Predicting Osteopathic Medical Student Performance on the United States Medical Licensing Examination Step 2 Clinical Knowledge From Results of the Comprehensive Osteopathic Medical Licensing Examination Level 2-Cognitive Evaluation.

Background To improve their standing in residency selection, many osteopathic medical students choose to take the United States Medical Licensing Examination (USMLE). Although scores on USMLE Step 1 and Level 1 of the Comprehensive Osteopathic Medical Licensing Examination (COMLEX-USA) are known to be highly correlated, scarce data exist on the association between COMLEX-USA Level 2-Cognitive Evaluation (CE) and USMLE Step 2 Clinical Knowledge (CK) scores. In this study, we aimed to determine the association between COMLEX-USA Level 2-CE and USMLE Step 2 CK scores and derive an equation to predict performance on USMLE Step 2 CK for applicants who have only taken COMLEX-USA. Methodology We reviewed COMLEX-USA Level 2-CE and USMLE Step 2 CK scores for all students at the Lake Erie College of Osteopathic Medicine from May 2020 to April 2021. Linear regression was used to evaluate the relationship between COMLEX-USA Level 2-CE and USMLE Step 2 CK scores. Results A total of 340 students took both COMLEX-USA Level 2-CE and USMLE Step 2 CK. There was a linear association between COMLEX-USA Level 2-CE and USMLE Step 2 CK scores such that every one-point increase in COMLEX-USA was associated with a 0.13-point increase in USMLE Step 2 CK score (standard error = 9.1; model R2 = 0.64). Conclusions Students or programs interested in predicting performance on USMLE Step 2 CK from performance on COMLEX-USA Level 2-CE can do so using the following equation: USMLE Step 2 CK = 0.13(COMLEX-USA Level 2-CE) + 163.5.

Predicting Osteopathic Medical Students' Performance on the United States Medical Licensing Examination From Results of the Comprehensive Osteopathic Medical Licensing Examination.

Introduction The reliance on the United States Medical Licensing Examination (USMLE) Step 1 scores in residency selection creates problems for osteopathic medical students and the programs that review their applications. Although many osteopathic students take the USMLE to improve their standing for residency selection, students who score poorly may harm their candidacy. Simultaneously, programs unfamiliar with the Comprehensive Osteopathic Medical Licensing Examination (COMLEX-USA) may struggle to evaluate applicants who have not taken USMLE. Objective To determine the association between COMLEX-USA Level 1 and USMLE Step 1 scores and derive an equation that could be used to predict USMLE performance or approximate USMLE scores for applicants who have only taken COMLEX-USA. Methods We reviewed COMLEX-USA Level 1 and USMLE Step 1 scores for all students at the Lake Erie College of Osteopathic Medicine (LECOM), Bradenton campus, from January 2012 until December 2016. Linear regression was used to evaluate the relationship between COMLEX-USA Level 1 and USMLE Step 1 scores. Results Overall, 2097 students took both examinations during the study period. Every one-point increase in COMLEX-USA was associated with a 0.15 point increase in USMLE Step 1 score (standard error 11.5; model R2 0.56). On average, students scored 30 percentile points lower on USMLE Step 1 than on COMLEX-USA, and 24% of students scoring <500 on COMLEX-USA Level 1 failed USMLE Step 1. Conclusions Students or programs interested in predicting performance on USMLE Step 1 from performance on COMLEX-USA Level 1 can do so with the following equation: USMLE Step 1 = 0.15 (COMLEX-USA Level 1) + 138.7.

Integrin αvß3 and fibronectin upregulate Slug in cancer cells to promote clot invasion and metastasis.

The blood clotting cascade is selectively involved in lung metastasis, but the reason for this selectivity is unclear. Here, we show that tumor cells that metastasize predominantly to the lung, such as renal cell carcinoma (RCC) and soft tissue sarcoma (STS), have an inherent capacity to generate extensive invadopodia when embedded in a blood clot. Compared with other metastatic cancer cells tested, RCC and STS cells exhibited increased levels of expression of fibronectin and an activated form of the integrin αvß3, which coordinately supported the generation of an elaborate fibronectin matrix and actin stress fibers in fibrin-embedded tumor cells. Together, fibronectin and αvß3 induced upregulation of the transcription factor Slug, which mediates epithelial-mesenchymal transition as well as fibrin invasion and lung metastasis. This mechanism is clinically significant, because primary cancer cells from patients with metastatic RCC strongly invaded fibrin and this correlated with fibronectin matrix formation and Slug expression. In contrast, tumor cells from patients with localized RCC were largely noninvasive. Together, our findings establish that activated integrin αvß3 and fibronectin promote lung metastasis by upregulating Slug, defining a mechanism through which cancer cells can colonize blood clots in the lung vasculature.

Association of the von Hippel-Lindau protein with AUF1 and posttranscriptional regulation of VEGFA mRNA.

The von Hippel-Lindau (VHL) tumor suppressor gene product is the recognition component of an E3 ubiquitin ligase and is inactivated in patients with VHL disease and in most sporadic clear-cell renal cell carcinomas (RCC). pVHL controls oxygen-responsive gene expression at the transcriptional and posttranscriptional levels. The VEGFA mRNA contains AU-rich elements (ARE) in the 3'-untranslated region, and mRNA stability or decay is determined through ARE-associated RNA-binding factors. We show here that levels of the ARE-binding factor, AUF1, are regulated by pVHL and by hypoxia. pVHL and AUF1 stably associate with each other in cells and AUF1 is a ubiquitylation target of pVHL. AUF1 and another RNA-binding protein, HuR, bind to VEGFA ARE RNA. Ribonucleoprotein (RNP) immunoprecipitations showed that pVHL associates indirectly with VEGFA mRNA through AUF1 and/or HuR, and this complex is associated with VEGFA mRNA decay under normoxic conditions. Under hypoxic conditions pVHL is downregulated, whereas AUF1 and HuR binding to VEGF mRNA is maintained, and this complex is associated with stabilized mRNA. These studies suggest that AUF1 and HuR bind to VEGFA ARE RNA under both normoxic and hypoxic conditions, and that a pVHL-RNP complex determines VEGFA mRNA decay. These studies further implicate the ubiquitin-proteasome system in ARE-mediated RNA degradation.

EAF2 loss enhances angiogenic effects of Von Hippel-Lindau heterozygosity on the murine liver and prostate.

Von Hippel-Lindau (VHL) disease results from the inactivation of the VHL gene and is characterized by highly vascular tumors. A consequence of VHL loss is the stabilization of hypoxia-inducible factor (HIF) alpha subunits and increased expression of HIF target genes, which include pro-angiogenic growth factors such as vascular endothelial growth factor (VEGF). In mice, homozygous deletion of VHL is embryonic lethal due to vascular abnormalities in the placenta; and, VHL(+/-) mice develop proliferative vascular lesions in several major organs, most prominently the liver. Loss of ELL-associated factor (EAF2) in murine models has also been shown to induce increased vascular density in the liver as well as the prostate. Previously, EAF2 was determined to be a binding partner of VHL and loss of EAF2 induced a reduction in pVHL levels and an increase in hypoxia induced factor 1α (HIF1α) levels in vitro. Here we characterized the cooperative effects of VHL- and EAF2-deficiency on angiogenesis in the liver and prostate of male mice. VHL deficiency consistently increased the incidence of hepatic vascular lesions across three mouse strains. These vascular lesions where characterized by an increase in microvessel density, and staining intensity of VHL target proteins HIF1α and VEGF. EAF2(-/-)VHL(+/-) mice had increased incidence of proliferative hepatic vascular lesions (4/4) compared to VHL(+/-) (10/18) and EAF2(-/-) (0/5) mice. Prostates of EAF2(-/-)VHL(+/-) mice also displayed an increase in microvessel density, as well as stromal inflammation and prostatic intraepithelial neoplasia. These results suggest that cooperation of VHL and EAF2 may be critical for angiogenic regulation of the liver and prostate, and concurrent loss of these two tumor suppressors may result in a pro-angiogenic phenotype.

Genotype-phenotype correlations of pheochromocytoma in two large von Hippel-Lindau (VHL) type 2A kindreds with different missense mutations.

Von Hippel-Lindau (VHL) disease type 2A is an inherited tumor syndrome characterized by predisposition to pheochromocytoma (pheo), retinal hemangioma (RA), and central nervous system hemangioblastoma (HB). Specific VHL subtypes display genotype-phenotype correlations but, unlike other familial syndromes such as MEN-2, the phenotype in VHL has not yet been stratified at the codon level. Over decades, we have managed two very large VHL type 2A regional kindreds with nearly adjacent but distinct VHL missense mutations. We determined the phenotype of Family 2 and compared the clinical and pathologic parameters of pheo between 30 members of Family 1 (Y112H mutation) and 33 members of Family 2 (Y98H mutation) with mean follow-up of 15.5 and 12.1 years, respectively (P = 0.24). In Family 2, pheo was the most frequent VHL manifestation (79%) and all pheo diagnoses occurred by age 50. Age at first diagnosis was younger in Family 2 than in Family 1 (mean 19.7 vs. 28.8 years; P = 0.02). Pheo expressivity differed by genotype: Family 1 pheo was more likely to be multifocal (P = 0.04), as well as malignant (P < 0.01) and lethal (P = 0.02). Family 1 pheo was also more likely to secrete vanillylmandelic acid (VMA) alone (P = 0.05). This analysis of 130 pheochromocytomas in 63 VHL type 2A patients demonstrates that mutation-specific malignancy and expression patterns exist within the VHL type 2A subtype, and provides information that may help tailor the screening and management algorithms of affected members and those at risk.

Lung cancer susceptibility in Fhit-deficient mice is increased by Vhl haploinsufficiency.

The FHIT gene plays important roles in cancer development, including lung cancers, in which the Fhit protein is frequently lost. To determine if Fhit-deficient mice exhibit increased susceptibility to carcinogen-induced lung cancer, mice were treated with the pulmonary carcinogen 4-methylnitrosamino-1-3-pyridyl-1-butanone. Wild-type and Fhit-deficient animals did not exhibit significantly different frequencies of lung lesions, but Fhit-/- mice showed significantly increased average tumor volume (1.62 mm3) and multiplicity in tumor-bearing mice, compared with wild-type mice (0.70 mm3). Tumors of Fhit-/- mice were all carcinomas, whereas Fhit+/+ mice did not develop carcinomas. To determine if Fhit absence, in combination with deficiency of an additional 3p tumor suppressor, would affect the frequency of tumor induction, we examined the spontaneous and dimethylnitrosamine-induced tumor phenotype of Fhit-/-Vhl+/- mice. Whereas no spontaneous lung tumors were observed in Fhit-/- or Vhl+/- mice, 44% of Fhit-/-Vhl+/- mice developed adenocarcinomas by 2 years of age. Dimethylnitrosamine (6 mg/kg body weight) induced lung tumors (adenomas and carcinomas) in 100% of Fhit-/-Vhl+/- mice and adenomas in 40% of Fhit-/- mice by 20 months of age. Thus, double deficiency in murine homologues of 3p suppressor genes, including haploinsufficiency of Vhl, predisposes to spontaneous and induced lung cancers, showing that Fhit-deficient mice will be useful, in combination with other 3p tumor suppressors, in recapitulating a pattern of lung cancer development similar to the human pattern; such double- or triple-deficient mice will be excellent lung cancer prevention and therapy models.

Susceptibility to vascular neoplasms but no increased susceptibility to renal carcinogenesis in Vhl knockout mice.

The von Hippel-Lindau (VHL) tumor suppressor gene plays a prominent role in the development of renal cell carcinoma (RCC) in humans. VHL functions as a ubiquitin E3 ligase, controlling the stability of hypoxia inducible factor (HIF) and tumor angiogenesis. Alterations in this tumor suppressor gene are rarely observed in spontaneous or chemically induced RCC that arise in conventional strains of rodents and Vhl knockout mice (Vhl+/-) do not develop spontaneous RCC. We tested whether Vhl knockout mice exhibited increased susceptibility to renal carcinogenesis using the well-characterized renal carcinogen streptozotocin. No differences were observed between wild-type and Vhl+/- animals in the frequency or type of renal lesions induced by 50-200 mg/kg streptozotocin. Carcinogen-induced RCC that developed in Vhl heterozygotes and wild-type mice did not contain mutations in the wild-type Vhl, as determined by direct sequencing of the primary tumors. While Vhl+/- mice exhibited no increase in renal lesions in response to streptozotocin, heterozygous animals did develop vascular proliferative lesions of the liver, uterus, ovary, spleen and heart. These lesions, ranging from angiectasis to hemangiosarcoma, were most prominent in the livers of Vhl+/- mice, where they were found in high incidence and high multiplicity. Wild-type mice developed a low-frequency of liver angiectasis (7-15%) only at the highest doses of carcinogen used (150 and 200 mg/kg, respectively) while Vhl+/- mice exhibited angiectasis, hemangioma and hemangiosarcomas with a frequency ranging from 19 to 46% at 50-200 mg/kg streptozotocin. Untreated Vhl+/- mice had a spontaneous incidence of hepatic vascular lesions of 21%. Furthermore, vascular lesions of the uterus, ovary, spleen and heart were observed only in Vhl+/- mice, with an incidence of (5-28%). Taken together, the data indicate that heterozygosity at the Vhl locus predisposes mice to a vascular phenotype ranging from angiectasis to hemangiosarcoma, consistent with the ability of this tumor suppressor gene to control the stability of HIF and regulate key proteins that participate in angiogenesis.

Loss of pVHL is sufficient to cause HIF dysregulation in primary cells but does not promote tumor growth.

Inactivation of the von Hippel-Lindau (VHL) gene is associated with the development of highly vascularized tumors. pVHL targets the alpha subunits of hypoxia inducible factor (HIF) for ubiquitin-mediated degradation in an oxygen-dependent manner. Although pVHL-deficient tumor cell lines demonstrate constitutive stabilization and activation of HIF, it has yet to be shown that loss of murine Vhl alone is sufficient to dysregulate HIF. We utilized a genetic approach to demonstrate that loss of Vhl is sufficient not only to stabilize HIF-alpha subunits under normoxia, but also fully activate HIF-mediated responses. These studies have implications for the hierarchy of signaling events leading to HIF stabilization, nuclear translocation, and target gene expression. We further demonstrate that loss of murine Vhl does not promote teratocarcinoma growth, indicating that other genetic changes must occur to facilitate Vhl-mediated tumorigenesis.

Induction of cell cycle arrest and apoptosis in human prostate carcinoma cells by a recombinant adenovirus expressing p27(Kip1).

BACKGROUND: Adp27(Kip1), a recombinant adenovirus, was evaluated for expression of p27, a cyclin-dependent kinase inhibitor (CDKI) and tumor suppressor protein, in human prostate carcinoma cells. Effects of p27(Kip1) on cell cycle and apoptosis were analyzed. METHODS: We evaluated the effects of overexpression of p27(Kip1) in the human prostate carcinoma cell lines LNCaP, DU-145, and PC-3 in vitro and in vivo. Growth curve studies, cell cycle analysis, terminal deoxynucleotidyl transferase-mediated nick end-labeling (TUNEL), and annexin V-fluorescein isothiocyanate apoptosis analyses were conducted to determine effects of p27(Kip1) on cell cycle. CDKI activity assays and Western blots were conducted to determine presence/activities of CDKIs. RESULTS: Adp27(Kip1)-induced protein levels increased in a dose-dependent manner; p27(Kip1) protein was detected within 6 hr of infection with Adp27(Kip1) and remained stable for at least 48 hr. The activities of Cdk2, Cdk4, and Cdc2 kinases were inhibited 24 hr after infection with Adp27(Kip1). Bromodeoxyuridine incorporation demonstrated a dose-dependent decrease in S-phase cells 24 hr postinfection. TUNEL analysis revealed an induction of apoptosis (10 pfu/cell) within 48 hr of infection in all cell lines. Growth curve analyses demonstrated that Adp27(Kip1) infection inhibited proliferation of all cell lines tested and decreased cell numbers for Adp27(Kip1)-infected LNCaP and PC-3 cells by 96 hr. Cell cycle analysis of DNA content demonstrated an accumulation of cells in G0/G1-phase 24-120 hr after Adp27(Kip1)-infection. In vivo studies demonstrated a reduction in LNCaP xenograft tumor growth rates in mice injected with Adp27(Kip1). CONCLUSION: Exogenous p27(Kip1) overexpression results in cell cycle regulation in the human prostate carcinoma cell lines tested, representing the first use of this vector on prostate cancer cell lines in vitro and in vivo. Moreover, p27(Kip1) expression is associated with an increase in early apoptosis, which represents a recently discovered function for this protein. It also represents the first time this association has been observed in prostate carcinoma cell lines. This study provides support for the further development of Adp27(Kip1) as a potential therapeutic vector in the treatment of adenocarcinoma of the prostate.

A recombinant adenovirus expressing p7(Kip1) induces cell cycle arrest and apoptosis in human 786-0 renal carcinoma cells.

PURPOSE: We evaluated the effects of the over expression of p27Kip1, a cyclin dependent kinase inhibitor and tumor suppressor protein, on the 786-0 human renal carcinoma cell line. MATERIALS AND METHODS: The recombinant adenovirus Adp27Kip1 was evaluated for the induction of p27 protein expression in 786-0 renal carcinoma cells. Expression time and optimal vector concentration were determined. Growth curve studies, cell cycle analysis and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling were done to determine the effects of p27Kip1 on the cell cycle. Cyclin dependent protein kinase (Cdk) inhibitor (CDKI) activity assays were done to determine the expression/activities of Cdks and Western blot analysis was performed to determine the presence of CDKIs and other cell cycle regulator proteins. Nude mouse xenografts were established to demonstrate the in vivo efficacy of Adp27Kip1. RESULTS: p27Kip1 protein expression was detected within 12 hours after Adp27Kip1 infection and it remained stable for at least 48 hours. Growth studies demonstrated that Adp27Kip1 infection resulted in the inhibition of proliferation by 3 days after infection and cell death was detected by day 5. Cell cycle analysis of DNA content indicated an accumulation of cells in the G1 phase of Adp27Kip1 infected cells and a corresponding decrease in S phase cells within 48 hours after infection. Cdk activity was determined, and Cdk2, Cdk4 and Cdc2 kinase activities were inhibited, consistent with p27Kip1 over expression. The levels of the CDKIs p16 and p18 were elevated 24 hours after Adp27Kip1 infection, while p21 levels remained unchanged. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling revealed that Adp27Kip1 infection but not infection by control virus induced detectable apoptosis within 24 hours. Adp27Kip1 significantly caused the reduction in the size of tumors of the renal cell carcinoma xenografts. CONCLUSIONS: This study demonstrates the potential effectiveness of Adp27Kip1 as a vector for gene therapy studies of renal cell carcinoma.