Expression and diagnostic values of calretinin and CK5/6 in cholangiocarcinoma.

BACKGROUND: Mesothelin, a mesothelial marker, has been found expressed in and as a potential treatment target of cholangioacarcinoma (CC). It is possible that CC may be derived from the cells sharing mesothelial markers. However, the expression of other mesothelial markers in CC is largely unknown. METHODS: Thirty CC cases (10 extrahepatic and 20 intrahepatic) were retrieved from our institutional archive. The immunohistochemical study of Calretinin (DC8), WT1 (6F-H2), Lymphatic Endothelial Marker (D2-40), CK5/6 (D5/16 B4) and CK19 (b170) was done on formalin fixed paraffin embedded sections for 2-3 blocks of each case. We compared the expression levels between CC and normal bile duct (NBD) on the same block. RESULTS: All of the CC and NBD are positive for CK19 (23/23) and negative for WT1 (0/23) and D2-40 (0/23), except one CC positive for D2-40(1/30, 3.3%) and one NBD positive for WT1 (1/23, 4.3%). Calretinin immunoreactivity was detected in 52.2% (12/23) of CC, but none in NBD (0/23). CK5/6 was also detectable in 73.3% (22/30) of CC and all NBD (30/30). Increased expression of calretinin and reduced expression of CK5/6 were more likely associated with CC than NBD (P < 0.001 and P = 0.002, respectively). The sequential staining pattern of positive calretinin and negative CK5/6 in calretinin negative cases has a sensitivity of 69.57% and a specificity of 100% for differentiating CC from NBD. CK5/6 expression was also more likely associated with well-differentiated CC (7/7 versus 12/20 in moderately differentiated, and 9/10 in poorly differentiated, P = 0.019) and extrahepatic CC (10/10 versus 12/20 in intrahepatic, P = 0.029), but there was no association between the calretinin expression and the CC grade or location. CONCLUSION: Calretinin and CK5/6 immunohistochemical stains may be useful for diagnosing a CC. Their immunohistochemical results should be interpreted with caution in the cases with differential diagnoses of mesothelioma and CC. A full mesothelioma panel, including WT1 and/or D2-40, is recommended to better define a mesothelial lineage. The biology of calretinin and CK5/6 expression in CC is unclear, but might shed light on identifying therapeutic targets for CC.

Expression of Notch signaling components in cutaneous foreign body and sarcoidal granulomas and fusing macrophages.

The evolutionarily conserved Notch signaling pathway affects tissue-specific cell differentiation, proliferation, and apoptosis. In the immune system, Notch has been implicated in the development and function of both adoptive and innate immune cells. Notch signaling is initiated by Notch receptor binding to cognate ligands, which results in the enzymatic cleavage and intranuclear translocation of the intracellular domain of Notch receptor (ICN). Recent murine models of chronic inflammation highlighted a critical role for a Notch ligand, Delta-like ligand (Dll)-4, in granuloma formation. In this study, we aimed to assess Notch-1 receptor activation and Dll4 expression in human cutaneous granulomas and in cultured human macrophages and multinucleated giant cells. ICN1 and Dll4 expression was evaluated by immunohistochemistry of cutaneous foreign body (n = 15) and sarcoidal (n = 19) granulomas. The results showed consistent intranuclear staining for ICN1 in foreign body but not in sarcoidal granulomas and strong cytoplasmic staining for Dll4 in mononuclear histiocytes and multinucleate giant cells in both types of granulomas. Additionally, immunofluorescence confocal microscopy showed ICN1 and Dll4 expression by cultured human macrophages undergoing fusion in the presence of granulocyte-macrophage colony-stimulating factor and interleukin-4. These findings indicate a potential role for the Notch-1-Dll4 signaling pathway in foreign body-induced granulomatous reactions and possibly distinct Notch pathway utilization in sarcoidal granulomas.

Lymphatic invasion predicts aggressive behavior in melanocytic tumors of uncertain malignant potential (MELTUMP).

Lymphatic invasion (LI) identified by immunohistochemical (IHC) staining is common in primary cutaneous melanoma, and LI has been shown to be an independent prognostic factor in melanoma. Its prognostic significance in melanocytic tumors of uncertain malignant potential (MELTUMPs) has not been well characterized. This study included 32 patients with provisional diagnoses of MELTUMP. Lesions were evaluated for tumor thickness, the presence of ulceration, mitotic figures, mitotic figures at the base, tumor infiltrating lymphocytes, as well as peritumoral and intratumoral lymphatic density. Dual IHC staining was used to microscopically detect lymphatic endothelium (podoplanin) containing melanoma cells (S100), with the aid of multispectral imaging in select cases. Univariate analysis was performed to identify associations between clinical and pathologic variables and melanoma-related events. The 32 patients had a median follow-up of 111 months. Two patients subsequently died of melanoma-related disease, 1 died of unknown causes, 5 developed nodal metastases, and the remainder showed no evidence of progressive disease. LI was identified in 8/32 patients (25%) by dual IHC staining, which included the 2 patients who died of melanoma-related disease, 1 patient with bulky nodal metastasis, 1/4 patients with microscopic nodal metastases, and 4 patients who showed no evidence of progressive disease. The presence of LI was associated with melanoma metastases or melanoma-related death (P=0.05). The presence of LI by dual IHC in MELTUMPs is associated with a poorer prognosis, specifically with melanoma metastasis, and may therefore serve as a useful prognostic factor for risk stratification of patients with these diagnostically challenging lesions.

Transoral robotic surgery and adjuvant therapy for oropharyngeal carcinomas and the influence of p16 INK4a on treatment outcomes.

OBJECTIVES/HYPOTHESIS: To determine the prognostic influence of p16(INK4a) immunohistochemistry on the survival of resectable oropharyngeal carcinomas (OPSCC). STUDY DESIGN: Retrospective pathologic evaluation of a prospective single-arm cohort study at a tertiary referral center. METHODS: There were 48 patients with resectable OPSCC who consented for transoral robotic surgery (TORS) and banked tissue specimen for assessment. TORS was with or without adjuvant radiation or chemoradiation. Main outcome measures were p16(INK4a) status, human papillomavirus status, local-regional disease control, and overall, disease-specific, and disease-free survival. RESULTS: p16(INK4a) and HPV positivity were identified in 73% and 74% of patients respectively. With a median follow-up of 38.8 months (2.5-63.3 months), only one local-regional relapse has occurred in both the p16(INK4a)-positive and p16(INK4a) -negative cohorts. No disease-specific, disease-free, and overall survival differences were observed between p16(INK4a) -positive and p16(INK4a)-negative patients (P = .446, P = .277, P = .643, respectively). CONCLUSIONS: p16(INK4a) was not prognostic in resectable OPSCC when treated with an initial TORS approach.

Is gene expression profiling of head and neck cancers ready for the clinic?

One of the major challenges in the head and neck oncology clinic is the need to identify biomarkers and/or gene expression signatures that complement, strengthen and increase the sensitivity and specificity of the current clinicopathologic analyses. Microarray analysis of head and neck tumors has demonstrated that the combined influence of many genes or biomarkers can make superior identifiers and/or predictors of tumor behavior and patient outcome. Here, an update of the recent literature on the prognostic and predictive value of microarrays for patients with head and neck squamous cell carcinomas is presented. Microarray technology has the potential for improved decision-making and corroboration within the clinical setting. However, further integration, standardization, validation and research are required before the use of microarray analysis is ready for routine clinical management of head and neck squamous cell carcinomas.

Lab-on-a-chip for oral cancer screening and diagnosis.

Oral squamous cell carcinoma (OSCC) is a disfiguring and deadly cancer. Despite advances in therapy, many patients continue to face a poor prognosis. Early detection is an important factor in determining the survival of patients with OSCC. No accurate, cost-efficient, and reproducible method exists to screen patients for OSCC. As a result, many patients are diagnosed at advanced stages of the disease. Early detection would identify patients, facilitating timely treatment and close monitoring. Mass screening requires a rapid oral cancer diagnostic test that can be used in a clinical setting. Current diagnostic techniques for OSCC require modern laboratory facilities, sophisticated equipment, and elaborate and lengthy processing by skilled personnel. The lab-on-chip technology holds the promise of replacing these techniques with miniaturized, integrated, automated, inexpensive diagnostic devices. This article describes lab-on-chip devices for biomarker-based identification of oral cancer. Similar methods can be employed for the screening of other types of cancers.

Activated Vav2 modulates cellular invasion through Rac1 and Cdc42 in oral squamous cell carcinoma.

The Rho family of GTPases regulates cellular adhesion and motility. Guanine nucleotide exchange factors (GEFs) in turn regulate GTPases by promoting nucleotide exchange from GDP to GTP. We have determined that GTP-bound Rac1 is elevated in invasive oral squamous cell carcinoma (OSCC) cell lines. Because of the critical role of invasion in the progression and metastasis of OSCC, we investigated if the GEF Vav2 modulated Rac1 and Cdc42 activation and thus influenced OSCC invasion. Expression levels of Vav2 did not correlate with invasion but phosphorylated or activated Vav2 was associated with the more invasive cell lines. Transfection of activated Vav2 into the immortalized keratinocyte cell line HaCat and a low-level expressing Vav2 invasive OSCC cell line resulted in increased GTP-bound Rac1 and Cdc42 and increased invasion. Thus, activation of Vav2 appears to modulate cellular invasion through specific regulation of Rac1 and Cdc42 activity in OSCC.

Identification of a gene signature for rapid screening of oral squamous cell carcinoma.

PURPOSE: Oral cancer is a major health problem worldwide and in the U.S. The 5-year survival rate for oral cancer has not improved significantly over the past 20 years and remains at approximately 50%. Patients diagnosed at an early stage of the disease typically have an 80% chance for cure and functional outcome, however, most patients are identified when the cancer is advanced. Thus, a convenient and an accurate way to detect oral cancer early will decrease patient morbidity and mortality. The ability to noninvasively monitor oral cancer onset, progression, and treatment outcomes requires two prerequisites: identification of specific biomarkers for oral cancers as well as noninvasive access to and monitoring of these biomarkers that could be conducted at the point of care (i.e., practitioner's or dentist's office) by minimally trained personnel. EXPERIMENTAL DESIGN: Here, we show that DNA microarray gene expression profiling of matched tumor and normal specimens can identify distinct anatomic site expression patterns and a highly significant gene signature distinguishing normal from oral squamous cell carcinoma (OSCC) tissue. RESULTS: Using a supervised learning algorithm, we generated a 25-gene signature for OSCC that can classify normal and OSCC specimens. This 25-gene molecular predictor was 96% accurate on cross-validation, averaging 87% accuracy using three independent validation test sets and failing to predict non-oral tumors. CONCLUSION: Identification and validation of this tissue-specific 25-gene molecular predictor in this report is our first step towards developing a new, noninvasive, microfluidic-based diagnostic technology for mass screening, diagnosis, and treatment of pre-OSCC and OSCC.

The extracellular matrix in oral squamous cell carcinoma: friend or foe?

Oral squamous cell carcinoma is a disfiguring, highly invasive and metastatic cancer. Despite advances in detection and therapy, many patients will continue to face a poor prognosis. It is well established that the predominate factor determining overall survival in patients with oral squamous cell carcinoma is lymph node involvement. Tumor growth and progression to invasive cancer requires tumor cell interactions with the extracellular matrix. An understanding of how the extracellular matrix influences tumor development and invasion is fundamental in the development of new prognostic indicators and treatment strategies for oral squamous cell carcinoma. In this review, we summarize how changes in the extracellular matrix contribute to oral cancer development.

Suppression of laminin-5 expression leads to increased motility, tumorigenicity, and invasion.

Laminin-5 (Ln-5) is expressed in several human carcinomas and hypothesized to contribute to tumor invasion. To understand the role of Ln-5 in human cancers, we stably delivered small interfering RNAs (siRNAs) directed against the Ln-5 gamma2 chain into JHU-022-SCC cells (022), a non-invasive oral squamous cell carcinoma (OSCC) cell line which secretes Ln-5. Lysates from gamma2 siRNA cells (022-sigamma2) had nearly undetectable levels of the gamma2 chain while the alpha3 and beta3 subunits of Ln-5 remained unchanged compared to parental and control. In conditioned medium from 022-sigamma2 cells, the gamma2 chain and the Ln-5 heterotrimer were barely detectable, similar to an invasive OSCC cell line. Conditioned medium from 022-sigamma2 cells contained less alpha3 and beta3 subunits than both parental and control. Although the proliferation and adhesive properties of the 022-sigamma2 cells remained similar to parental and control cells, 022-sigamma2 cells showed increased detachment and a fibroblastic morphology similar to invasive cells. Moreover, migration, in vitro invasion, and in vivo tumorigenicity were enhanced in 022-sigamma2 cells. Our results suggest that the Ln-5 gamma2 chain regulates the secretion of the alpha3 and beta3 subunits. More importantly, suppression of Ln-5 results in a phenotype that is representative of invasive tumor cells.

Dysregulation of hypoxia inducible factor-1alpha in head and neck squamous cell carcinoma cell lines correlates with invasive potential.

OBJECTIVES/HYPOTHESIS: Tumor hypoxia appears to be closely associated with tumor propagation, malignant progression, and resistance to radiotherapy. Hypoxia inducible factor-1alpha (HIF-1alpha) is a transcription factor that is upregulated under hypoxic conditions and activates hypoxic adaptation pathways which include neovascularization, erythropoiesis, and glycolysis. Hypoxia inducible factor-1alpha is under tight regulation with undetectable levels of expression in normoxia and robust expression in hypoxia. Mutations that activate oncogenes or inactivate tumor suppressor genes increase the expression of HIF-1alpha. Furthermore, it has been demonstrated that HIF-1alpha is overexpressed in head and neck squamous cell carcinoma and that the degree of expression has predictive and prognostic significance for patients undergoing radiotherapy. The study investigated whether overexpression of HIF-1alpha in head and neck squamous cell carcinoma results from a physiological response to local hypoxia or from oncogenic mutational progression. STUDY DESIGN: Expression of HIF-1alpha under normoxic and hypoxic conditions was evaluated in cell lines derived from head and neck squamous cell carcinoma. Cell lines that were used displayed varying degrees of in vitro invasiveness. METHODS: Hypoxia inducible factor-1alpha expression was detected by Western blot analysis. Cells were treated for 3 hours in 1% oxygen, then re-exposed to normoxia for varying times before lysis and detection of HIF-1alpha. RESULTS: Under normoxic conditions, HIF-1alpha expression was upregulated in invasive cells compared with noninvasive cells, and the degradation of HIF-1alpha following a hypoxic stimulus was blunted in invasive cells as compared with noninvasive cells. CONCLUSION: The authors presented evidence that dysregulation of HIF-1alpha may play a role in the malignant progression of head and neck squamous cell carcinoma. It is likely that dysregulated expression of the transcription factor HIF-1alpha contributes to the invasive properties associated with hypoxia and advanced head and neck squamous cell carcinoma.

c-Met expression in tall cell variant papillary carcinoma of the thyroid.

BACKGROUND: Tall cell variant papillary carcinoma of the thyroid demonstrates unusually aggressive clinical behavior compared with the usual form of papillary thyroid carcinoma. The proto-oncogene c-met encodes a tyrosine kinase receptor known to influence cell invasion. This current study examined c-Met expression in tall cell variant tumors compared with other types of papillary thyroid carcinoma and benign thyroid disease. METHODS: c-Met expression in 60 archived thyroid specimens was evaluated by immunohistochemical staining. RESULTS: Tall cell variant specimens expressed significantly greater levels of c-Met than other forms of papillary thyroid carcinoma and benign thyroid disease (P < 0.0001). c-Met expression was significantly different for the following pairs of histologies: tall cell variant versus usual papillary carcinoma of the thyroid (P < 0.0001), tall cell variant versus follicular variant papillary thyroid carcinoma (P < 0.0001), tall cell variant versus benign thyroid (P < 0.0001), and usual papillary carcinoma of the thyroid versus benign thyroid (P = 0.005). In addition, for all types of papillary carcinomas evaluated, c-Met expression was significantly higher in specimens with extracapsular spread (P = 0.01) and skeletal muscle invasion (P = 0.02), and approached significance for specimens with lymphatic invasion (P = 0.06). After adjusting for extracapsular spread, c-Met expression was still found to be associated significantly with tall cell histology (P < 0.0001). CONCLUSIONS: c-Met expression is a significant marker for tall cell variant papillary carcinoma of the thyroid and its invasive behavior. This finding may explain the unusually aggressive behavior of this tumor and suggests a role for c-Met in the early identification of patients with tall cell variant thyroid disease.

Integrins in head and neck squamous cell carcinoma invasion.

OBJECTIVE: To relate the invasive properties of different squamous cell cancer cell lines to the function and expression of the integrins. STUDY DESIGN: A series of in vitro and in vivo experiments were designed to assess and compare integrin expression and function in two different head and neck squamous cell carcinoma cell lines. METHODS: Invasive properties of two squamous cell carcinoma cell lines (UM-SCC-1 and JHU-022-SCC) were assessed using an in vitro artificial matrix assay as well as an in vivo system with orthotopically implanted tumor cells in mice. Whole cell and surface expression levels of integrin subunits (alpha2, alpha3, alpha5, alpha6, beta1, and beta4) were determined for each cell line using Western blot analysis and flow cytometry. We compared the ability of JHU-022-SCC and UM-SCC-1 cells to bind the extracellular matrix elements collagen IV, fibronectin, laminin 5, and laminin10 using an in vitro adhesion assay. Contributions of the different integrins to the adhesive properties were determined by selective antibody blocking of different subunits. RESULTS: The UM-SCC-1 cell line is 50% more invasive in vitro and displays a greater propensity for perineural and lymphatic invasion in vivo. The UM-SCC-1 cells exhibited greater adherence to fibronectin than JHU-022-SCC cells. Alpha6 and beta4 expression is approximately twofold greater in the JHU-022-SCC cells. Alpha2, alpha3, and beta1 expression appears to be upregulated in UM-SCC-1 cells. CONCLUSION: The UM-SCC-1 carcinoma cells are more invasive than JHU-022-SCC cells and may be related to differential expression of the integrins alpha6beta4, alpha3beta1, and alpha2beta1.