Monitoring mouse papillomavirus-associated cancer development using longitudinal Pap smear screening.

A substantial percentage of the population remains at risk for cervical cancer due to pre-existing human papillomavirus (HPV) infections, despite prophylactic vaccines. Early diagnosis and treatment are crucial for better disease outcomes. The development of new treatments heavily relies on suitable preclinical model systems. Recently, we established a mouse papillomavirus (MmuPV1) model that is relevant to HPV genital pathogenesis. In the current study, we validated the use of Papanicolaou (Pap) smears, a valuable early diagnostic tool for detecting HPV cervical cancer, to monitor disease progression in the MmuPV1 mouse model. Biweekly cervicovaginal swabs were collected from the MmuPV1-infected mice for viral DNA quantitation and cytology assessment. The Pap smear slides were evaluated for signs of epithelial cell abnormalities using the 2014 Bethesda system criteria. Tissues from the infected mice were harvested at various times post-viral infection for additional histological and virological assays. Over time, increased viral replication was consistent with higher levels of viral DNA, and it coincided with an uptick in epithelial cell abnormalities with higher severity scores noted as early as 10 weeks after viral infection. The cytological results also correlated with the histological evaluation of tissues harvested simultaneously. Both immunocompromised and immunocompetent mice with squamous cell carcinoma (SCC) cytology also developed vaginal SCCs. Notably, samples from the MmuPV1-infected mice exhibited similar cellular abnormalities compared to the corresponding human samples at similar disease stages. Hence, Pap smear screening proves to be an effective tool for the longitudinal monitoring of disease progression in the MmuPV1 mouse model. IMPORTANCE: Papanicolaou (Pap) smear has saved millions of women's lives as a valuable early screening tool for detecting human papillomavirus (HPV) cervical precancers and cancer. However, more than 200,000 women in the United States alone remain at risk for cervical cancer due to pre-existing HPV infection-induced precancers, as there are currently no effective treatments for HPV-associated precancers and cancers other than invasive procedures including a loop electrosurgical excision procedure (LEEP) to remove abnormal tissues. In the current study, we validated the use of Pap smears to monitor disease progression in our recently established mouse papillomavirus model. To the best of our knowledge, this is the first study that provides compelling evidence of applying Pap smears from cervicovaginal swabs to monitor disease progression in mice. This HPV-relevant cytology assay will enable us to develop and test novel antiviral and anti-tumor therapies using this model to eliminate HPV-associated diseases and cancers.

Immune Responses in Oral Papillomavirus Clearance in the MmuPV1 Mouse Model.

Human papillomavirus (HPV)-induced oropharyngeal cancer now exceeds HPV-induced cervical cancer, with a noticeable sex bias. Although it is well established that women have a more proficient immune system, it remains unclear whether immune control of oral papillomavirus infections differs between sexes. In the current study, we use genetically modified mice to target CCR2 and Stat1 pathways, with the aim of investigating the role of both innate and adaptive immune responses in clearing oral papillomavirus, using our established papillomavirus (MmuPV1) infection model. Persistent oral MmuPV1 infection was detected in Rag1ko mice with T and B cell deficiencies. Meanwhile, other tested mice were susceptible to MmuPV1 infections but were able to clear the virus. We found sex differences in key myeloid cells, including macrophages, neutrophils, and dendritic cells in the infected tongues of wild type and Stat1ko mice but these differences were not observed in CCR2ko mice. Intriguingly, we also observed a sex difference in anti-MmuPV1 E4 antibody levels, especially for two IgG isotypes: IgG2b and IgG3. However, we found comparable numbers of interferon-gamma-producing CD8 T cells stimulated by E6 and E7 in both sexes. These findings suggest that males and females may use different components of innate and adaptive immune responses to control papillomavirus infections in the MmuPV1 mouse model. The observed sex difference in immune responses, especially in myeloid cells including dendritic cell (DC) subsets, may have potential diagnostic and prognostic values for HPV-associated oropharyngeal cancer.

AACC Guidance Document on Cervical Cancer Detection: Screening, Surveillance, and Diagnosis.

BACKGROUND: Persistent genital infection with high-risk human papilloma virus (hrHPV) causes the vast majority of cases of cervical cancer. Early screening, ongoing surveillance, and accurate diagnosis are crucial for the elimination of cervical cancer. New screening guidelines for testing in asymptomatic healthy populations and management guidelines for managing abnormal results have been published by professional organizations. CONTENT: This guidance document addresses key questions related to cervical cancer screening and management including currently available cervical cancer screening tests and the testing strategies for cervical cancer screening. This guidance document introduces the most recently updated screening guidelines regarding age to start screening, age to stop screening, and frequencies of routine screening as well as risk-based management guidelines for screening and surveillance. This guidance document also summarizes the methodologies for the diagnosis of cervical cancer. Additionally, we propose a report template for human papilloma virus (HPV) and cervical cancer detection to facilitate interpretation of results and clinical decision-making. SUMMARY: Currently available cervical cancer screening tests include hrHPV testing and cervical cytology screening. The screening strategies can be primary HPV screening, co-testing with HPV testing and cervical cytology, and cervical cytology alone. The new American Society for Colposcopy and Cervical Pathology guidelines recommend variable frequencies of screening and surveillance based on risk. To implement these guidelines, an ideal laboratory report should include the indication for the test (screening, surveillance, or diagnostic workup of symptomatic patients); type of test (primary HPV screening, co-testing, or cytology alone); clinical history of the patient; and prior as well as current testing results.

Comparison of Performance between the Elecsys 4th and 5th Generation STAT Cardiac Troponin T Immunoassays.

OBJECTIVE: To evaluate the analytical and clinical performance of the 5th GEN cTnT assay by comparing it with the 4th GEN assay. METHODS: Imprecision, analytical measurement range (AMR), reference interval, and quantitative comparison were studied. Qualitative comparisons of the two assays for randomly selected patients with the cTnT test orders and patients with elevated N-terminal proB-type natriuretic peptide (NT-proBNP), decreased estimated glomerular filtration rate (eGFR), and increased procalcitonin were performed. RESULTS: Within-run and between-run CVs of the 5th GEN assay were 0.6-2.3% and 3.1-5.0% with AMR of 8-10,000 ng/L and reference interval of <19 ng/L. Regression analysis of the two assays showed linear relationship: y (5th GEN)=0.899x (4th GEN)+26, r=0.9993 with a positive bias of the 5th GEN assay in samples with cTnT <150 ng/L and a negative bias in samples with cTnT >500 ng/L compare to the 4th GEN assay. Agreement was 88.1% (95% CI: 80.4 to 93.1%) between the two methods according to the 99th percentile threshold. In patients with elevated NT-proBNP, decreased eGFR, and increased procalcitonin, agreements were 79.4%, 92.7%, and 100%. CONCLUSION: The 5th GEN cTnT assay demonstrates excellent precision and acceptable AMR. Compared to the 4th GEN assay, the 5th GEN assay shows a negative bias for samples with cTnT >500 ng/L but a positive bias for samples with cTnT <150 ng/L and identifies more patients with elevated cTnT.

Passive Immunization with a Single Monoclonal Neutralizing Antibody Protects against Cutaneous and Mucosal Mouse Papillomavirus Infections.

We have established a mouse papillomavirus (MmuPV1) model that induces both cutaneous and mucosal infections and cancers. In the current study, we use this model to test our hypothesis that passive immunization using a single neutralizing monoclonal antibody can protect both cutaneous and mucosal sites at different time points after viral inoculation. We conducted a series of experiments involving the administration of either a neutralizing monoclonal antibody, MPV.A4, or control monoclonal antibodies to both outbred and inbred athymic mice. Three clinically relevant mucosal sites (lower genital tract for females and anus and tongue for both males and females) and two cutaneous sites (muzzle and tail) were tested. At the termination of the experiments, all tested tissues were harvested for virological analyses. Significantly lower levels of viral signals were detected in the MPV.A4-treated female mice up to 6 h post-viral inoculation compared to those in the isotype control. Interestingly, males displayed partial protection when they received MPV.A4 at the time of viral inoculation, even though they were completely protected when receiving MPV.A4 at 24 h before viral inoculation. We detected MPV.A4 in the blood starting at 1 h and up to 8 weeks postadministration in some mice. Parallel to these in vivo studies, we conducted in vitro neutralization using a mouse keratinocyte cell line and observed complete neutralization up to 8 h post-viral inoculation. Thus, passive immunization with a monoclonal neutralizing antibody can protect against papillomavirus infection at both cutaneous and mucosal sites and is time dependent. IMPORTANCE This is the first study testing a single monoclonal neutralizing antibody (MPV.A4) by passive immunization against papillomavirus infections at both cutaneous and mucosal sites in the same host in the mouse papillomavirus model. We demonstrated that MPV.A4 administered before viral inoculation can protect both male and female athymic mice against MmuPV1 infections at cutaneous and mucosal sites. MPV.A4 also offers partial protection at 6 h post-viral inoculation in female mice. MPV.A4 can be detected in the blood from 1 h to 8 weeks after intraperitoneal (i.p.) injection. Interestingly, males were only partially protected when they received MPV.A4 at the time of viral inoculation. The failed protection in males was due to the absence of neutralizing MPV.A4 at the infected sites. Our findings suggest passive immunization with a single monoclonal neutralizing antibody can protect against diverse papillomavirus infections in a time-dependent manner in mice.

Elevated hCG Concentration in a Patient after an Unsuccessful in vitro Fertilization.

A patient had a positive serum human chorionic gonadotropin (hCG) 22 days after a failed in vitro fertilization (IVF). The result was confirmed by repeating the test using quantitative and qualitative assays after 48 hours, but the quantitative result did not double compared to the previous concentration. Heterophilic antibody interference was ruled out. The above results indicated true-positive hCG, but inconsistent with normal pregnancy. Medical history excluded hCG produced by pituitary gland, malignancy and exogenous hCG use. Ectopic pregnancy (EP) was suspected and methotrexate was initiated. Ultrasound showed periadnexal fluid suggesting separation phenomenon on the right adnexal EP and hCG was decreased one weeks after the treatment. Two weeks later, hCG became negative. The above data suggest that the elevated hCG was most likely due to EP following IVF.

Pseudohyperphosphatemia in a patient with incidentally identified progression of smoldering myeloma.

BACKGROUND: Pseudohyperphosphatemia is a rare laboratory finding in MM, especially in patients with smoldering myeloma (SMM) progressing to symptomatic multiple myeloma (MM). Laboratorians and clinicians should be aware of this phenomenon and take necessary actions to avoid misdiagnosis. METHODS: Specimens from a monoclonal IgG kappa SMM patient with extremely high serum phosphorus concentrations measured by the Roche phosphomolybdate assay were re-evaluated using serial dilutions and the ORTHO VITROS assay free from monoclonal gammaglobulin interference. Serum free kappa/lambda chain ratio was also assessed. RESULTS: Both serial dilutions and the ORTHO VITROS assay normalized serum phosphorus concentrations, suggesting the extremely high serum phosphorus concentrations measured by the Roche assay is due to interference from monoclonal gammaglobulin. Additionally, the patient's serum free kappa/lambda ratio was >100. Based on serum free kappa/lambda ratio, disease progression from SMM to MM was diagnosed. CONCLUSIONS: Prompt and appropriate laboratory investigations ensure correct diagnosis of pseudohyperphosphatemia and help clinicians properly manage patients. To our knowledge, this patient is the first reported case of pseudohyperphosphatemia in patients with progression from SMM to MM.

Human Papillomavirus and Its Testing Assays, Cervical Cancer Screening, and Vaccination.

Human papillomavirus (HPV) was found to be the causative agent for cervical cancer in the 1980s with almost 100% of cervical cancer cases testing positive for HPV. Since then, many studies have been conducted to elucidate the molecular basis of HPV, the mechanisms of carcinogenesis of the virus, and the risk factors for HPV infection. Traditionally, the Papanicolaou test was the primary screening method for cervical cancer. Because of the discovery and evolving understanding of the role of HPV in cervical dysplasia, HPV testing has been recommended as a new method for cervical cancer screening by major professional organizations including the American Cancer Society, American Society for Colposcopy and Cervical Pathology, and the American Society for Clinical Pathology. In order to detect HPV infections, many sensitive and specific HPV assays have been developed and used clinically. Different HPV assays with various principles have shown their unique advantages and limitations. In response to a clear causative relationship between high-risk HPV and cervical cancer, HPV vaccines have been developed which utilize virus-like particles to create an antibody response for the prevention of HPV infection. The vaccines have been shown in long-term follow-up studies to be effective for up to 8 years; however, how this may impact screening for vaccinated women remains uncertain. In this chapter, we will review the molecular basis of HPV, its pathogenesis, and the epidemiology of HPV infection and associated cervical cancer, discuss the methods of currently available HPV testing assays as well as recent guidelines for HPV screening, and introduce HPV vaccines as well as their impact on cervical cancer screening and treatments.

Challenges of inpatient blood glucose monitoring: standards, methods, and devices to measure blood glucose.

Glucose control in the hospital setting is very important. There is a high incidence of hyperglycemia, hypoglycemia, and glycemic variability in hospitalized patients. Safe insulin delivery and glucose control is dependent on reliable glucose meters and monitoring systems in the hospital. Different glucose monitoring systems use arterial, venous, central venous, and capillary blood samples. It is important for clinicians to be aware that there are limitations of specific point-of-care (POC) glucose meters and that situations exist whereby POC glucose meters as the sole measurement device should be avoided. POC meter devices are not approved by the Food and Drug Administration for use in critical care, although POC meter devices are commonly used in critical care settings and elsewhere. This review focuses on glucose assay principles, instrument technology, influences on glucose measurement, standards for glucose measurement, and an evaluation of different methods to measure blood glucose in the hospital setting.

Differences identified between serum and urine immunofixation electrophoresis.

BACKGROUND: Different patterns between serum immunofixation electrophoresis (SIFE) and urine immunofixation electrophoresis (UIFE) happen occasionally and laboratorians should understand the mechanisms behind the differences and additional tests required for complicated cases. METHODS: We investigated a complicated multiple myeloma case that showed inconsistent patterns of SIFE and UIFE. To differentiate monoclonal proteins (M-proteins), the urine sample was treated with dithiothreitol to open up the IgA molecule and urine free light chain assay was used for free light chain analysis. RESULTS: The patient's SIFE indicated 2 IgA λ and 1 IgM κ M-proteins, while UIFE revealed monoclonal free λ light chains and a suspicious monoclonal IgA κ protein. Subsequent treatment of the urine sample with dithiothreitol and urine free light chain assay demonstrated that the suspicious monoclonal IgA κ protein was actually a monoclonal IgA λ and a free κ light chain that had similar electrophoretic mobility. CONCLUSIONS: The differences identified between SIFE and UIFE in this case are due to the limitation of immunofixation electrophoresis on different specimen types and intra-molecular disulfide bonds formation in IgA. The laboratorians must be cognizant of the strengths and limitations of the procedure being performed and the ancillary testing techniques available to solve the problem.

Detection and monitoring of ovarian cancer.

Ovarian cancer is the fifth leading cause of death among women in the United States and remains the most common cause of death from a gynecologic malignancy. Most ovarian cancers are diagnosed at an advanced stage in which 5-year survival rate is approximately 30%. Given that the 5-year survival rate is greater than 90% for women diagnosed at an early stage, early detection in women presenting with vague symptoms is crucial to improve outcome. Diagnosis of ovarian cancer is largely based on symptoms, imaging, and laboratory biomarkers. Overall, more than 200 potential biomarkers differentially expressed in ovarian cancer have been identified (Lokshin, 2012). However, no single marker has been found useful for the diagnosis of ovarian cancer. Increased sensitivity and specificity for the diagnosis of ovarian cancer are observed when multiple markers are used in combination. The Food and Drug Administration (FDA) recently cleared two algorithms to evaluate the risk of ovarian cancer for women who present with pelvic mass. In this review, we will summarize the most recent serum biomarkers and clinical applications of biomarkers for the early detection and treatment monitoring of ovarian cancers. We will also discuss the algorithms for predicting the risk of ovarian cancers.

Telomerase RNA accumulates in Cajal bodies in human cancer cells.

Telomerase synthesizes telomeric DNA repeats at the ends of eukaryotic chromosomes. The RNA component of the enzyme (hTR) provides the template for telomere synthesis, which is catalyzed by telomerase reverse transcriptase (hTERT). Little is known regarding the subcellular localization of hTR and hTERT and the pathway by which telomerase is assembled. Here we report the first glimpse of the detailed subcellular localization of endogenous hTR in human cells, which we obtained by fluorescence in situ hybridization (FISH). Our studies have revealed a distinctive hTR localization pattern in cancer cells. We have found that hTR accumulates within intranuclear foci called Cajal bodies in all typical tumor-derived cell lines examined (in which telomerase is active), but not in primary or ALT cells (where little or no hTERT is present). Accumulation of hTR in the Cajal bodies of primary cells is induced when hTERT is ectopically expressed. Moreover, we report that hTERT is also found in Cajal bodies. Our data suggest that Cajal bodies are involved in the assembly and/or function of human telomerase.

The nucleolar localization domain of the catalytic subunit of human telomerase.

Telomerase is the enzyme essential to complete the replication of the terminal DNA of most eukaryotic chromosomes. In humans, this enzyme is composed of the telomerase reverse transcriptase (hTERT) and telomerase RNA (hTR) subunits. hTR has been found in the nucleolus, a site of assembly of ribosomes as well as other ribonucleoproteins (RNPs). We therefore tested whether the hTERT component is also found in the nucleolus, where it could complex with the hTR RNA to form a functional enzyme. We report here that hTERT does indeed localize to the nucleolus, and we mapped the domain responsible for this localization to the hTR-binding region of the protein by deletion analysis. Substitution mutations in two of the three conserved hTR-binding domains in this nucleolar localization domain (NoLD) abolished nucleolar localization. However, another mutation that impeded hTR binding did not alter this subcellular localization. Additionally, wild type hTERT was detected in the nucleolus of cells that failed to express hTR. Taken together, we propose that the nucleolar localization of hTERT involves more than just the association with the hTR subunit. Furthermore, the coincidental targeting of both the hTR and hTERT subunits to the nucleolus supports the premise that the assembly of telomerase occurs in the nucleolus.