Christina S. Kong

Lysyl oxidase is essential for hypoxia-induced metastasis

Metastasis is a multistep process responsible for most cancer deaths, and it can be influenced by both the immediate microenvironment (cell–cell or cell–matrix interactions) and the extended tumour microenvironment (for example vascularization). Hypoxia (low oxygen) is clinically associated with metastasis and poor patient outcome, although the underlying processes remain unclear. Microarray studies have shown the expression of lysyl oxidase (LOX) to be elevated in hypoxic human tumour cells. Paradoxically, LOX expression is associated with both tumour suppression and tumour progression, and its role in tumorigenesis seems dependent on cellular location, cell type and transformation status. Here we show that LOX expression is regulated by hypoxia-inducible factor (HIF) and is associated with hypoxia in human breast and head and neck tumours. Patients with high LOX-expressing tumours have poor distant metastasis-free and overall survivals. Inhibition of LOX eliminates metastasis in mice with orthotopically grown breast cancer tumours. Mechanistically, secreted LOX is responsible for the invasive properties of hypoxic human cancer cells through focal adhesion kinase activity and cell to matrix adhesion. Furthermore, LOX may be required to create a niche permissive for metastatic growth. Our findings indicate that LOX is essential for hypoxia-induced metastasis and is a good therapeutic target for preventing and treating metastases.

Galectin-1: A Link Between Tumor Hypoxia and Tumor Immune Privilege

PURPOSE To identify a 15-KDa novel hypoxia-induced secreted protein in head and neck squamous cell carcinomas (HNSCC) and to determine its role in malignant progression. METHODS We used surface-enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF-MS) and tandem MS to identify a novel hypoxia-induced secreted protein in FaDu cells. We used immunoblots, real-time polymerase chain reaction (PCR), and enzyme-linked immunoabsorbent assay to confirm the hypoxic induction of this secreted protein as galectin-1 in cell lines and xenografts. We stained tumor tissues from 101 HNSCC patients for galectin-1, CA IX (carbonic anhydrase IX, a hypoxia marker) and CD3 (a T-cell marker). Expression of these markers was correlated to each other and to treatment outcomes. RESULTS SELDI-TOF studies yielded a hypoxia-induced peak at 15 kDa that proved to be galectin-1 by MS analysis. Immunoblots and PCR studies confirmed increased galectin-1 expression by hypoxia in several cancer cell lines. Plasma levels of galectin-1 were higher in tumor-bearing severe combined immunodeficiency (SCID) mice breathing 10% O2 compared with mice breathing room air. In HNSCC patients, there was a significant correlation between galectin-1 and CA IX staining (P = .01) and a strong inverse correlation between galectin-1 and CD3 staining (P = .01). Expression of galectin-1 and CD3 were significant predictors for overall survival on multivariate analysis. CONCLUSION Galectin-1 is a novel hypoxia-regulated protein and a prognostic marker in HNSCC. This study presents a new mechanism on how hypoxia can affect the malignant progression and therapeutic response of solid tumors by regulating the secretion of proteins that modulate immune privilege.

The relationship between human papillomavirus status and other molecular prognostic markers in head and neck squamous cell carcinomas.

PURPOSE To evaluate the relationship between human papillomavirus (HPV) status and known prognostic makers for head and neck cancers including tumor hypoxia, epidermal growth factor receptor (EGFR) expression and intratumoral T-cell levels and to determine the prognostic impact of these markers by HPV status. METHODS AND MATERIALS HPV status in 82 evaluable head and neck squamous cell carcinomas patients was determined by pyrosequencing and related to p16(INK4a) staining and treatment outcomes. It was correlated with tumor hypoxia (tumor pO(2) and carbonic anhydrase [CAIX] staining), EGFR status, and intratumoral lymphocyte expression (CD3 staining). RESULTS Forty-four percent of evaluable tumors had strong HPV signal by pyrosequencing. There was a significant relationship between strong HPV signal and p16(INK4a) staining as well as oropharynx location. The strong HPV signal group fared significantly better than others, both in time to progression (TTP, p = 0.008) and overall survival (OS, p = 0.004) for all patients and for the oropharyngeal subset. Positive p16(INK4a) staining was associated with better TTP (p = 0.014) and OS (p = 0.00002). There was no relationship between HPV status and tumor pO(2) or CAIX staining. However, HPV status correlated inversely with EGFR reactivity (p = 0.0006) and directly with CD3(+) T-lymphocyte level (p = 0.03). Whereas CAIX and EGFR overexpression were negative prognostic factors regardless of HPV status, CD3(+) T-cell levels was prognostic only in HPV(-) tumors. CONCLUSION HPV status was a prognostic factor for progression and survival. It correlated inversely with EGFR expression and directly with T-cell infiltration. The prognostic effect of CAIX and EGFR expression was not influenced by HPV status, whereas intratumoral T-cell levels was significant only for HPV(-) tumors.

An Evaluation of Tumor Oxygenation and Gene Expression in Patients with Early Stage Non–Small Cell Lung Cancers

Background: To directly assess tumor oxygenation in resectable non–small cell lung cancers (NSCLC) and to correlate tumor pO2 and the selected gene and protein expression to treatment outcomes. Methods: Twenty patients with resectable NSCLC were enrolled. Intraoperative measurements of normal lung and tumor pO2 were done with the Eppendorf polarographic electrode. All patients had plasma osteopontin measurements by ELISA. Carbonic anhydrase-IX (CA IX) staining of tumor sections was done in the majority of patients (n = 16), as was gene expression profiling (n = 12) using cDNA microarrays. Tumor pO2 was correlated with CA IX staining, osteopontin levels, and treatment outcomes. Results: The median tumor pO2 ranged from 0.7 to 46 mm Hg (median, 16.6) and was lower than normal lung pO2 in all but one patient. Because both variables were affected by the completeness of lung deflation during measurement, we used the ratio of tumor/normal lung (T/L) pO2 as a reflection of tumor oxygenation. The median T/L pO2 was 0.13. T/L pO2 correlated significantly with plasma osteopontin levels (r = 0.53, P = 0.02) and CA IX expression (P = 0.006). Gene expression profiling showed that high CD44 expression was a predictor for relapse, which was confirmed by tissue staining of CD44 variant 6 protein. Other variables associated with the risk of relapse were T stage (P = 0.02), T/L pO2 (P = 0.04), and osteopontin levels (P = 0.001). Conclusions: Tumor hypoxia exists in resectable NSCLC and is associated with elevated expression of osteopontin and CA IX. Tumor hypoxia and elevated osteopontin levels and CD44 expression correlated with poor prognosis. A larger study is needed to confirm the prognostic significance of these factors.

p16 Protein Expression and Human Papillomavirus Status As Prognostic Biomarkers of Nonoropharyngeal Head and Neck Squamous Cell Carcinoma

PURPOSE Although p16 protein expression, a surrogate marker of oncogenic human papillomavirus (HPV) infection, is recognized as a prognostic marker in oropharyngeal squamous cell carcinoma (OPSCC), its prevalence and significance have not been well established in cancer of the oral cavity, hypopharynx, or larynx, collectively referred as non-OPSCC, where HPV infection is less common than in the oropharynx. PATIENTS AND METHODS p16 expression and high-risk HPV status in non-OPSCCs from RTOG 0129, 0234, and 0522 studies were determined by immunohistochemistry (IHC) and in situ hybridization (ISH). Hazard ratios from Cox models were expressed as positive or negative, stratified by trial, and adjusted for clinical characteristics. RESULTS p16 expression was positive in 14.1% (12 of 85), 24.2% (23 of 95), and 19.0% (27 of 142) and HPV ISH was positive in 6.5% (six of 93), 14.6% (15 of 103), and 6.9% (seven of 101) of non-OPSCCs from RTOG 0129, 0234, and 0522 studies, respectively. Hazard ratios for p16 expression were 0.63 (95% CI, 0.42 to 0.95; P = .03) and 0.56 (95% CI, 0.35 to 0.89; P = .01) for progression-free (PFS) and overall survival (OS), respectively. Comparing OPSCC and non-OPSCC, patients with p16-positive OPSCC have better PFS and OS than patients with p16-positive non-OPSCC, but patients with p16-negative OPSCC and non-OPSCC have similar outcomes. CONCLUSION Similar to results in patients with OPSCC, patients with p16-negative non-OPSCC have worse outcomes than patients with p16-positive non-OPSCC, and HPV may also have a role in outcome in a subset of non-OPSCC. However, further development of a p16 IHC scoring system in non-OPSCC and improvement of HPV detection methods are warranted before broad application in the clinical setting.

MYB Expression and Translocation in Adenoid Cystic Carcinomas and Other Salivary Gland Tumors With Clinicopathologic Correlation

Background Adenoid cystic carcinoma is a locally aggressive salivary gland neoplasm, which has a poor long-term prognosis. A chromosomal translocation involving the genes encoding the transcription factors, MYB and NFIB, has been recently discovered in these tumors. Methods MYB translocation and protein expression were studied in 37 adenoid cystic carcinomas, 112 other salivary gland neoplasms, and 409 nonsalivary gland neoplasms by fluorescence in situ hybridization and immunohistochemistry. MYB translocation and expression status in adenoid cystic carcinoma was correlated with clinicopathologic features including outcome, with a median follow-up of 77.1 months (range, 23.2 to 217.5 mo) for living patients. Results A balanced translocation between MYB and NFIB is present in 49% of adenoid cystic carcinomas but is not identified in other salivary gland tumors or nonsalivary gland neoplasms. There is no apparent translocation of MYB in 35% of the cases. Strong Myb immunostaining is very specific for adenoid cystic carcinomas but is only present in 65% of all cases. It is interesting to note that Myb immunostaining is confined to the basal cell component although the translocation is present in all the cells. Neoplasms with MYB translocation show a trend toward higher local relapse rates, but the results are not statistically significant with the current number of cases. Conclusions MYB translocation and expression are useful diagnostic markers for a subset of adenoid cystic carcinomas. The presence of the translocation may be indicative of local aggressive behavior, but a larger cohort may be required to show statistical significance.

p16INK4A immunohistochemistry is superior to HPV in situ hybridization for the detection of high-risk HPV in atypical squamous metaplasia.

In situ hybridization (ISH) assays for high-risk human papillomavirus (HR-HPV) and immunohistochemical (IHC) assays for surrogate markers such as p16INK4A can be useful in detecting HR-HPV in cervical dysplasia, but the use of these markers in problematic cervical biopsies has not been well-established. We evaluated 3 chromogenic ISH assays (Ventana INFORM HPVII and HPVIII and DakoCytomation GenPoint) in conjunction with p16INK4A IHC and HPV polymerase chain reaction in a study set consisting of 12 low-grade squamous intraepithelial lesions, 16 high-grade squamous intraepithelial lesions, and 30 benign cervix samples. A test set of 28 cases of atypical squamous metaplasia were also evaluated withVentana HPVIII ISH and p16INK4A IHC. In the study set, the sensitivity of the DakoCytomation ISH assay (which detects HPV subtypes 16, 18, 31, 33, 35, 39, 45, 52, 56, 58, 59, and 68) was similar to the Ventana HPVII assay but less than that of the Ventana HPVIII ISH assay (both of which detect HPV subtypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 66) and less than p16INK4A IHC (55.6% vs. 53.6 vs. 69.2% vs. 82.1%). All HPV ISH assays exhibited 100% specificity. p16INK4A reactivity consisted of 2 patterns: focal strong and diffuse strong. Because focal strong p16INK4A reactivity was identified in benign squamous epithelium (6.7% cases) and dysplastic epithelium, it was considered an equivocal result and only diffuse strong reactivity was considered to be specific for the presence of HR-HPV. In the squamous intraepithelial lesions study set, the difference in sensitivity between Ventana HPVIII ISH and p16INK4A was not statistically significant. However, in the atypical squamous metaplasia test set cases, p16INK4A reactivity (focal strong and diffuse strong) was significantly more sensitive than Ventana HPVIII ISH in correlating with the presence of human papillomavirus as detected by polymerase chain reaction (83.3% vs. 33.3% P=0.004). Because focal strong p16INK4A reactivity is less specific, cases with this staining pattern are considered atypical and require further evaluation by other means. Overall, p16INK4A IHC is considered the best candidate for the initial assessment of cervical biopsies that are histologically indeterminate for dysplasia given its wide availability, comparative ease of interpretation, and high sensitivity and specificity.

Tumor galectin-1 mediates tumor growth and metastasis through regulation of T-cell apoptosis

Galectin-1 (Gal-1), a carbohydrate-binding protein whose secretion is enhanced by hypoxia, promotes tumor aggressiveness by promoting angiogenesis and T-cell apoptosis. However, the importance of tumor versus host Gal-1 in tumor progression is undefined. Here we offer evidence that implicates tumor Gal-1 and its modulation of T-cell immunity in progression. Comparing Gal-1-deficient mice as hosts for Lewis lung carcinoma cells where Gal-1 levels were preserved or knocked down, we found that tumor Gal-1 was more critical than host Gal-1 in promoting tumor growth and spontaneous metastasis. Enhanced growth and metastasis associated with Gal-1 related to its immunomodulatory function, insofar as the benefits of Gal-1 expression to Lewis lung carcinoma growth were abolished in immunodeficient mice. In contrast, angiogenesis, as assessed by microvessel density count, was similar between tumors with divergent Gal-1 levels when examined at a comparable size. Our findings establish that tumor rather than host Gal-1 is responsible for mediating tumor progression through intratumoral immunomodulation, with broad implications in developing novel targeting strategies for Gal-1 in cancer.

Validation that metabolic tumor volume predicts outcome in head-and-neck cancer.

PURPOSE We have previously reported that metabolic tumor volume (MTV) obtained from pretreatment (18)F-fluorodeoxydeglucose positron emission tomography (FDG PET)/ computed tomography (CT) predicted outcome in patients with head-and-neck cancer (HNC). The purpose of this study was to validate these results on an independent dataset, determine whether the primary tumor or nodal MTV drives this correlation, and explore the interaction with p16(INK4a) status as a surrogate marker for human papillomavirus (HPV). METHODS AND MATERIALS The validation dataset in this study included 83 patients with squamous cell HNC who had a FDG PET/CT scan before receiving definitive radiotherapy. MTV and maximum standardized uptake value (SUV(max)) were calculated for the primary tumor, the involved nodes, and the combination of both. The primary endpoint was to validate that MTV predicted progression-free survival and overall survival. Secondary analyses included determining the prognostic utility of primary tumor vs. nodal MTV. RESULTS Similarly to our prior findings, an increase in total MTV of 17 cm(3) (difference between the 75th and 25th percentiles) was associated with a 2.1-fold increase in the risk of disease progression (p = 0.0002) and a 2.0-fold increase in the risk of death (p = 0.0048). SUV(max) was not associated with either outcome. Primary tumor MTV predicted progression-free (hazard ratio [HR] = 1.94; p < 0.0001) and overall (HR = 1.57; p < 0.0001) survival, whereas nodal MTV did not. In addition, MTV predicted progression-free (HR = 4.23; p < 0.0001) and overall (HR = 3.21; p = 0.0029) survival in patients with p16(INK4a)-positive oropharyngeal cancer. CONCLUSIONS This study validates our previous findings that MTV independently predicts outcomes in HNC. MTV should be considered as a potential risk-stratifying biomarker in future studies of HNC.

Diagnostic problems in anal pathology.

Anal squamous cell carcinoma and its precursor lesions are increasing in incidence in the United States and Europe. This trend predates human immunodeficiency virus/acquired immune deficiency syndrome and has been associated with persistent high-risk human papilloma virus (HPV) genotype infection, previous lower genital tract dysplasia/carcinoma, high frequency anoreceptive intercourse, heavy cigarette smoking, immunosuppression in solid organ transplant and immune disorders, and human immunodeficiency virus seropositivity. Screening protocols for at-risk patients are under active investigation and pathologists are often asked to assess anal canal and perianal biopsies for the presence of dysplasia and/or invasive carcinoma. Because underdiagnosis and overdiagnosis of anal cancer and precancer may lead to inappropriate treatment, it is important for the pathologist to be aware of current screening strategies, specific risk lesions, and the role of pathology in initial diagnosis and evaluation of anal biopsy and/or resection specimens. Standardized histologic criteria and uniform terminology should be used for reporting all anal canal and perianal squamous intraepithelial lesions. HPV subtyping, anal cytology, and recently identified biomarkers, such as p16INK4a and Becton Dickinson ProEx C may provide additional information in problematic cases, but it is important to be aware of the limitations of these assays. HPV has been linked to all the major histologic subtypes of anal carcinoma (eg, basaloid, cloacogenic, transitional, etc.) and this association is strongest for anal canal lesions. With the possible exception of the microcystic pattern, histologic subtype does not seem to predict prognosis; and anal squamous cell carcinomas should be classified as either keratinizing or nonkeratinizing. Poorly differentiated squamous cell carcinomas have a worse prognosis and should be distinguished from poorly differentiated adenocarcinoma, melanoma, and neuroendocrine tumors. Very well differentiated squamous cell carcinoma with pushing margins (so-called giant condyloma of Buschke and Lowenstein) should be classified as verrucous carcinoma; this tumor shows aggressive local infiltration but does not metastasize. As all anal condylomata may harbor foci of high-grade dysplasia or invasive carcinoma, careful sectioning and complete histologic examination is required.