In a recent study, a unique gene expression signature was observed when comparing esophageal squamous cell carcinoma (ESCC) epithelial cells to normal esophageal epithelial cells using laser capture microdissection (LCM) and cDNA microarray technology. To validate the expression of several intriguing genes from that study (KRT17, cornulin, CD44, and EpCAM), we employed two new technologies, expression microdissection (xMD) for high-throughput microdissection facilitating protein analysis and RNAscope for the evaluation of low abundant transcripts in situ. For protein measurements, xMD technology was utilized to specifically procure sufficient tumor and normal epithelium from frozen human tissue for immunoblot analysis of KRT17 (CK17) and cornulin. A novel in situ hybridization method (RNAscope) was used to determine the transcript level of two relatively low expressed genes, CD44 and EpCAM in both individual formalin-fixed paraffin-embedded (FFPE) tissue sections and in an ESCC tissue microarray (TMA). The results successfully confirmed the initial expression pattern observed for all four genes, potentially implicating them in the pathogenesis of ESCC. Additionally, the study provides important methodological information on the overall process of candidate gene validation.
Expression microdissection; esophageal squamous cell carcinoma; RNAscope; immunoblot
Current technologies for measuring protein expression across a tissue section are based on mass spectrometry or in situ detection such as immunohistochemistry. However, due to the inherent molecular complexity of tissue samples and the large dynamic range of protein expression in cells, current approaches are often unable to measure moderate- and low-abundant proteins. In addition, they do not provide information on the physico- chemical properties of the proteins studied. To address these problems, we are developing a new pre-analytic methodology termed Layered Electrophoretic Transfer (LET) that selectively separates and processes proteins from an intact tissue section without compromising important two-dimensional histological information. LET offers two potential advantages over standard techniques: 1) A reduced complexity of the tissue proteome for subsequent analysis; 2) An opportunity to assess the biochemical status of proteins as they exist in situ. As an initial proof-of-concept, we demonstrate here that the protein content from a mixture of molecular weight standards, human tissue lysates, and tissue sections can be successfully transferred and separated using LET, and further demonstrate that the method can be coupled with immunoblotting or mass spectrometry for downstream measurements. LET technology represents a new pre-analytic tool for interrogating the proteome in tissue sections while preserving valuable spatial information.
2D protein separation; layered electrophoretic transfer; tissue protein mapping; proteomics
Testicular fibroma of gonadal stromal origin is a rare benign tumor of testis which usually presents as a slow growing testicular mass. Only 25 cases of testicular fibroma have been reported in the literature. Presence of minor sex cord elements in this tumor is even rarer. We report a case of testicular fibroma with minor sex cord elements that involved almost the entire testis and tunica vaginalis. The patient presented with hydrocele, a rare presentation for this entity. The rarity of the diagnosis and the clinical presentation prompted this case report.
testicular fibroma; minor sex cord elements; gonadal stromal tumor
Teratoid Wilms’ tumor is an unusual variant of nephroblastoma in which heterologous tissue predominates. We report a case of teratoid Wilms’ tumor in a 2-year-old male. Right sided abdominal mass was the presenting complaint. Ultrasonography of the abdomen showed a mass in the right kidney. Histopathological examination revealed blastemal, epithelial, and mesenchymal components along with areas presenting heterologous elements. More than 75% predominance of squamous differentiation with the keratin pearl formation was observed. The patient underwent nephrectomy and was followed post-operatively for 1 year and was normal.
Nephroblastoma; squamous differentiation; teratoid Wilms’ tumor
Hepatoblastoma (HBL) is a rare primary malignant liver tumor affecting mainly pediatric patients in the age group 6 months to 3 years. Presentation of HBL in the neonatal period is rare. HBL can be diagnosed on cytology along with subtyping. Estimation of serum alpha-fetoprotein (AFP) is essential as a tumor marker. Fetal type HBL usually shows high AFP level. In this report, diagnosis of HBL in a 10-day-old baby with low serum AFP is being described for its unusual presentation.
Alpha-fetoprotein; fine needle aspiration cytology; hepatoblastoma
Hemangioendothelioma is a rare vascular tumor of intermediate malignancy. Cytologically, it can simulate a non-vascular malignant tumor. We report two cases of this tumor, which were misdiagnosed at cytology. In the first case, a 27-year-old man presented with an anterior abdominal wall tumor. Fine needle aspiration cytology (FNAC) of the tumor showed polygonal cells with vacuolated cytoplasm in clusters having moderate nuclear atypia in a background of necrosis. A diagnosis of metastatic carcinoma was made. The histological examination showed features of epithelioid hemangioendothelioma. In the second case, a 13-year-old female child presented with unilateral enlargement of the right tonsil. At ultrasound-guided FNAC, a diagnosis of, ‘small round cell tumor, could be consistent with alveolar rhabdomyosarcoma,’ was made. The histological examination showed features of papillary intralymphatic angioendothelioma (Dabska's tumor). We conclude that epithelioid hemangioendothelioma should be considered in the differential diagnosis of metastatic carcinoma and small round cell tumor even at unusual sites.
Carcinoma; epithelioid hemangioendothelioma; small round cell tumor
Esophageal squamous cell carcinomas (ESCC) are usually asymptomatic and go undetected until they are incurable. Cytological screening is one strategy to detect ESCC at an early stage and has shown promise in previous studies, although improvement in sensitivity and specificity are needed. Proteases modulate cancer progression by facilitating tumor invasion and metastasis. In the current study, matrix metalloproteinases (MMPs) were studied in a search for new early detection markers for ESCC.
Protein expression levels of MMPs were measured using zymography in 24 cases of paired normal esophagus and ESCC, and in the tumor-associated stroma and tumor epithelium in one sample after laser capture microdissection (LCM). MMP-3 and MMP-10 transcripts in both the epithelium and stroma in five cases were further analyzed by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR).
Gelatin zymography showed bands corresponding in size to MMP-2, MMP-3, MMP-9, and MMP-10 enzymes in each of the 24 cancer cases. MMP levels tended to be higher in tumors than paired normal tissue; however, only the 45 kDa band that corresponds to the activated form of MMP-3 and MMP-10 was strongly expressed in all 24 tumors with little or no expression in the paired normal foci. LCM-based analysis showed the 45 kDA band to be present in both the stromal and epithelial components of the tumor microenvironment, and that MMP-3 and MMP-10 mRNA levels were higher in tumors than paired normal tissues for each compartment.
Increased levels of MMPs occur in ESCC suggesting their up-regulation is important in esophageal tumorigenesis. The up-regulated gene products have the potential to serve as early detection markers in the clinic.
Glomus tumors are uncommon, with an estimated incidence of 1.6%. Cytological descriptions of this tumor are few. We report a 15-year-old boy presenting with a painful subungual swelling. Fine needle aspiration cytology showed uniform cells with homogeneous chromatin and scanty cytoplasm. Cytology was reported as “suggestive of glomus tumor”. Histopathological examination confirmed the diagnosis. Careful cytomorphological examination supported by appropriate clinical history should suggest the diagnosis of glomus tumor and help in preoperative diagnosis.
Fine needle aspiration cytology; glomus tumor; subungal
The purpose of this study was to examine solid tumor heterogeneity on a cellular basis using tissue proteomics that relies on a functional relationship between Laser Capture Microdissection (LCM) and biological mass spectrometry (MS). With the use of LCM, homogeneous regions of cells exhibiting uniform histology were isolated and captured from fresh frozen tissue specimens, which were obtained from a human lymph node containing breast carcinoma metastasis. Six specimens ∼50 000 cell each (three from tumor proper and three from tumor stroma) were collected by LCM. Specimens were processed directly on LCM caps, using sonication in buffered methanol to lyse captured cells, solubilize, and digest extracted proteins. Prepared samples were analyzed by LC/MS/MS resulting in more than 500 unique protein identifications. Decoy database searching revealed a false-positive rate between 5 and 10%. Subcellular localization analysis for stromal cells revealed plasma membrane 14%, cytoplasm 39%, nucleus 11%, extracellular space 27%, and unknown 9%; and tumor cell results were 5%, 58%, 26%, 4%, and 7%, respectively. Western blot analysis confirmed specific linkage of validated proteins to underlying pathology and their potential role in solid tumor heterogeneity. With continued research and optimization of this method including analysis of additional clinical specimens, this approach may lead to an improved understanding of tumor heterogeneity, and serve as a platform for solid tumor biomarker discovery.
laser capture microdissection (LCM); mass spectrometry (MS); solid tumor heterogeneity
Fine needle aspiration cytology (FNAC) may be diagnostic in candidates with indeterminate solitary pulmonary nodules (SPNs) suspicious of bronchogenic carcinoma.
The study was performed to evaluate the usefulness of computed tomography (CT)-guided FNAC in our centre.
Materials and Methods:
All the cases had a strong clinical suspicion of lung cancer, negative bronchoscopy, negative sputum cytology for malignant cells and acid fast bacilli. A thorough radiological evaluation was made to rule out primary malignancy elsewhere.
A total of 94 patients were studied in one year. May-Grünwald-Giemsa stain was used for the smears. The cytological diagnosis was correlated with clinical-radiological follow-up and biopsy to arrive at a final diagnosis. The procedure had a high sensitivity and specificity. Chi-square test was used to calculate statistical significance. Tumor of more than three centimeter and immediate cytological assessment significantly increased the yield. Review of slides added two cases of malignancy that were missed initially. There were very few complications.
CT-guided FNAC was an accurate and safe procedure for SPNs.
Bronchogenic carcinoma; computed tomography; fine needle aspiration cytology