Luis Chiriboga

Infrared Spectroscopy of Cells and Tissues: Shining Light onto a Novel Subject

T his focal point article aims to introduce the readers to a new spectroscopic endeavor, infrared spectroscopy of entire cells and tissues, and to present an overview of the results from, and the potential of, this research. The ® eld has seen quite a bit of activity, as judged by the number of papers published and patents ® led. But unfortunately, the level of activity in a ® eld is no measure of the quality of the work published: the earlier reports of ``detecting cancer’ ’ by comparing infrared spectra of healthy and diseased samples were sometimes based on rather naive interpretations of the observations and bypassed all ground

Stromal Cell–Derived Factor-1α and CXCR4 Expression in Hemangioblastoma and Clear Cell-Renal Cell Carcinoma: von Hippel-Lindau Loss-of-Function Induces Expression of a Ligand and Its Receptor

The genetic hallmark of hemangioblastomas and clear cell-renal cell carcinomas (CC-RCCs) is loss-of-function of the von Hippel-Lindau (VHL) tumor suppressor protein. VHL is required for oxygen-dependent degradation of hypoxia-inducible factor-1alpha (HIF-1alpha). In hemangioblastomas and CC-RCCs, HIF-1alpha is constitutively overexpressed leading to increased transcription of HIF-1-regulated genes, including vascular endothelial growth factor (VEGF). Because loss of VHL function is associated with increased expression of the chemokine receptor CXCR4 in CC-RCCs, we investigated the expression of HIF-1alpha, CXCR4, and its ligand stromal cell-derived factor-1alpha (SDF-1alpha) in hemangioblastomas and CC-RCCs. Immunohistochemistry revealed overexpression of both CXCR4 and SDF-1alpha within tumor cells and endothelial cells of hemangioblastomas and CC-RCCs. HIF-1alpha was detected in tumor cell nuclei of both hemangioblastomas and CC-RCCs. A specific ELISA showed that hemangioblastomas and CC-RCCs expressed SDF-1alpha protein at levels that were significantly higher than those found in normal tissue. Analysis of the VHL-null RCC line 786-0 revealed that SDF-1alpha mRNA levels were 100-fold higher than in a subclone transfected with the wild-type VHL gene. Expression of CXCR4 and SDF-1alpha mRNA was significantly decreased in HIF-1alpha-null compared with wild-type mouse embryo fibroblasts (MEFs). ELISA and Western blot studies for SDF-1alpha and CXCR4 protein expression confirmed the RNA findings in RCC lines and MEFs. These results suggest that loss-of-function of a single tumor suppressor gene can up-regulate the expression of both a ligand and its receptor, which may establish an autocrine signaling pathway with important roles in the pathogenesis of hemangioblastoma and CC-RCC.

Immunization of Malignant Melanoma Patients with Full-Length NY-ESO-1 Protein Using TLR7 Agonist Imiquimod as Vaccine Adjuvant

T cell-mediated immunity to microbes and to cancer can be enhanced by the activation of dendritic cells (DCs) via TLRs. In this study, we evaluated the safety and feasibility of topical imiquimod, a TLR7 agonist, in a series of vaccinations against the cancer/testis Ag NY-ESO-1 in patients with malignant melanoma. Recombinant, full-length NY-ESO-1 protein was administered intradermally into imiquimod preconditioned sites followed by additional topical applications of imiquimod. The regimen was very well tolerated with only mild and transient local reactions and constitutional symptoms. Secondarily, we examined the systemic immune response induced by the imiquimod/NY-ESO-1 combination, and show that it elicited both humoral and cellular responses in a significant fraction of patients. Skin biopsies were assessed for imiquimod’s in situ immunomodulatory effects. Compared with untreated skin, topical imiquimod induced dermal mononuclear cell infiltrates in all patients composed primarily of T cells, monocytes, macrophages, myeloid DCs, NK cells, and, to a lesser extent, plasmacytoid DCs. DC activation was evident. This study demonstrates the feasibility and excellent safety profile of a topically applied TLR7 agonist used as a vaccine adjuvant in cancer patients. Imiquimod’s adjuvant effects require further evaluation and likely need optimization of parameters such as formulation, dose, and timing relative to Ag exposure for maximal immunogenicity.

Sox10: a pan-schwannian and melanocytic marker.

S100 protein is a sensitive marker for melanomas and peripheral nerve sheath tumors. It is, however, expressed by other mesenchymal and epithelial tumors. Despite its low specificity, S100 protein is valuable for the diagnosis of desmoplastic melanomas and peripheral nerve sheath tumors, for which no specific marker is available. Sox10 is a neural crest transcription factor crucial for specification, maturation, and maintenance of Schwann cells and melanocytes. Anti-Sox10 antibody was applied to a variety of neural crest-derived tumors, mesenchymal and epithelial neoplasms, and normal tissues. Sox10 nuclear expression was found in 76 of 78 melanomas (97%) and 38 of 77 malignant peripheral nerve sheath tumors (49%) whereas S100 protein was expressed in 71 melanomas (91%) and 23 malignant peripheral nerve sheath tumors (30%). Sox10 was diffusely expressed in schwannomas and neurofibromas. Sox10 reaction was seen only in sustentacular cells of pheochromocytomas/paragangliomas, and occasionally carcinoid tumors from various organs, but it was not seen in the tumor cells. In normal tissues, Sox10 was expressed in Schwann cells, melanocytes, and myoepithelial cells of salivary, bronchial, and mammary glands. Sox10 reaction was not identified in any other mesenchymal and epithelial tumors except for myoepitheliomas and diffuse astrocytomas. Sox10 was expressed by metastatic melanomas and nodal capsular nevus in sentinel lymph nodes, but not by other lymph node components such as dendritic cells. Our results indicate that Sox10 will serve as a more sensitive and specific marker for the diagnosis of melanocytic and schwannian tumors than S100 protein.

Intrahepatic levels of CXCR3‐associated chemokines correlate with liver inflammation and fibrosis in chronic hepatitis C

Chemokines, chemotactic cytokines, may promote hepatic inflammation in chronic hepatitis C virus (HCV) infection through the recruitment of lymphocytes to the liver parenchyma. We evaluated the association between inflammation and fibrosis and CXCR3‐associated chemokines, interferon‐γ (IFN‐γ)–inducible protein 10 (IP‐10/CXCL10), monokine induced by IFN‐γ (Mig/CXCL9), and interferon‐inducible T cell α chemoattractant (I‐TAC/CXCL11), in HCV infection. Intrahepatic mRNA expression of these chemokines was analyzed in 106 chronic HCV‐infected patients by real‐time PCR. The intrahepatic localization of chemokine producer cells and CXCR3+ lymphocytes was determined in selected patients by immunohistochemistry. We found elevated intrahepatic mRNA expression of all three chemokines, most markedly CXCL10, in chronic HCV‐infected patients with higher necroinflammation and fibrosis. By multivariable multivariate analysis, intrahepatic CXCL10 mRNA expression levels were significantly associated with lobular necroinflammatory grade and HCV genotype 1. In the lobular region, CXCL10‐expressing and CXCL9‐expressing hepatocytes predominated in areas with necroinflammation. Strong CXCL11 expression was observed in almost all portal tracts, whereas CXCL9 expression varied considerably among portal tracts in the same individual. Most intrahepatic lymphocytes express the CXCR3 receptor, and the number of CXCR3+ lymphocytes was increased in patients with advanced necroinflammation. Conclusion: These findings suggest that the CXCR3‐associated chemokines, particularly CXCL10, may play an important role in the development of necroinflammation and fibrosis in the liver parenchyma in chronic HCV infection. (Hepatology 2008.)

Expression of pax8 as a useful marker in distinguishing ovarian carcinomas from mammary carcinomas.

The ovary is a common site of involvement for metastasis and the breast is one of the most common sources. Metastatic breast carcinoma can mimic a primary ovarian carcinoma. Pax8 is a crucial transcription factor for organogenesis of the thyroid gland, kidney, and Müllerian system, and it also regulates Wilms tumor suppressor gene (WT1) expression. A total of 124 cases of ovarian carcinomas (84 serous papillary, 18 endometrioid, 12 mucinous, 10 clear cell) and 243 cases of invasive breast carcinomas (178 ductal, 65 lobular) were immunostained with Pax8 and WT1 by tissue microarrays to see the differential expression. Pax8 reaction was found in 108 of 124 ovarian carcinomas (87.1%) generally in diffuse staining, including 81 of 84 serous papillary carcinomas (96.4%), 16 of 18 endometrioid carcinomas (88.9%), 10 of 10 clear cell carcinomas (100%), and 1 of 12 mucinous carcinomas (8.3%), whereas WT1 expression was seen in 78 of 124 ovarian carcinomas (62.9%), including 73 of 84 serous papillary carcinomas (86.9%), and 5 of 18 endometrioid carcinomas (27.8%), with no expression in all 10 clear cell carcinomas and 12 mucinous carcinomas. All the mammary carcinomas were completely negative for Pax8, but WT1 expression was seen in 5 of 243 cases (2.1%). Pax8 is a useful marker in the differential diagnosis of ovarian and breast carcinomas, and it seems to be superior to WT1 for the diagnosis of all types of nonmucinous ovarian carcinomas, notably clear cell and endometrioid types where WT1 expression is generally negative or only focal.

Diagnostic utility of thyroid transcription factors Pax8 and TTF-2 (FoxE1) in thyroid epithelial neoplasms

Thyroid-specific transcription factors, Pax8, TTF-1, and TTF-2, are crucial for thyroid organogenesis and differentiation. Compared with TTF-1, the other two markers have scarcely been investigated in surgical pathology. The goal of this study is to evaluate the expressions of these markers in thyroid tumors of the full spectrum of differentiation, with special emphasis on anaplastic carcinomas. A total of 94 cases of thyroid neoplasms were studied: 17 papillary carcinomas, 18 follicular adenomas, 16 follicular carcinomas, 7 poorly differentiated carcinomas, 28 anaplastic carcinomas, and 8 medullary carcinomas. Immunostains for these three markers were performed. The antibodies to Pax8 and TTF-2 were also applied on 147 lung carcinomas as well as a variety of normal tissues and malignant tumors. All three markers were seen in papillary carcinomas, follicular adenomas and carcinomas, and poorly differentiated carcinomas in a diffuse manner, whereas their expressions in medullary carcinomas were variable. Pax8 was expressed in 79% of anaplastic carcinomas to a variable extent, whereas TTF-1 and TTF-2 were seen only in 18 and 7% of anaplastic carcinomas, respectively. TTF-2 was negative in all other neoplastic and non-neoplastic tissues including those of the lung. Pax8 was expressed in renal tubules, fallopian tubes, ovarian inclusion cysts, and lymphoid follicles as well as renal carcinoma, nephroblastoma, seminoma, and ovarian carcinoma, but not in normal tissue and carcinomas of the lung. Pax8 is a useful marker for the diagnosis of anaplastic carcinomas, particularly when the differential diagnosis includes pulmonary carcinoma. In differentiated thyroid neoplasms, no significant difference in expression was seen in all the three transcription factors.

Infrared spectroscopy of human tissue. I. Differentiation and maturation of epithelial cells in the human cervix

Infrared spectral results for the different epithelial layers of human cervical squamous tissue are reported. The layers, representing different cellular maturation stages, exhibit quite different spectral patterns. Thus, infrared spectroscopy presents a powerful tool to monitor cell maturation and differentiation. Furthermore, a detailed understanding of the spectra of the individual layers of tissue permit a proper interpretation of the state of health of cells exfoliated from such tissue. Part II of this series describes the use of the spectral information presented here to interpret the infrared spectra of exfoliated cells.

Hypoxia- and Vascular Endothelial Growth Factor-Induced Stromal Cell-Derived Factor-1α/CXCR4 Expression in Glioblastomas : One Plausible Explanation of Scherer’s Structures

The morphological patterns of glioma cell invasion are known as the secondary structures of Scherer. In this report, we propose a biologically based mechanism for the nonrandom formation of Scherers secondary structures based on the differential expression of stromal cell-derived factor (SDF)-1alpha and CXCR4 at the invading edge of glioblastomas. The chemokine SDF-1alpha was highly expressed in neurons, blood vessels, subpial regions, and white matter tracts that form the basis of Scherers secondary structures. In contrast, the SDF-1alpha receptor, CXCR4, was highly expressed in invading glioma cells organized around neurons and blood vessels, in subpial regions, and along white matter tracts. Neuronal and endothelial cells exposed to vascular endothelial growth factor up-regulated the expression of SDF-1alpha. CXCR4-positive tumor cells migrated toward a SDF-1alpha gradient in vitro, whereas inhibition of CXCR4 expression decreased their migration. Similarly, inhibition of CXCR4 decreased levels of SDF-1alpha-induced phosphorylation of FAK, AKT, and ERK1/2, suggesting CXCR4 involvement in glioma invasion signaling. These studies offer one plausible molecular basis and explanation of the formation of Scherers structures in glioma patients.

Histologic features are important prognostic indicators in early stages lung adenocarcinomas.

This study attempts to evaluate the clinicopathologic features of mixed subtype adenocarcinomas and the prognostic implications of histopathology classifications. Surgical specimens from 141 patients with clinical stage I or II lung adenocarcinoma during the period 1992–2004 were included. These cases were classified into four groups defined by the extent of the bronchioloalveolar carcinoma component: group I: pure bronchioloalveolar carcinoma; group II: mixed subtype with predominant bronchioloalveolar carcinoma component and ≤5 mm invasive component; group III: mixed subtype with bronchioloalveolar carcinoma component and >5 mm invasive component; group IV: invasive carcinoma with no bronchioloalveolar carcinoma component. Descriptive statistics were used to examine the groups with respect to age, tumor size, lymph node metastasis, and Ki-67 and p53 expression levels. Death rate for the groups was obtained by patients charts and from the National Death Index database. The population was similar in age, tumor size and lymph node metastasis. Immunohistochemical results showed that the mean Ki-67 labeling and the amount of p53 overexpression had the same trend of increasing mean values or positive results from groups I to IV. The reported proportion of deaths ranged from 0% for groups I and II, 20% in patients with predominant invasive component with bronchioloalveolar carcinoma (group III), and 18% in patients with invasive carcinomas and no bronchioloalveolar carcinoma component (group IV). The difference between the proportion of patients with reported deaths in the time period of this study in the combined greater than 5 mm+pure invasive groups (groups III, IV), and the <5 mm+noninvasive groups (groups I, II) is statistically significant. These results suggest that histological features may be useful in defining categories of lung adenocarcinomas with differing survival and prognostic features. These results are helpful in defining a subcategory of ‘minimally invasive adenocarcinoma’, which has features similar to bronchioloalveolar carcinoma.