Henry F. Frierson

Activating c-kit Gene Mutations in Human Germ Cell Tumors

The c-kit gene encodes a tyrosine kinase receptor (KIT) that is required in normal spermatogenesis and is expressed in seminomas and dysgerminomas, a subset of human germ cell tumors (GCTs). To determine whether activating mutations of the c-kit gene occur in GCTs, primary tissue samples of 33 testicular and ovarian tumors were examined for mutations in the juxtamembrane and phosphotransferase domains by polymerase chain reaction amplification and DNA sequencing. A novel missense mutation (D816H) was found in the phosphotransferase domain in tumors of seminoma/dysgerminoma differentiation. The c-kit alleles in nonneoplastic tissues from these patients were wild type, suggesting that the mutant alleles were acquired and selected for during malignant transformation. In cell transfection experiments, the D816H mutant protein was a constitutively activated kinase and was constitutively phosphorylated on tyrosine residues. This is the first description of an activating c-kit mutation in GCTs and is evidence that the KIT signal transduction pathway is important in the pathogenesis of neoplasms with seminoma differentiation.

Discovery and validation of new protein biomarkers for urothelial cancer: a prospective analysis

BACKGROUND Non-invasive methods for diagnosis of urothelial carcinoma have reduced specificity in patients with non-malignant genitourinary disease or other disorders. We aimed to use mass spectrometry and bioinformatics to define and validate a cancer-specific proteomic pattern. METHODS We used capillary-electrophoresis-coupled mass spectrometry to obtain polypeptide patterns from urine samples of 46 patients with urothelial carcinoma and 33 healthy volunteers. From signatures of polypeptide mass, we established a model for predicting the presence of cancer. The model was refined further by use of 366 urine samples obtained from other healthy volunteers and patients with malignant and non-malignant genitourinary disease. We estimated the proportion of correct classifications from the refined model by applying it to a masked group containing 31 patients with urothelial carcinoma, 11 healthy individuals, and 138 patients with non-malignant genitourinary disease. We also sequenced several diagnostic polypeptides for urothelial carcinoma. FINDINGS We identified a diagnostic urothelial-carcinoma pattern of 22 polypeptide masses. On masked assessment, prediction models based on these polypeptides correctly classified all samples of urothelial carcinoma (sensitivity 100% [95% CI 87-100) and all healthy samples (specificity 100% [84-100]). Correct identification of patients with urothelial carcinoma from those with other malignant and non-malignant genitourinary disease ranged from 86% to 100%. A prominent polypeptide from the diagnostic pattern for urothelial carcinoma was identified as fibrinopeptide A-a known biomarker of ovarian cancer and gastric cancer. INTERPRETATION Validation of a highly specific biomarker pattern for urothelial carcinoma in a large group of patients with various urological disorders could be used in the diagnosis of other diseases that are identified in urine samples or in other body fluids.

Large-scale delineation of secreted protein biomarkers overexpressed in cancer tissue and serum

Genetic alterations in tumor cells often lead to the emergence of growth-stimulatory autocrine and paracrine signals, involving overexpression of secreted peptide growth factors, cytokines, and hormones. Increased levels of these soluble proteins may be exploited for cancer diagnosis and management or as points of therapeutic intervention. Here, we combined the use of controlled vocabulary terms and sequence-based algorithms to predict genes encoding secreted proteins from among ≈12,500 sequences represented on oligonucleotide microarrays. Expression of these genes was queried in 150 carcinomas from 10 anatomic sites of origin and compared with 46 normal tissues derived from the corresponding sites of tumor origin and other body tissues and organs. Of 74 different genes identified as overexpressed in cancer tissues, several encode proteins with demonstrated clinical diagnostic application, such as α-fetoprotein in liver carcinoma, and kallikreins 6 and 10 in ovarian cancer, or therapeutic utility, such as gastrin-releasing peptide/bombesin in lung carcinomas. We show that several of the other candidate genes encode proteins with high levels of tumor-associated expression by immunohistochemistry on tissue microarrays and further demonstrate significantly elevated levels of another novel candidate protein, macrophage inhibitory cytokine 1, a distant member of the tranforming growth factor-β superfamily, in the serum of patients with metastatic prostate, breast, and colorectal carcinomas. Our results suggest that the combination of annotation/protein sequence analysis, transcript profiling, immunohistochemistry, and immunoassay is a powerful approach for delineating candidate biomarkers with potential clinical significance and may be broadly applicable to other human diseases.

Comprehensive analysis of HE4 expression in normal and malignant human tissues

The HE4 (WFDC2) gene encodes a WAP-type four disulphide core domain-containing protein with a presumptive role in natural immunity. Multiple studies have consistently identified upregulation of HE4 gene expression in carcinomas of the ovary; however, the expression in normal and malignant adult tissues has not been examined in detail. Here, we examined the expression of the HE4 gene and protein in a large series of normal and malignant adult tissues by oligonucleotide microarray and tissue microarray, respectively. HE4 gene expression was highest in normal human trachea and salivary gland, and to a lesser extent, lung, prostate, pituitary gland, thyroid, and kidney. In a series of 175 human adult tumors, gene expression was highest in ovarian serous carcinomas. However, adenocarcinomas of the lung, and occasional breast, transitional cell and pancreatic carcinomas had moderate or high levels of HE4 expression. Using tissue microarrays and full tissue sections of normal and 448 neoplastic tissues, HE4 immunoreactivity was found in normal glandular epithelium of the female genital tract and breast, the epididymis and vas deferens, respiratory epithelium, distal renal tubules, colonic mucosa, and salivary glands, consistent with HE4 gene expression. In addition to consistent positivity in ovarian carcinoma, some pulmonary, endometrial, and breast adenocarcinomas, mesotheliomas, and less often, gastrointestinal, renal and transitional cell carcinomas were also positive. Knowledge of the expression patterns of HE4 in our survey is useful for application in histopathologic diagnosis, and should be taken into consideration in future studies that examine the role of HE4 as a serological tumor biomarker or as a target for gene-based therapy.

Basaloid squamous cell carcinoma of the head and neck. A clinicopathologic and immunohistochemical study of 40 cases.

In this study of 40 cases of basaloid squamous cell carcinoma, 83% arose in the pyriform sinus, base of tongue, tonsil, and larynx. The 35 men and five women ranged in age from 27 to 88 years (median 62). In patients for whom social habits were recorded, 24 of 26 patients were smokers and 22 of 25 drank ethanol. Most presented with stage III or IV disease. Twenty-seven patients had regional metastases at the time of presentation and 15 developed distant metastases. Seventeen patients died with disease (median survival 18 months). The tumors were composed of moderately pleomorphic basaloid cells forming nests, cords, and frequent cribriform patterns. Squamous dysplasia of surface mucosa, focal squamous differentiation within invasive basaloid squamous cell carcinoma, or foci of conventional squamous cell carcinoma were present, alone or in combination. All studied neoplasms were immunohistochemically positive for keratins with the 34(3E12 antibody. Approximately 80% were immunoreactive using AE1/AE3 or CAM 5.2. Epithelial membrane antigen, carcinoembryonic antigen, and SI00 protein were found in 83%, 53%, and 39%, respectively, of the cases. Diffuse, weak immunoreactivity for neuronspecific enolase was seen in 75% of tumors. Synaptophysin, chromogranin, muscle-specific actin, and glial fibrillary acidic protein were absent. Basaloid squamous cell carcinoma has been confused with adenoid cystic carcinoma and small cell undifferentiated carcinoma, but is usually distinguishable in routine hematoxylin and eosin- stained sections, or, in rare problematic cases, with the aid of immunohistochemical studies. Distinction is warranted because the biologic behavior of basaloid squamous cell carcinoma differs from that of both of these lesions.

Cdx2 as a marker of epithelial intestinal differentiation in the esophagus

The histologic diagnosis of Barretts esophagus requires the presence of goblet cells, but this finding may not be the earliest indicator of intestinal metaplasia. We used immunohistochemistry to detect Cdx2, a transcriptional regulator important in the early differentiation and maintenance of intestinal epithelium, in 134 esophageal biopsy or resection specimens, including 62 with junctional-type epithelium (13 of which had equivocal histologic features of Barretts epithelium), 34 with Barretts epithelium without dysplasia, and 38 with Barretts epithelium and dysplasia or carcinoma (13 low-grade dysplasias, 19 high-grade dysplasias, and 6 adenocarcinomas). We also performed PAS-alcian blue staining (pH 2.5) on adjacent sections. Cdx2 was observed in all cases of Barretts epithelium. In some dysplasias (chiefly high-grade) and adenocarcinomas, there was diminution or focal loss of detectable protein. Cdx2 was detected in 20 of 62 cases (30%) of junctional-type epithelium, including 10 of 13 (77%) with equivocal histologic features of Barretts epithelium. Acid mucin was present in goblet cells and non-goblet columnar cells in all cases of Barretts esophagus and in non-goblet columnar cells in 48 of 62 cases (77%) with junctional-type epithelium only, including 17 of 20 (85%) that were Cdx2 positive and 31 of 42 (74%) that were Cdx2 negative. These results provide evidence that Cdx2 protein is a sensitive marker of intestinal metaplasia in the upper gastrointestinal tract and may be useful in detecting histologically equivocal cases of Barretts esophagus. Cdx2 is present in dysplasia and adenocarcinoma, with some loss of protein primarily in high-grade dysplasia and adenocarcinoma. Acid mucin in non-goblet columnar cells is a common feature of Barretts and junctional-type epithelium and may not always be indicative of intestinal metaplasia.

Sinonasal undifferentiated carcinoma. An aggressive neoplasm derived from schneiderian epithelium and distinct from olfactory neuroblastoma.

Eight cases of a highly aggressive undifferentiated carcinoma of the nasal cavity and paranasal sinuses are described. The patients, who ranged in age from 30-77 years, had multiple sinonasal symptoms, and each had involvement of the nasal cavity, maxillary antrum, and ethmoid sinus. Six tumors extended into the orbital bones, and five penetrated the cranial cavity. Five patients died of disease from 1 to 41 months after diagnosis (median: 4 months), and three are alive with tumor less than 1 year following diagnosis. Microscopically, the neoplasms formed nests, trabeculae, and sheets containing medium-sized cells with small to moderate amounts of eosinophilic cytoplasm. A high mitotic rate, tumor necrosis, and prominent vascular permeation were characteristic. Seven neoplasms were immunoreactive for cytokeratin, five for epithelial membrane antigen, and four for neuron-specific enolase. Ultrastructurally, occasional small desmosomes and rare membrane-bound, dense-core granules were observed. Sinonasal undifferentiated carcinoma is a distinctive clinicopathologic entity that must be distinguished from other, less aggressive sinonasal neoplasms.

Prognostic factors in squamous cell carcinoma of the lower lip

One hundred eighty-seven squamous cell carcinomas of the lower lip were examined microscopically to identify parameters that might predict metastasis and patient outcome. Excision specimens of 157 nonmetastasizing carcinomas (group I) were compared with specimens from 30 tumors that had metastasized (group II). The following features were recorded: architectural pattern; microscopic thickness (mm); cytologic grade; presence of muscle, nerve, or vessel invasion; inflammatory response; and mitotic rate. The mean thickness was 2.5 mm for group I tumors and 7.5 mm for group II tumors. Seventy-six per cent of the group I tumors were 3 mm thick or less, whereas only one group II lesion (3 per cent) was less than 3 mm thick. Five per cent of the group I neoplasms, compared with 77 per cent of the group II tumors, were at least 6 mm thick. Perineural invasion was seen in 5 per cent of the group I and 41 per cent of the group II lesions. Three per cent of the group I carcinomas had a dispersed pattern, compared with 57 per cent of those in group II. One group I lesion (0.6 per cent) and 37 per cent of the group II tumors were grade 4. Each of these differences was statistically significant (P less than 0.0001). For all lesions studied, metastases had occurred in 60 per cent with perineural invasion, 74 per cent measuring 6 mm or more, 77 per cent with a dispersed pattern, and 92 per cent that were grade 4. These important prognostic variables were best evaluated in the deeper portions of the lesions.

Cdx2 Protein Expression in Normal and Malignant Human Tissues: An Immunohistochemical Survey Using Tissue Microarrays

Cdx2 has been identified as a marker of colon cancer in RNA-profiling experiments. We show here that the detection of Cdx2 protein by immunohistochemistry correlates well with RNA transcript levels as detected by oligonucleotide microarrays. Using tissue microarrays containing most normal tissue types and an antibody to the Cdx2 protein, strong diffuse Cdx2 staining was only seen in the nuclei of small and large intestinal epithelium and portions of the pancreatic duct system. In tissue microarrays containing 745 cancers from many anatomic sites, colonic adenocarcinomas showed strong extensive staining in 90% of cases, with adenocarcinomas of the stomach, esophagus, and ovary (endometrioid and mucinous types) showing extensive staining in only 20–30% of cases. Other types of carcinomas showed extensive staining in only ≤1% of cases. Of 30 neuroendocrine tumors examined, carcinoids of the midgut and hindgut had the most cases with extensive staining (73% and 44%, respectively), thus paralleling the distribution of Cdx2 expression in adenocarcinomas. Cdx2 shows a limited range of expression in the spectrum of human tissues and neoplasia and thus may have utility in determining the site of origin of tumors in certain clinical situations.

Analysis of MYB expression and MYB-NFIB gene fusions in adenoid cystic carcinoma and other salivary neoplasms.

Recent studies have shown that the recurrent t(6;9)(q22–23;p23–24) translocation in adenoid cystic carcinoma results in a novel fusion of the MYB proto-oncogene with the transcription factor gene NFIB. To determine the frequency of this finding, we used RT-PCR assays of the MYB and MYB-NFIB fusion transcripts, and immunohistochemistry for the MYB protein, to study adenoid cystic carcinomas and other epithelial tumors of the salivary glands, and head and neck region. MYB-NFIB fusion transcript was detected in 25 of 29 (86%) frozen adenoid cystic carcinoma tumor samples, and in 14 of 32 (44%) formalin-fixed paraffin-embedded adenoid cystic carcinoma tumor specimens. In contrast, the MYB-NFIB fusion was not expressed in non-adenoid cystic carcinoma neoplasms of the head and neck, confirming the high specificity of the MYB-NFIB fusion. Adenoid cystic carcinomas from various anatomic sites, including salivary gland, sinonasal cavity, tracheobronchial tree, larynx, breast, and vulva were repeatedly fusion-positive, indicating that adenoid cystic carcinomas located in different anatomic sites not only have important morphologic features in common, but also probably evolve through activation of the same molecular pathways. Studies of the expression of MYB revealed that 89% of the tumors, including both fusion-positive and fusion-negative cases, overexpressed MYB RNA. Similarly, 82% of adenoid cystic carcinomas stained positive for MYB protein, compared with 14% of non-adenoid cystic carcinoma neoplasms, indicating that MYB immunostaining may be useful for the diagnosis of adenoid cystic carcinoma, but that neoplasms sometimes in the differential diagnosis are also labeled. The latter are, however, fusion-negative. In summary, our studies show that MYB activation through gene fusion or other mechanisms is a major oncogenic event in adenoid cystic carcinoma occurring at various anatomic sites. In addition to being a diagnostically useful biomarker for adenoid cystic carcinoma, MYB and its downstream effectors are also novel potential therapeutic targets.