8+ cells in vitro

Human thymoma is a thymic epithelial cell tumour which often contains a large number of immature T cells and is frequently associated with autoimmune diseases. Since thymic epithelial cells play key roles in the development and selection of T cells in the normal thymus, we hypothesized that the neoplastic thymic epithelial cells of thymoma may support T cell differentiation in the tumour. We characterized CD4^?CD8^? cells in thymoma and applied an in vitro reconstitution culture system using the CD4^?CD8^? cells and the neoplastic epithelial cells isolated from thymoma. CD34, a stem cell marker, was expressed on 29.9 ± 12.2% of CD4^?CD8^? cells in thymoma. TCRγδ was expressed on 27.4 ± 15.1% of CD4^?CD8^? cells and CD19, a B cell marker, was expressed on 14.1 ± 23.1% of CD4^?CD8^? cells. CD4^?CD8^? cells expressed both IL-7R α-chain and common γ-chain. Purified CD4^?CD8^? cells from thymomas were cultured with the neoplastic epithelial cells, and their differentiation into CD4⁺CD8⁺ cells via CD4 single-positive intermediates was observed within 9 days' co-culture in the presence of recombinant IL-7. Furthermore, we examined the reconstitution culture using CD34⁺CD4^?CD8^? cells purified from normal infant thymus. The CD34⁺CD4^?CD8^? cells in normal thymus also differentiated to CD4⁺CD8⁺ cells in the allogeneic co-culture with the neoplastic epithelial cells of thymoma. These results indicate that the tumour cells of thymoma retain the function of thymic epithelial cells and can induce differentiation of T cells in thymoma.     

Interobserver variation in the classification of thymic tumours--a multicentre study using the WHO classification system

Aims:  To test the reproducibility of the current World Health Organization (WHO) classification of thymic epithelial tumours and to determine the level of interobserver variation within a group of pathologists, all with experience and expertise in thoracic pathology.
Methods and results:  Ninety-five thymic tumours were circulated to a group of 17 pathologists in the UK and The Netherlands over a 1-year period. Participants were asked to classify them according to WHO criteria. The diagnoses were subjected to statistical analysis and κ values calculated. The overall level of agreement was moderate (κ 0.45). When the categories were reduced in number by creating two groups, (A + AB + B1 + B2 and B3 + C), the level of agreement increased to 0.62. An alternative grouping (A + AB + B1 and B2 + B3 + C) increased it slightly further. The best agreement was in tumour types A and AB. Difficulties arose in distinguishing B1 tumours from B2 tumours and B2 tumours from B3 tumours.
Conclusions:  Although the WHO system describes a number of well-defined tumour types with clear diagnostic criteria, the overall level of agreement was moderate and improved if some groups were amalgamated.     

CD117 expression in operable oesophageal squamous cell carcinomas predicts worse clinical outcome

Aims

To investigate the aberrant expression of CD117 in oesophageal squamous cell carcinoma (SCC) and its prognostic significance.

Methods and results

Immunohistochemical staining for CD117 was performed on tissue microarray and routine tissue sections from 157 oesophageal SCC patients and 10 normal oesophageal epithelia adjacent to tumour. The positive rate of CD117 expression was 29.9% in oesophageal SCC tissues, whereas no CD117 expression was detected in the 10 normal oesophageal epithelia. CD117 expression was significantly associated with T stage (P < 0.001), distant metastasis (P = 0.015), lymph node metastasis (P = 0.019), and clinical stage (P = 0.021). Progression‐free survival in the patients with CD117‐positive tumours was shorter than that in the patients with CD117‐negative tumours (P = 0.010). In univariate analyses, CD117 expression was the most significant factor for overall survival of oesophageal SCC patients (P < 0.001), followed by lymph node metastasis (P = 0.001), T stage (P = 0.002), clinical stage (P = 0.006), distant metastasis (P = 0.020), and histological grade (P = 0.027). Multivariate analyses verified that CD117 expression was an independent prognostic marker for oesophageal SCC patients (P = 0.002). In addition, CD117 expression predicted poorer survival in patients without distant metastases.

Conclusions

CD117 expression in operable oesophageal SCC may be a valuable prognostic marker, and detection of its expression in clinical samples may be useful in defining a subclass of oesophageal SCCs with extremely poor clinical outcome, which may require a specially targeted treatment modality.     

Expression of apoptosis-related markers and HER-2/neu in thymic epithelial tumours

AIMS: To correlate the expression of a series of apoptotic and oncogene markers (including p53, Bcl-2, BAX, Bcl-XL, p21WAF,1/CIP1, cyclin D1, HER-2/neu) in thymic epithelial tumours with histological type, stage and resectability and to determine whether the information on HER-2/neu would be valuable in identifying patients who are eligible for anti-HER-2/neu treatment. METHODS AND RESULTS: Immunohistochemical stains were performed on 16 cases of non-neoplastic thymus, 63 thymomas and 17 thymic carcinomas. Fluorescence in-situ hybridization (FISH) for HER2 was performed to validate the gene amplification. Eighteen thymomas were positive for p53 and 14 of them were low-expressors, with positive cells below 10%. All thymic carcinomas revealed over-expression of p53 with positive cells either between 10% and 50% or >50%. The expression of p53 correlated with histological type and stage in thymoma. In both thymoma and thymic carcinoma, there was a statistically significant correlation between p53 status and resectability, with low expressors having a higher likelihood of being resectable. Thymic carcinomas, regardless of the histological subtypes, uniformly expressed Bcl-2, while thymomas showed no or only weak cytoplasmic immunoreactivity. Most thymomas and thymic carcinomas were negative for Bcl-XL, p21WAF,1/CIP1 and cyclin D1. The expression of BAX was inconsistent among different histological types. Nine thymic carcinomas revealed membranous positivity for HER-2/neu, but no HER2 gene amplification could be demonstrated by FISH in any of the cases. CONCLUSIONS: p53 and Bcl-2 are more implicated in the development of thymic carcinoma than thymoma. The higher level of p53 expression and the strong immunopositive pattern of Bcl-2 in thymic carcinomas have potential value in the differential diagnosis and prediction of aggressiveness and resectability. On account of the absence of HER2 amplification, patients would probably not benefit from anti-HER-2/neu treatment.     

KIT immunohistochemistry and mutation status in gastrointestinal stromal tumours (GISTs) evaluated for treatment with imatinib

AIMS: With the availability of effective but expensive treatment in the form of imatinib, accurate diagnosis of gastrointestinal stromal tumour (GIST) is extremely important. The aims of this study were: to describe the clinicopathological, immunohistochemical and molecular features of cases referred to a cancer centre with a possible diagnosis of GIST; to identify pitfalls in the performance and interpretation of KIT immunohistochemistry; to define the role of KIT mutation testing in making a diagnosis of GIST. METHODS AND RESULTS: Morphological review, KIT immunohistochemistry and mutation testing were performed on all cases referred with a diagnosis of GIST or where the diagnosis was under serious consideration on the basis of KIT immunopositivity with a view to treating with imatinib. Thirty-seven cases met the inclusion criteria. Of these, 26 were classified as GIST and 11 as non-GIST. Most GISTs showed strong diffuse membranous, cytoplasmic or paranuclear KIT immunopositivity. Some non-GISTs demonstrated patchy cytoplasmic KIT immunopositivity related to the immunohistochemical protocol used in the external laboratory, which led to erroneous diagnoses of GIST in nine (24%) cases. KIT mutations involving exons 11 or 9 were identified in 22 (88%) GISTs tested and none of the non-GISTs. CONCLUSIONS: An accurate diagnosis of GIST can be made on clinicopathological and immunohistochemical criteria without the need for mutational analysis in most cases, provided proper attention is paid to the immunohistochemical protocol used and, most importantly, control material. False-positive diagnoses of GIST potentially leading to inappropriate treatment with imatinib are more common than missed diagnoses.     

Immunohistochemical and mutational analysis of apoptosis-inducing factor (AIF) in colorectal carcinomas

Apoptosis-inducing factor (AIF) has a principal role in caspase-independent apoptosis. In addition, AIF has a vital oxidoreductase activity that is required for cell survival. It may be important to identify the alterations of the AIF gene and AIF protein expression to see the function of AIF in human cancers. In this study we analyzed the expression of AIF protein in 103 colorectal carcinomas by immunohistochemistry. We also analyzed the mutation in exons 10-15 of AIF encoding the region that possesses the cell death function of AIF by single-strand conformation polymorphism (SSCP) in 48 colorectal, 48 gastric, 48 breast and 48 hepatocellular carcinomas, and 48 leukemias. By immunohistochemistry, AIF protein expression was detected in both cancer cells and normal mucosal epithelial cells in all of the 103 colorectal carcinoma tissues. However, the cancer cells showed higher intensities of AIF immunostaining than the normal cells in 83 cases (80.5%). AIF immunoreactivities were observed in the cancers irrespective of their location or the depth of invasion. Mutational analysis detected one AIF mutation in the colorectal carcinomas, but none in the other cancers. The AIF mutation detected was a two-base deletion mutation in intron 15. The increased expression of AIF in the malignant colorectal epithelial cells compared to the normal mucosal epithelial cells suggests that AIF expression may play a role in colorectal tumorigenesis. The data also suggest that somatic mutation of AIF is a rare event in common human cancers.