Jonathan M. Hernandez

Expression of the Antiapoptotic Protein Survivin in Colon Cancer

BACKGROUND The antiapoptotic protein survivin has been demonstrated to play an important role in colorectal carcinogenesis. However it is unclear whether the upregulation of survivin is maintained through progressive stages of disease, or if other apoptosis-related genes are coexpressed and/or repressed. We sought to evaluate survivin expression in colonic neoplasia and identify relationships with additional regulators of apoptosis. PATIENTS AND METHODS Tissue samples from 168 patients with primary colorectal cancer were profiled using the GeneChip Human Genome U133 Plus 2.0 Array (Affymetrix, Santa Clara, CA) and evaluated for survivin expression. Immunohistochemical staining for survivin and a panel of apoptosis-associated proteins were used in 86 patients with tissue microarray (TMA) blocks; scoring was by stain intensity and percentage of positive cells (range, 0-9). RESULTS Survivin mRNA was upregulated (1.8-fold increase) in primary colon cancers- irrespective of American Joint Committee on Cancer (AJCC) stage- and metastases compared with normal colonic tissue (P < .0001). Survivin staining was positive in 93% of adenocarcinomas (median immunohistochemistry [IHC] score: 2 [range, 1-6]), 100% of adenomas (1 [range,1-2]), and 43% of normal colonic mucosa (1, [range 1-2]) (P = .006). Survivin expression increased with worsening tumor grade (P < .05). In colon cancers, survivin expression positively correlated with the coexpression of PUMA (P < .001), TACE (P = .003), and MCL1 (P = .01), and trended toward an inverse correlation with BAX (P = .058). CONCLUSIONS Survivin expression increases during the normal mucosa-adenoma-carcinoma sequence and is maintained throughout progression of disease, which strengthens its appeal as a therapeutic target. Furthermore, we have demonstrated co-overexpression of several other apoptosis-related genes, which may in turn serve as additional and potentially synergistic therapeutic targets.

Complementary Strand MicroRNAs Mediate Acquisition of Metastatic Potential in Colonic Adenocarcinoma

BackgroundAltered expression of specific microRNAs (miRNA) is known to occur during colorectal carcinogenesis. However, little is known about the genome-wide alterations in miRNA expression during the neoplastic progression of primary colorectal cancers.MethodsUsing a miRNA array platform, we evaluated the expression of 668 miRNA in primary colonic adenocarcinomas. Biological functions of selected miRNA were evaluated with in vitro invasion assays.ResultsRNA was extracted for miRNA analysis from 65 primary colon cancers. We identified a seven-miRNA expression signature that differentiated stage I and stage IV primary colon cancers. We then demonstrated this signature was able to discriminate between stage II and III primary colon cancers. Six differentially expressed miRNA were downregulated in association with the development of metastases, and all 7 miRNA were complementary strand miRNA. We transfected HCT-116, a highly invasive colon cancer cell line, with corresponding downregulated miRNA and demonstrated that overexpression of three miRNA (miR200c*, miR143*, and miR424*) significantly abrogated invasive potential.ConclusionWe have identified a seven-miRNA signature that is associated with metastatic potential in the primary tumor. Forced overexpression of three downregulated miRNA resulted in attenuation of in vitro invasion, suggesting direct tumor suppressive function and further supporting the biological importance of complementary strand miRNA.

DNA Methylation Profiling across the Spectrum of HPV-Associated Anal Squamous Neoplasia

Background Changes in host tumor genome DNA methylation patterns are among the molecular alterations associated with HPV-related carcinogenesis. However, there is little known about the epigenetic changes associated specifically with the development of anal squamous cell cancer (SCC). We sought to characterize broad methylation profiles across the spectrum of anal squamous neoplasia. Methodology/Principal Findings Twenty-nine formalin-fixed paraffin embedded samples from 24 patients were evaluated and included adjacent histologically normal anal mucosa (NM; n = 3), SCC-in situ (SCC-IS; n = 11) and invasive SCC (n = 15). Thirteen women and 11 men with a median age of 44 years (range 26–81) were included in the study. Using the SFP10 LiPA HPV-typing system, HPV was detected in at least one tissue from all patients with 93% (27/29) being positive for high-risk HPV types and 14 (93%) of 15 invasive SCC tissues testing positive for HPV 16. Bisulfite-modified DNA was interrogated for methylation at 1,505 CpG loci representing 807 genes using the Illumina GoldenGate Methylation Array. When comparing the progression from normal anal mucosa and SCC-IS to invasive SCC, 22 CpG loci representing 20 genes demonstrated significant differential methylation (p<0.01). The majority of differentially methylated gene targets occurred at or close to specific chromosomal locations such as previously described HPV methylation “hotspots” and viral integration sites. Conclusions We have identified a panel of differentially methlylated CpG loci across the spectrum of HPV-associated squamous neoplasia of the anus. To our knowledge, this is the first reported application of large-scale high throughput methylation analysis for the study of anal neoplasia. Our findings support further investigations into the role of host-genome methylation in HPV-associated anal carcinogenesis with implications towards enhanced diagnosis and screening strategies.

Increased age is an independent risk factor for radiographic aspiration and laryngeal penetration after thoracotomy for pulmonary resection

OBJECTIVES Aspiration is an increasingly recognized complication after thoracotomy for pulmonary resection, but mechanisms of postoperative aspiration are poorly characterized. This study sought to evaluate risk factors to better define postthoracotomy aspiration. METHODS Three hundred twenty-one consecutive patients underwent clinical bedside swallowing evaluations after thoracotomy for pulmonary resection on postoperative day 1. Results of videofluoroscopic swallowing studies were independently reviewed by 2 speech pathologists and were assigned aspiration-penetration scores of either 1 (normal) or greater than 1 (abnormal) based on the worst swallow. Operative, demographic, and outcomes data were abstracted for each patient, and multivariate regression analysis was performed. RESULTS Seventy-three (22.7%) patients failed bedside evaluation and proceeded to undergo videofluoroscopic swallowing studies. Forty-four (60.3%) patients had an abnormal videofluoroscopic swallowing study result with a mean aspiration-penetration score of 3.9 +/- 0.3. Multivariate analysis showed that older age (68.8 vs 56.2 years, P = .002), prior premature spillage (P = .0006), and vallecular residuals after the swallow (P < .0002) were all associated with aspiration. Interestingly, certain variables were not independently associated with aspiration, including presence of gastroesophageal reflux disease, operative approach or degree of resection, mediastinal lymphadenectomy, preoperative thoracic radiation, same hospitalization reoperation, and pathology. CONCLUSIONS Postoperative risk of aspiration after thoracotomy for pulmonary resection is characterized by repeatable episodes of oropharyngeal discoordination on videofluoroscopic swallowing studies. We recommend routine videofluoroscopic swallowing studies for all patients older than 67 years before the initiation of oral intake to diminish the incidence of postoperative aspiration.

Paraneoplastic hypercalcemia caused by an invasive squamous cell carcinoma arising from a giant anal condyloma acuminatum

Dear Editor, Buschke and Lowenstein first described the morphologic characteristics of a giant condyloma acuminatum in 1925. In 1948, Akerman described the morphology of a condylomatous lesion in the oral cavity with the term verrucous carcinoma. Since that time there has been considerable ambiguity with the use of these terms that remains unresolved. Whether or not the Bushke–Lowenstein tumor represents a variant of verrucous carcinoma or a clinically distinct entity will likely remain a subject of esoteric debate. To the contrary, it is now apparent that human papilloma virus-(HPV) related lesions of the anogenital squamous epithelium range in continuum from condyloma to invasive squamous cell carcinoma (SCC) and that degeneration toward the latter can occur. This malignant transformation was first described in 1975 by Sturm et al. in a patient who developed invasive squamous cell carcinoma of the anus. At present, giant condyloma acuminatum of the anogenital tract is known to be a locally aggressive tumor with a high rate of recurrence and a rate malignant transformation of 30%. We present a case of giant condyloma acuminatum that developed into an invasive squamous cell carcinoma that in turn, was associated with paraneoplastic hypercalcemia. A 40-year-old HIV-negative female presented to the emergency department at an outside facility with complaints of severe dizziness and syncopal episodes for the past 3 months confining her to bed rest. The patient was found to be hypotensive and tachycardic with a leukocytosis of 56,000. Her laboratory values were otherwise remarkable for a hemoglobin of 9.8 and serum calcium level of 15.8. On examination, the patient was found to have a large anal condylomatous lesions with cephalad extension to involve the vulva. A large erythematous, fluctuant mass was identified on the left buttock adjacent to the left lesions. Purulent drainage was noted and the patient was taken to the operating room where the abscess was drained and a diverting transverse loop colostomy was performed. Multiple biopsies were obtained. The final pathologic diagnosis was invasive squamous cell carcinoma arising in a giant anal condyloma acuminatum. Hypercalcemia was treated by saline diuresis. The patient was then transferred to our facility for definitive management. At our institution, the patient was re-examined under general anesthesia. She was found to have a large exophytic, cauliflower-like mass involving both buttocks. The tumor was locally destructive replacing much of the normal tissue architecture. Numerous fistulous sinus tracts were evident. The tumor extended cephalad to involve the labia. There was no extension into the vagina and the cervix appeared to be uninvolved. Multiple biopsies were again obtained and confirmed the development of invasive squamous cell carcinoma in the background of a giant anal condyloma. An extensive hypercalcemia workup was performed which was remarkable for an isolated elevation of parathyroid hormone related peptide (PTHrp). Metastatic workup including computerized tomography scans of the head, chest abdomen, and pelvis as well as bone density scans were unremarkable. The patient was then referred for neoadjuvant therapy. She underwent a complete course of 5-fluorouracil, mitomycin C, and external beam radiation. Following completion of her chemoradiation treatment, the Int J Colorectal Dis (2009) 24:359–360 DOI 10.1007/s00384-008-0554-5

Survival after pancreatectomy for pancreatic adenocarcinoma is not impacted by performance status

BACKGROUND Patients with the best performance status have the best prognosis after resection for pancreatic adenocarcinoma. This study was undertaken to determine the impact of performance status on survival after pancreatectomy for adenocarcinoma. METHODS Patients with a Karnofsky Performance Score (KPS) status (KPS) ≥60 after pancreatectomy for adenocarcinoma were evaluated, and the impact of the KPS at 6 weeks after pancreatectomy on survival was determined using survival curve analysis. RESULTS Recurrence was experienced by 84% of patients and negatively impacted patient survival. The median overall survival was 12 months, and the 2-year overall survival was 35%. The KPS after pancreatectomy did not impact survival when using survival curve analysis (P = .5740). CONCLUSIONS Performance status for patients with a KPS ≥60 after pancreatectomy does not impact survival. Patients with pancreatic adenocarcinoma without adjuvant therapy have poor overall survival, and KPS after pancreatectomy for adenocarcinoma should not be used to withhold therapy for these patients.

Perturbation and candidate analysis to combat overfitting of gene expression microarray data

Analysis of gene expression microarray datasets presents the high risk of over-fitting (spurious patterns) because of their feature-rich but case-poor nature. This paper describes our ongoing efforts to develop a method to combat over-fitting and determine the strongest signal in the dataset. A GA-SVM hybrid along with Gaussian noise (manual noise gain) is used to discover feature sets of minimal size that accurately classifies the cases under cross-validation. Initial results on a colorectal cancer dataset shows that the strongest signal (modest number of candidates) can be found by a binary search.

The Tumor Suppressive Effects of HPP1 Are Mediated Through JAK-STAT-Interferon Signaling Pathways.

HPP1, a novel tumor suppressive epidermal growth factor (EGF)-like ligand, mediates its effects through signal transducer and activators of transcription (STAT) activation. We previously demonstrated the importance of STAT1 activation for HPP1 function; however the contribution of STAT2 remains unclear. We sought to delineate the components of JAK-STAT-interferon (IFN) signaling specifically associated with HPP1s biological effects. Using stable HPP1-HCT116 transfectants, expression analyses were performed by polymerase chain reaction (PCR)/western blotting while expression knockdowns were achieved using siRNA. Growth parameters evaluated included proliferation, cell cycle distribution, and anchorage-independent growth. STAT dimerization, translocation, and DNA binding were examined by reporter assays, fluorescent microscopy, and chromatin immunoprecipitation (ChIP), respectively. Forced expression of HPP1 in colon cancer cell lines results in the upregulation of total and activated levels of STAT2. We have also determined that JAK1 and JAK2 are activated in response to HPP1 overexpression, and are necessary for subsequent STAT activation. Overexpression of HPP1 was associated with significant increases in STAT1:STAT1 (p=0.007) and STAT1:STAT2 (p=0.036) dimer formation, as well as subsequent nuclear translocation. By ChIP, binding of activated STAT1 and STAT2 to the interferon-signaling regulatory element promoter sites of the selected genes, protein kinase RNA-activated (PKR), IFI44, and OAS1 was demonstrated. STAT2 knockdown resulted in partial abrogation of HPP1s growth suppressive activity with increased proliferation (p<0.0001), reduced G1/G0 phase cell cycle fraction, and a restoration of growth potential in soft agar (p<0.01). Presumably as a consequence of upregulation of IFN signaling elements, HPP1 overexpression resulted in an acquisition of exogenous IFN sensitivity. Physiologic doses of IFN-α resulted in a significant reduction in proliferation (p<0.001) and increase in G1/G0 cell cycle arrest in HPP1 transfectants. STAT2 is necessary for HPP1-associated growth suppression, and mediates these effects through activation of IFN-α pathways. Given the interest in therapeutic targeting of oncogenic erbB proteins, further understanding of HPP1s role as a tumor suppressive EGF-like ligand is warranted.

Anal Carcinoma Surveillance Counterpoint: USA

Anal cancer accounts for 4 % of all lower gastrointestinal tract malignancies in the United States [1]. The incidence of anal cancer appears to be on the rise, with increases in incidence rates of 2.6 % per year reported between 1992 and 2000 [2]. Over 5,000 new cases of anal cancer are diagnosed in the U.S. annually [3]. The overall 5-year survival rate is 66.5 % [4]. This varies by stage at diagnosis (82 % for local disease; 59 % for regional disease and 19 % for distant disease) [3]. The vast majority (65–85 %) of anal malignancies are squamous cell carcinomas, which include various histological subtypes such as cloacogenic, basaloid, and transitional cell cancers [2]. Cancers of the anus have been categorized as those arising in the anal canal, or intraanal, and those arising at the anal margin, or perianal region [5].

Su2021 The Tumor Suppressive Effects of HPP1 via Stat Signaling in Colon Cancer are Abrogated by Site-Directed Mutation of Its EGF-Like Domain

Introduction: Variability of percentage excess weight loss (%EWL) in LAGB patients can be influenced by many preoperative factors, such as gender, race/ethnicity, and age. We hypothesize that race/ethnicity plays an important predictor in the post-operative weight loss. Methods: A retrospective analysis of 428 patients using electronic medical record was performed to assess differential %EWL for patients across a period of three years post-band implantation, with an average of 1.53 years for all groups and no significant differences between ethnic groups. Average initial BMI is 42 ± 5. ANOVA was used to analyze data and P<0.05 considered significant. Results: Percent excess weight loss (EWL) are reported for the following racial groups. Asians lost the most, followed by Caucasians, the Hispanics and finally African Americans. The Caucasian group lost 66.33% ± 2.4% %EWL (N=209); the Asian group lost 88.6% ± 7.7 %EWL (N=23); the Hispanic group (50.2% ± 2.3%; P= 1.5E-05, N=123), and African American group lost 44.4% ± 3.3%; P=9.96E-06, (N=73). Furthermore, the average number of adjustments was 6 for the Caucasian group, 5 for the Asian group, and 4 for the Hispanic and African American groups. Conclusion: Our findings suggest that weight loss outcome for LAGB may be related to a patients race/ethnicity. Patients in the Caucasian group have significantly more adjustments than any other group. Excess weight loss is correlated with race and number of adjustments as well as willingness to diet and exercise. Cultural differences in types of food and exercise is also important in weight loss outcome.