Cytology and direct human papillomavirus testing on fine needle aspirates from cervical lymph node metastases of patients with oropharyngeal squamous cell carcinoma or occult primary.

OBJECTIVE: Cervical lymph node fine needle aspirates (FNAs) may represent the only specimens available for an initial characterisation of patients with lymphadenopathy. Morphology and human papillomavirus (HPV) DNA presence were evaluated in FNAs collected from patients with oropharyngeal squamous cell carcinoma (OPSCC) or cancer of unknown primary (CUP). FNA HPV results were compared with those of the respective formalin-fixed paraffin-embedded (FFPE) primary cancer. METHODS: Liquid-based cytology was performed on FNAs collected in PreservCyt. HPV-DNA was analysed by the INNO-LiPA HPV genotyping Extra II on both cytological and FFPE samples. The CINtec® Histology Kit was used to assess p16 expression in cancer tissues. RESULTS: Forty-seven FNAs were collected from OPSCC and 16 from CUP patients. Cancer cells were found in 35/47 cases (74.5%), while 11 (23.4%) showed only necrosis and one (2.1%) was negative for malignancy. HPV-DNA was detected in 30/47 FNAs (63.8%), mostly harbouring HPV16 (90.0%). An excellent agreement was observed between the FNA and corresponding FFPE HPV status (raw agreement: 97.5%; Cohen κ: 0.94). The HPV test result on the necrotic FNAs completely matched that of the respective primary cancer. FNA HPV testing correctly identified 26/27 HPV-driven OPSCCs (96.3%). HPV was detected in nine of 16 FNAs (56.2%) from CUP patients. CONCLUSIONS: HPV status of metastatic cervical lymph node FNAs reflects that of the corresponding primary OPSCCs even when cell integrity in the FNA is not preserved and only necrotic debris are present. In patients with initial CUP, HPV-positivity on the FNA may guide the diagnostic workup and therapeutic management, since it suggests an oropharyngeal origin.

Noninferiority of 99mTc-Ethylenedicysteine-Glucosamine as an Alternative Analogue to 18F-Fluorodeoxyglucose in the Detection and Staging of Non-Small Cell Lung Cancer.

Objective.99mTc-ethylenedicysteine-glucosamine (99mTc-EC-G) was developed as a potential alternative to 18F-FDG for cancer imaging. A Phase 2 study was conducted to compare 18F-FDG PET/CT and 99mTc-EC-G SPECT/CT in the detection and staging of patients with non-small cell lung cancer (NSCLC). This study was aimed to demonstrate that 99mTc-EC-G SPECT/CT was not inferior to 18F-FDG PET/CT in patients with confirmed NSCLC. Methods. Seventeen patients with biopsy proven NSCLC were imaged with 99mTc-EC-G and 18F-FDG to detect and stage their cancers. Imaging with PET/CT began 45-60 minutes after injection of 18F-FDG. Imaging with 99mTc-EC-G began at two hours after injection (for 5 patients) or three hours (for 12 patients). SPECT/CT imaging devices from the three major vendors of SPECT/CT systems were used at 6 participating study sites. The image sets were blinded to all clinical information and interpreted by independent PET and SPECT expert readers at a central independent core laboratory. Results. 100% concordance between 99mTc-EC-G and 18F-FDG for primary lesion detection, lesion location and size, and confidence that the biopsied lesion was malignant. There was 70% agreement between 99mTc-EC-G and 18F-FDG for metastatic lesion detection, location and size, and confidence that the suspicious lesions were malignant. Conclusions. Evaluation of primary and suspicious metastatic lesions detected by 99mTc-EC-G and 18F-FDG on 17 patients resulted in excellent agreement for detection of primary and metastatic lesions. The study results indicated that 99mTc-EC-G SPECT/CT has the potential to be a clinically viable alternative to 18F-FDG PET/CT and 99mTc-EC-G is not inferior to 18F-FDG PET/CT.

Anal human papillomavirus in HIV-uninfected men who have sex with men: incidence and clearance rates, duration of infection, and risk factors.

Little is known regarding the natural history of anal human papillomavirus (HPV) infection. We aimed to evaluate incidence and clearance rates, their risk factors, and duration of anal HPV infection in HIV-uninfected men who have sex with men (MSM). A longitudinal study was conducted. Anal samples were analysed using the Linear Array HPV Genotyping test. Incidence and clearance rates, and corresponding risk factors, were estimated using a two-state Markov model. Overall, 155 MSM (median age 33.4 years) attending the largest sexually transmitted infection (STI) centre in Rome, Italy, were followed for a median of 12.2 months (Q1-Q3: 7.0-18.1). Incidence and clearance rates for any HPV were 85.6 (95% CI: 58.4-125.4) and 35.6 (95% CI: 24.7-51.5) × 1000 person-months, respectively; the median duration of infection was 9.4 months (Q1-Q3: 7.5-12.1). Receptive anal sex emerged as the only risk factor for the acquisition of any HPV (Hazard Ratio, HR = 2.65, 95% CI: 1.16-6.06). The incidence rates for carcinogenic and non-carcinogenic types were 42.3 (95% CI: 29.2-61.4) and 29.2 (95% CI: 19.5-43.7) × 1000 person-months, respectively (p = 0.13); their clearance rates were 62.9 (95% CI: 45.1-87.7) and 65.7 (95% CI: 47.4-91.0) × 1000 person-months, respectively (p = 0.83). HPV16 showed the lowest clearance rate among carcinogenic types (59.7 × 1000 person-months), and a duration of infection of 16.8 months. In conclusion, a higher incidence rate was observed for carcinogenic compared to non-carcinogenic HPV types, although the difference was not significant. HPV16 emerged as the type with the longest duration of infection and the lowest clearance rate among carcinogenic types.

High risk human papillomavirus genotyping in clinical samples: evaluation of different commercial tests.

The aim of the present study is to compare the performance of several commercial human papillomavirus (HPV) tests in a cohort of 281 women. The hybrid capture II, the PreTect-HPV-Proofer, the linear array, and DR.HPVTMIVD were utilized to detect and type HPV in parallel with in-house PCR tests followed by direct automated sequencing or by sub-cloning and sequencing. The concordance levels along with other tests were evaluated with a Cohen's K value varying between 0.60 to 0.88, indicating good correlation with nearly perfect agreement between hybrid capture II, (HCII) and the linear array test. High sensitivity was recorded by the linear array and HCII with 100% (95% CI, 0.8021 to 1.0000) detection of cervical intraepithelial neoplasia (CIN) III by both methods. Conversely, the PreTect-HPV-Proofer showed high specificity with 12% (95% CI, 0.7966 to 0.9163) positivity on normal samples. The genotyping analysis showed that agreement among tests was only low to moderate with great differences between different HPV types. Multiple infections were detected with poor concordance and sub-cloning assays revealed the presence of a lower number of HPV in comparison to the other methods. In summary, the use of different HPV tests applied to the same group of cervical smears may possibly lead to incongruent results, suggesting the need to standardize type-specific sensitivity of genotyping methods and the need to evaluate their accuracy in detecting multiple HPV infections. This would be a prerequisite for the use of genotyping assays in cervical cancer screening programs.

Predictors of human papilloma virus (HPV) infection in Italian women.

HPV infection is a "necessary cause" of cervical cancer and it is sexually transmitted. Due to upcoming mass vaccination investigation on risk factors for infection is the basis to implement prophylactic strategy even in older women. The aim of the study was to evaluate predictors of high-risk (HR) HPV infection in adult women. Between 2006 and 2008, 100 women aged >18 years, with no previous treatment for cervical lesions, were screened for HR HPV infection in Rome, Italy. Risk factors for HPV infection were investigated through a questionnaire including: ethnicity, religion, education, marital status, sexual behavior, gynecological and obstetrical history, smoking and alcohol intake. Multivariate analysis identified the "never married-separated/divorced" status (OR: 3.38; 95% CI: 1.14-10.12) as predictor of HPV infection, while having a higher age at the first sexual intercourse (FSI) shows a protective effect (OR: 0.84; 95% CI: 0.71-1.00). A trend for the association between the infection and having more than three lifetime partners was also observed (OR: 2.57; 95% CI: 0.86-7.71). No significant association was found for other demographic characteristics investigated. These findings provide a contribution in the knowledge of an adult population defining a "high-risk" sexual behavioral profile and could be helpful to target prophylactic strategies in older woman.

HPV genotypes concordance between sex partners.

The HPV genotype concordance in the sexual couples could support the sexual viral transmission of HPV infection. The present study contains a case-report of a stable Italian sex couple harbouring the same five HPV genotypes in their genital samples. The female partner, affected by vulvar condilomatosis, evidenced positivity in her cervicovaginal scraping with high risk HPV DNA Hybrid Capture 2 test and was negative at liquid-based performed Pap Test and at colposcopic examination. The male partner was clinically healthy regarding his external genitalia. In both male and female genital scrapings, the following HPV genotypes were detected by means of a PCR-based assay: 6, 16, 53, 73 and 84. This considerably high genotype concordance does not appear to be casual and supports, in our opinion, the hypothesis that genital HPV types are sexually transmitted agents

Molecular imaging: an overview and clinical applications.

Molecular imaging is a new medical discipline that integrates cell biology, molecular biology and diagnostic imaging. Clinical applications of molecular imaging include the use of nuclear medicine, magnetic resonance imaging (MRI) and ultrasound (US). The nuclear medicine applications utilize devices such as single photon emission computerized tomography (SPECT) and positron emission tomography (PET). Molecular imaging has two basic applications. The first is diagnostic imaging, which is used to determine the location and extent of targeted molecules specific to the disease being assessed. The second is therapy, which is used to treat specific disease-targeted molecules. The basic principle of the diagnostic imaging application is derived from the ability of cell and molecular biologists to identify specific receptor sites associated with target molecules that characterize the disease process to be studied. The biology teams then develop molecular imaging agents, which will bind specifically to the target molecules of interest. The principle for using molecular targeting therapy is based on an extension of the diagnostic imaging principle. Basically, it is assumed that if the molecular probe does target the specific disease molecules of interest, the same molecular agent can be loaded with an agent that will deliver therapy to the targeted cells. Patients and physicians have the clinical expectation that molecular imaging, when used for diagnostic purposes, will significantly improve the time-liness as well as the accuracy of detecting the presence and extent of disease. When applied to therapy, the expectation is that FDA-approved agents will have been shown in clinical trials to provide a significant improvement in clinical outcomes over traditional therapy methods. The eventual clinical owners of molecular imaging may be a specialty group that is a hybrid by conventional measures. For example, the clinical owner should have fundamental knowledge in basic cellular and molecular biology but must also be certified as well as competent in the specific diagnostic imaging specialty applied (i.e. nuclear, MR or ultrasound). If the owner is also to be involved with therapy, experience and appropriate certification will also be required. Another issue relates specifically to the therapy applications in oncology. It is conceivable that traditional chemotherapy and radiotherapy may be replaced in part with molecular imaging therapy that utilizes target-specific agents to treat cancer on a non-toxic, outpatient basis. The issue to be addressed by the radiology administrator is whether this new discipline will be performed in the radiology department or oncology and radiotherapy departments. Clearly, radiology and its associated diagnostic imaging subspecialties are the most logical owner of molecular imaging. However, to make this ownership a reality will require major shifts in training requirements, as well as exertion of political influence from the radiology administrators against other specialties that have much to lose in terms of patient populations and revenue to their practice.

Molecular paleontology.

Molecular paleontology, i.e., the recovery of DNA from ancient human, animal, and plant remains is an innovative research field that has received progressively more attention from the scientific community since the 1980s. In the last decade, the field was punctuated by claims which aroused great interest but eventually turned out to be fakes--the most famous being the sequence of dinosaur DNA later shown to be of human origin. At present, the discipline is characterized by some certainties and many doubts. We know, for example, that we have reasonable chances to recover authentic DNA from a mammoth carcass, while our chances are negligible (or nonexistent) in the case of a dynastic mummy from Egypt. On the other hand, though we are developing convincing models of DNA decay in bone, we are not yet able to predict whether a certain paleontological or archeological site will yield material amenable to DNA analysis. This article reviews some of the most important and promising investigations using molecular paleontology approaches, such as studies on the conservation of DNA in human bone, the quest for ancient DNA in permafrost-frozen fauna, the Tyrolean iceman, and the Neandertals.

Sequence analysis of bacterial DNA in the colon and stomach of the Tyrolean Iceman.

The male human body found in an Alpine glacier on September 19, 1991 ("Tyrolean Iceman") has, for the first time in history, given scientists a chance to perform detailed anatomical, histological, and molecular investigations on the organs of a person from the Neolithic Age (5350-5100 B.P.). In the present study, tissue samples aseptically taken from the stomach and the colon of the mummy were utilized for DNA extraction, and the DNA was PCR-amplified, using primer pairs designed to bind to fragments of the 16s ribosomal RNA gene (16s rDNA) of a broad range of bacteria. The PCR products were cloned in plasmid vectors, and the recombinant clones (amplicons) were sequenced. The sequence data were finally used for scanning data libraries containing the corresponding sequences of present-day bacteria, to infer the putative ecophysiology of the ancient ones. The same procedure was repeated on some fragments of grass from the clothing found near the corpse. These fragments were taken as a control of the microbiological situation of the glacier. The results show that the flora of the Iceman's stomach is entirely composed of Burkholderia pickettii, an organism commonly found in aquatic habitats. The colon, on the other hand, contains several members of the fecal flora of humans, such as Clostridium perfringens, C. ghonii, C. sordellii, Eubacterium tenue, and Bacteroides sp. The Iceman's colon, however, was found to contain, rather unexpectedly, also some members of the genus Vibrio. The results are discussed in light of what is known about the preservation of microbial DNA at the Iceman's site and of previous parasitological studies performed on the Iceman himself and on human coprolites.

Analysis of bacterial DNA in skin and muscle of the Tyrolean iceman offers new insight into the mummification process.

About 80 sequences (16s ribosomal RNA gene) of bacterial DNA in samples of skin and muscle taken directly from the Tyrolean iceman (3350-3100 years B.C.) or recovered during the 1992 archaeological expedition at the Alpine site were analyzed to obtain clues to the natural mummification process that allowed the corpse of the Neolithic shepherd/hunter to be preserved for more than 5,000 years. The investigation was made more complex by the fact that the surface of the mummy had been swabbed with phenol soon after the discovery (September 19, 1991). Our results show that no trace of microbial DNA is left on the actual surface of the body, while the untreated skin still bears the remains of large numbers of bacteria belonging to the genera Sphingomonas, Afipia, Curtobacterium, Microbacterium, Agromyces, and others. Compared to the untreated skin, the iceman's muscle is also very rich in bacterial DNA. However, this DNA comes, with few exceptions, from the species Clostridium algidicarnis. The sharp difference in the bacterial DNA composition of skin and muscle suggests that the remains of the original cadaveric microflora of the latter have not disappeared during the iceman's taphonomic history. On the other hand, the massive presence of C. algidicarnis, a cold-adapted sporigenous, the DNA of which was previously (Ubaldi et al. [1998] Am. J. Phys. Anthropol. 107:285-295) found in the soft tissue of a naturally desiccated Andean mummy, indicates that the hypothesis that the iceman's corpse underwent rapid dehydration by the effect of a warm wind (föhn) is no longer plausible. The results best fit with the hypothesis (Bereuter et al. [1997] Chem. Eur. J. 7:1032-1038) that the body was first covered by snow and ice, and then underwent thawing and, finally, desiccation.

The small-subunit rRNA gene sequences of venerids and the phylogeny of bivalvia.

The complete nucleotide sequence of the 18S subunit of ribosomal DNA (rDNA) was determined for the venerid clams Callista chione (Pitarinae) and Venus verrucosa (Venerinae). Comparison of the new sequences with the published sequences of 1 annelid, 2 gastropods, 2 polyplacophorans, and 19 bivalves showed that when the annelids are used as outgroup the gastropods diverge from the bivalves, which form a cluster including the polyplacophorans. When the gastropods alone were compared with the bivalves, the latter split in two groups corresponding to the two subclasses of Heterodonta and Pteriomorpha. The former include two taxa that diverged early, Galeomma and Tridacna, while the Veneridae and Mactridae form two sister groups. In contrast to previous reports and in line with morphological data, the Ostreidae are included in the Pteriomorphia and form a monophyletic group.

How microbial ancient DNA, found in association with human remains, can be interpreted.

The analysis of the DNA of ancient micro-organisms in archaeological and palaeontological human remains can contribute to the understanding of issues as different as the spreading of a new disease, a mummification process or the effect of diets on historical human populations. The quest for this type of DNA, however, can represent a particularly demanding task. This is mainly due to the abundance and diffusion of bacteria, fungi, yeasts, algae and protozoans in the most diverse environments of the present-day biosphere and the resulting difficulty in distinguishing between ancient and modern DNA. Nevertheless, at least under some special circumstances, by using rigorous protocols, which include an archaeometric survey of the specimens and evaluation of the palaeoecological consistency of the results of DNA sequence analysis, glimpses of the composition of the original microbial flora (e.g. colonic flora) can be caught in ancient human remains. Potentials and pitfalls of this research field are illustrated by the results of research works performed on prehistoric, pre-Columbian and Renaissance human mummies.

Sequence analysis of bacterial DNA in the colon of an Andean mummy.

We have isolated DNA from 14 tissue samples from the internal organs of an Andean human mummy (10th-11th century A.D.) and have checked the persistence of the original human and bacterial templates using the following main approaches: 1) amino acid racemization test; 2) quantification of mitochondrial DNA copy number; 3) survey of bacterial DNA in the different organs; 4) sequence analysis of bacterial amplicons of different lengths. The results demonstrate that both the original human DNA and the DNA of the bacteria of the mummy gut are preserved. In particular, sequence analysis of two (respectively 100 and 196 bp in length) libraries of bacterial 16s ribosomal RNA gene amplicons from the mummy colon shows that while the shortest amplicons give only modest and biased indications about the bacterial taxa, the longer amplicons allow the identification several species of the genus Clostridium which are typical of the human colon. This work represents a first example of a methodological approach which is applicable, in principle, to many other natural and artificial mummies and might open the way to the study of the structure of the human microbial ecosystem from prehistory to present.

Phylogenetic analysis of Veneridae (Bivalvia): comparison of molecular and palaeontological data.

An approximately 400-bp-long portion of the 16s rRNA gene sequence has been determined for the venerid clams Chamelea gallina (Chioninae), Dosinia lupinus (Dosiniinae), Pitar rudis, Callista chione (Pitarinae), Tapes decussatus, T. philippinarum, Venerupis (= Paphia) aurea (Tapetinae), and Venus verrucosa (Venerinae). Neighbor-joining and maximum parsimony trees support the results of traditional classification methods at the subfamily level but do not support the concept of a genus Tapes. The transversion divergence rate estimated on the basis of the palaeontological record for the C. gallina/V. verrucosa separation and for the Pitarinae is very close (0.14-0.16% per Myr, respectively) to that of ungulates and cetaceans, while the Tapetinae exhibit a much higher (0.36% per Myr) rate.

Molecular phylogeny of the fungi of the Iceman's grass clothing.

To investigate the origin of the fungal hyphae that cover the grass clothing (cloak, boots) found near the neolithic mummy known as the Tyrolean Iceman, two radiocarbon-dated samples of grass were submitted to DNA extraction. The DNA was then PCR amplified using, respectively, primers specific for the region containing the internal transcribed spacers and the 5.8s rDNA (ITS), and primers specific for an approximately 600-bp long fragment of the nuclear small-subunit ribosomal DNA (SSU rDNA) repeat units of eukaryotes. The amplification products were cloned and sequenced. Sequence analysis of 20 individual ITS clones and of ten SSU rDNA clones indicated that three types of fungal DNA can be extracted from the grass. Phylogenetic analyses, using 5.8s and SSU rDNA fungal reference sequences from EMBL and GenBank databases, suggest that the DNAs come, respectively, from a psychrophilic basidiomycetous yeast, phylogenetically close to Leucosporidium scottii, and from two ascomycetes, one of which is possibly related to the Eurotiales.

Nucleic acids in mummified plant seeds: biochemistry and molecular genetics of pre-Columbian maize.

Nucleic acids fractions were isolated from pre-Columbian maize seeds and characterized using different approaches such as polyacrylamide gel electrophoresis, anti-DNA antibody binding, HPLC fractionation, molecular hybridization with cloned genes, and DNA amplification by the polymerase chain reaction. The nucleic acids were found to be very depolymerized (less than or equal to 140 base pairs in length) and composed mainly of ribosomal RNA. Despite the very low amount and degree of polymerization of seed DNA, specific maize nuclear Mu1, Mu4, Mu8 and, possibly, Mu5 element components could be detected, thanks to the use of amplification systems as short as 90 bp. The results suggest that evaluation of the relative proportions of Mu-type element components and, possibly, other maize genomic components in single mummified kernels, may offer a new key to the study of ancient maize populations.