A complementary role of multiparameter flow cytometry and high-throughput sequencing for minimal residual disease detection in chronic lymphocytic leukemia: an European Research Initiative on CLL study.

In chronic lymphocytic leukemia (CLL) the level of minimal residual disease (MRD) after therapy is an independent predictor of outcome. Given the increasing number of new agents being explored for CLL therapy, using MRD as a surrogate could greatly reduce the time necessary to assess their efficacy. In this European Research Initiative on CLL (ERIC) project we have identified and validated a flow-cytometric approach to reliably quantitate CLL cells to the level of 0.0010% (10(-5)). The assay comprises a core panel of six markers (i.e. CD19, CD20, CD5, CD43, CD79b and CD81) with a component specification independent of instrument and reagents, which can be locally re-validated using normal peripheral blood. This method is directly comparable to previous ERIC-designed assays and also provides a backbone for investigation of new markers. A parallel analysis of high-throughput sequencing using the ClonoSEQ assay showed good concordance with flow cytometry results at the 0.010% (10(-4)) level, the MRD threshold defined in the 2008 International Workshop on CLL guidelines, but it also provides good linearity to a detection limit of 1 in a million (10(-6)). The combination of both technologies would permit a highly sensitive approach to MRD detection while providing a reproducible and broadly accessible method to quantify residual disease and optimize treatment in CLL.

Mechanism and requirements for bovine papillomavirus, type 1, E1 initiator complex assembly promoted by the E2 transcription factor bound to distal sites.

DNA replication of papillomavirus requires the viral initiator E1 and the transcription factor E2. Bovine papillomavirus, type 1 (BPV-1), E1, and E2 bind cooperatively as dimers to proximal sites in the viral replicator generating a sequence-specific E1E2-ori complex. This complex is critical for replication and can be converted to a multimeric E1-ori initiator complex by displacement of E2 in the presence of hydrolyzable ATP. However, E2 can function over extended distances, and E2 at a distal position 33 base pairs upstream of the E1-binding site also loads an E1 dimer onto ori. Under these conditions, neither displacement of E2 nor ATP hydrolysis are required for E1-ori formation, consistent with a need for ATP hydrolysis in E2 displacement from E1E2-ori. However, ATP is required for stabilization of the resulting E1-ori complex. These results indicate that BPV (with a proximal E2-binding site) and human papillomaviruses (with distal E2-binding sites) utilize the same general mechanism for E1 loading but suggest that E1E2-ori, which forms preferentially on ori, may perform an additional role in BPV replication.

Structure of the intact transactivation domain of the human papillomavirus E2 protein.

Papillomaviruses cause warts and proliferative lesions in skin and other epithelia. In a minority of papillomavirus types ('high risk, including human papillomaviruses 16, 18, 31, 33, 45 and 56), further transformation of the wart lesions can produce tumours. The papillomavirus E2 protein controls primary transcription and replication of the viral genome. Both activities are governed by a approximately 200 amino-acid amino-terminal module (E2NT) which is connected to a DNA-binding carboxy-terminal module by a flexible linker. Here we describe the crystal structure of the complete E2NT module from human papillomavirus 16. The E2NT module forms a dimer both in the crystal and in solution. Amino acids that are necessary for transactivation are located at the dimer interface, indicating that the dimer structure may be important in the interactions of E2NT with viral and cellular transcription factors. We propose that dimer formation may contribute to the stabilization of DNA loops which may serve to relocate distal DNA-binding transcription factors to the site of human papillomavirus transcription initiation.

Transcription factor-dependent loading of the E1 initiator reveals modular assembly of the papillomavirus origin melting complex.

Replication of bovine papillomavirus type 1 DNA absolutely requires the viral transcription factor E2 as well as the initiator E1, although E1 alone has all the activities expected of an initiator protein. E1 assembles on the DNA in a stepwise fashion and undergoes a transition in activities from site-specific DNA-binding protein to mobile helicase. Complex assembly is assisted by the viral transcription factor E2 at two levels. E2 acts generally as a specificity factor, which through cooperative binding with E1 generates an initial E1 complex containing three E1 dimers bound to ori on one face of the DNA, E1-ori. Furthermore, E2 can promote the transition to an ori melting complex by recruiting additional E1 molecules to ori, effectively reducing the E1 concentration required for ori melting. This reaction is dependent on an E2-binding site positioned distal to the precursor E1-ori complex. The final origin melting complex has two subunits that each encircle the DNA and function independently to melt ori. The assembly pathway we describe has implication for understanding DNA melting and unwinding reactions, which are generally poorly understood.

Automated homogeneous immunoassay for gentamicin on the dimension clinical chemistry system.

BACKGROUND: Monitoring of the concentration of gentamicin in serum and plasma during therapy is widely recommended and practiced in hospitals. Our aim was to develop a homogeneous immunoassay based on particle-enhanced turbidimetric inhibition immunoassay technology to quantify gentamicin on the Dimension clinical chemistry system. METHODS: Assay performance was assessed on each of the Dimension models in a 15-instrument interlaboratory comparison study. A split-sample comparison (n = 1171) was also performed between the gentamicin methods on the Dimension system and the Abbott TDx analyzer, using multiple reagent and calibrator lots on multiple instruments. RESULTS: The Dimension method was linear to 25.1 micromol/L (12.0 microg/mL) with a detection limit of 0.63 micromol/L (0.3 microg/mL). Calibration was stable for 30 days. The within-run imprecision (CV) was <1.3%, and total imprecision ranged from 1.8% to 3.2% between 4.2 micromol/L (2.0 microg/mL) and 16.7 micromol/L (8.0 microg/mL) gentamicin. Linear regression analysis of the results on the Dimension method (DM) vs the Abbott TDx yielded the following equation: DM = 0.98TDx - 0.42; r = 0.987. Minimal interference was observed from structurally related compounds such as sagamicin, netilmicin, and sisomicin. CONCLUSION: The monoclonal antibody used in this method has similar reactivities toward the individual gentamicin subspecies C1, C1a, and C2, thus providing analytical recovery not significantly dependent on relative subspecies concentrations.

Recruitment and loading of the E1 initiator protein: an ATP-dependent process catalysed by a transcription factor.

Initiation of DNA replication critically depends on ori recognition as well as on catalytic activities of the initiator complex. For replication of papillomaviruses the catalytic activities for initiation are provided by the E1 protein. Here, we show that the transcription factor E2 acts to assemble E1 into a complex active for ori distortion in two steps. First, cooperative DNA binding of E1 and E2 generates a sequence-specific ori recognition complex. In the second ATP-dependent step, E2 is displaced and additional E1 molecules are incorporated. The net result is a final complex with low sequence specificity deposited onto a specific sequence in the DNA. This may be a general strategy to accomplish specific positioning of protein complexes with low sequence specificity.

Characterization of the interactions of human papillomavirus type 16 E6 with p53 and E6-associated protein in insect and human cells.

Human papillomavirus (HPV) 16 E6 induces the degradation of the tumour suppressor protein p53 by the ubiquitin-dependent proteolysis pathway. In vitro, this process involves the formation of a trimolecular complex between E6, p53 and a cellular protein E6-associated protein (E6-AP). However, an analysis of their potential interactions in vivo has not been carried out. We have established a model for the expression and analysis of the interactions of these three proteins in insect cells, a eukaryotic system where potentially crucial modifications of the proteins will occur. In baculovirus-infected cells the degradation of p53 can occur. However, p53 is only degraded early in the infectious cycle due to a lack of ATP at later times. Consequently, substantial quantities of material can be produced in this system for further analysis. Evidence is also provided that, in vivo, E6 can interact with p53 in the absence of E6-AP and that E6-AP can interact with p53 in the absence of E6. Furthermore, analysis of the subcellular localization of the proteins using both biochemical fractionation and indirect immunofluorescence suggests that the degradation of p53 occurs in the perinuclear region of the cell.

Expression, crystallization and preliminary X-ray analysis of the E2 transactivation domain from papillomavirus type 16.

The N-terminal transactivation domain of the E2 protein from human papillomavirus type 16 has been crystallized by vapour diffusion. Crystals belong to the space group P3121 (or P3221) with unit-cell dimensions a = b = 54.3, c = 155.5 A. There is one molecule per asymmetric unit with a solvent content of 55%. Crystals diffract to at least 2.5 A resolution and complete X-ray data to 3.4 A have been collected on a conventional laboratory source. This 201 amino-acid domain of the E2 protein has been shown to interact functionally with both the HPV E1 protein and at least three cellular transcription factors, to fulfil its role in the control of viral transcription and replication. A knowledge of the structural basis of these multiple interactions should lead to a fuller understanding of the mechanism of action of this key regulator of the HPV life cycle.

Expression patterns of the human papillomavirus type 16 transcription factor E2 in low- and high-grade cervical intraepithelial neoplasia.

Specific antibodies against the C-terminus of E2, produced by affinity purification of polyclonal antisera, have been used to identify the cellular populations which express the HPV 16 E2 transcription factor, in a series of formalin-fixed, paraffin-embedded cervical tissues. Cases were selected for both the presence of HPV 16 DNA (confirmed by multiple gene-specific PCR detections) and the presence of multiple grades of cervical intraepithelial neoplasia (CIN). The data indicate that E2 expression is highest in CIN I and in koilocytic lesions. Lower expression was observed in CIN II and little in CIN III lesions. In contrast, there was some restoration of E2 expression in invasive carcinomas, although the intracellular distribution was much more diffuse. The location of E2 expression to the superficial layers of the cervical epithelium, as well as the occurrence of some basal expression in CIN I, suggests that antibodies against HPV 16 E2 could be a useful adjunct to standard histological techniques for the detection of 'at-risk' patients as part of a cervical screening programme.

Molecular analysis of the interaction between HPV type 16 E6 and human E6-associated protein.

The complex formed between the human papillomavirus type 16 E6 protein and human E6-associated protein, which combine to ubiquitylate and degrade p53, has been studied by chemical crosslinking. Analysis of the interactions of proteins purified from Escherichia coli as well as proteins expressed in insect cells indicates that, while E6 has the capacity to form dimers, E6 and E6-associated protein interact as two monomers to form a heterologous dimer.

Differences in serological IgA responses to recombinant baculovirus-derived human papillomavirus E2 protein in the natural history of cervical neoplasia.

Infection with certain types of human papillomavirus (HPV) presents a high risk for the subsequent development of cervical intraepithelial neoplasia (CIN) and cervical carcinoma. Immunological mechanisms are likely to play a role in control of cervical HPV lesions. The HPV E2 protein has roles in virus replication and transcription, and loss of E2 functions may be associated with progression of cervical neoplasia. Accordingly, it is of interest to monitor immune responses to the E2 protein, and previous studies have reported associations between serological reactivity to E2 peptide antigens and cervical neoplasia. In order to investigate serological responses to native, full-length E2 protein, we expressed HPV-16 E2 proteins with and without an N-terminal polyhistidine tag using the baculovirus system. Purified HPV-16 E2 protein was used to develop enzyme-linked immunosorbent assays to detect serological IgG and IgA responses in cervical neoplasia patients and controls. We found that serum IgA levels against the E2 protein were elevated in CIN patients relative to normal control subjects but were not elevated in cervical cancer patients. Moreover, there appeared to be a gradient of response within cervical neoplasia such that the highest antibody levels were seen in lower grades of neoplasia up to CIN 2, whereas lower levels were observed in CIN 3 and still lower levels in cervical carcinoma. These findings suggest that the IgA antibody response to E2 may associate with stage and progression in cervical neoplasia.

Characterization of human papillomavirus type 16 E2 protein and subdomains expressed in insect cells.

The E2 open reading frame of human papillomavirus type 16 (HPV-16) encodes a DNA-binding protein which modulates papillomavirus transcription and replication. To investigate the biological and biochemical properties of the HPV-16 E2 protein, we have constructed recombinant baculoviruses which express the full-length molecule and individual N- and C-terminal domains in Sf21 insect cells. In this system the full-length E2 protein was phosphorylated and targeted to the insect cell nucleus. A 93 amino acid C-terminal fragment encompassing the DNA binding and dimerization functions of E2 was also translocated to the nucleus but was not modified by phosphorylation. The E2 N-terminal protein accumulated in the insect cell cytoplasm but was not efficiently phosphorylated. The formation of heterodimers between full-length and N-terminally truncated E2 species was observed when Sf21 cells were co-infected with recombinant viruses and when homodimers were mixed in vitro, suggesting that the dimer interface is not sufficiently stable to prevent subunit exchange in vivo. Both homo- and heterodimeric E2 species were able to bind specifically and in any combination to tandem E2 binding sites from the HPV-16 regulatory region. Furthermore, the HPV-16 E2 protein bound to DNA exhibited a distinct susceptibility profile to pronase digestion, potentially contrasting with that reported for BPV-1 E2. These observations suggest that significant structural and functional differences may exist between the BPV/HPV E2 proteins and have implications for understanding E2-dependent regulation of transcription and replication.

Human papillomavirus DNA and TP53 mutations in lung cancers from butchers.

To investigate whether the high frequency of human papillomavirus infection in butchers may be linked to their higher than average incidence of lung cancer, we have examined lung cancers from 40 butchers and 26 controls for the presence of DNA from both HPV type 7, which is found almost uniquely in hand warts from butchers and fishermen, and for those HPV types associated with laryngeal and genital cancers. No HPV 7, and only a low frequency of HPV DNA was found, suggesting that HPV infection does not make an important contribution to the elevated levels of lung cancer in meat handlers. In addition, the frequency of p53 mutation was shown to be slightly lower than previously reported in lung cancers.

A polymerase chain reaction (PCR) investigation of oral verrucae which contain HPV types 2 and 57 by in situ hybridization.

The polymerase chain reaction (PCR) and direct DNA sequencing have been used to identify strain variants of HPV types 2a/57 in formalin-fixed sections of human oral verrucae, where the virus had previously been detected by both immunofluorescence and in situ hybridization. By employing type-specific and type-common PCR primers we show that these lesions contain a mixture of viral DNAs which vary by up to 27% in DNA sequence, in a region where the variation between HPV types 2a and 57 is only 4%. The extra discriminatory power of fluorescent sequencing indicates that the lesions may also contain wild-type HPV2a/57 DNA which could provide a helper function for defective viral DNA molecules or indicate a mosaic origin for the lesions.

Kinetic and equilibrium binding studies of the human papillomavirus type-16 transcription regulatory protein E2 interacting with core enhancer elements.

The human papillomaviruses (HPVs) are a family of DNA viruses which cause benign tumours of the skin and mucosa that infrequently progress to malignant carcinoma. The E2 open reading frame of HPV is thought to encode a papillomavirus-specific transcription factor which also has a role in viral replication. The E2 proteins of all papillomaviruses studied to date have been shown to bind specifically to the common conserved sequence ACC(N)6GGT found at multiple locations in their genomes. In the case of HPV-16, a 'high risk' genital papillomavirus, the E2 protein is thought to negatively regulate expression of the major viral transforming genes E6 and E7, which have been directly implicated in the oncogenic process. However, little information exists concerning the relative or absolute affinities of the native HPV-16 protein for its palindromic recognition sequences; moreover, interpretation of any transcription or replication phenomena attributed to this protein is more complicated in the absence of such data. Here we describe the overexpression, purification and characterisation of the C-terminal 89 amino acids of the protein encompassing the DNA binding/dimerisation domain. We show that the recombinant protein purified from E.coli by a combination of non-group-specific chromatography steps retains high biological activity and is able to bind to all sites in the HPV-16 genome with high affinity (approximately 8 x 10(-11) M). In addition, kinetic studies show that the E2-DNA complexes are very stable, with half-lives ranging from 2.15 to greater than 240 min, and that nucleotides internal and external to the conserved palindrome appear to influence stability.

Butchers' warts: no evidence for person to person transmission of HPV7.

The distribution of warts due to HPV7 in workers in six abattoirs and 103 retail and wholesale butcheries has been studied to determine whether the high prevalence of HPV7 in the meat trade is the result of enhanced person to person transmission, or whether it is a ubiquitous virus which is activated by an unknown factor in meat. Warts were detected in 164 of 486 men. Scrapings were taken from 156 men, and HPV DNA was found in 112 samples, 74 of which contained HPV7. HPV7 was found in 36 workplaces, and there was no evidence of clustering of cases, as would be expected if person to person transmission was occurring in the workplace. This suggests that HPV7 is widely distributed in the community, but only causes clinical disease under specific conditions. We suggest that some unknown factor in meat enhances viral replication.

Cutaneous warts in butchers.

Several studies have indicated a high prevalence of hand warts in meat handlers, although the reasons for this are not clear. The high prevalence may be partly due to HPV7, a virus found almost exclusively in meat handlers, but the source of HPV7 is not known. We have carried out a cross-sectional survey of hand warts in male meat workers and controls from other occupational groups, to investigate the reasons for the high prevalence of warts, and particularly of HPV7, in butchers. We studied 240 abattoir workers, 246 retail and wholesale butchers, 308 engineering fitters and 292 office workers. Each subject was interviewed using a standard questionnaire, and his hands were examined by a dermatologist. Scrapings from the warts were tested for HPV1, HPV2 and HPV7 by a polymerase chain reaction method. The prevalence of hand warts was 33.3% in the abattoir workers, 34.1% in the butchers, 19.5% in the engineers and 14.7% in the office workers. Scrapings were taken from 247 of 267 subjects with warts, and HPV DNA was detected in 151 samples. The most common viruses were HPV2 (94 men) and HPV7 (76 men). The excess of warts in meat workers was largely due to HPV7, which was found in only two of the office workers, and was not found in any of the engineers. Logistic regression analysis showed no association between the prevalence of hand warts (or HPV2 and HPV7 specifically) and hand trauma, cold and wet working conditions, smoking, atopy, or handling any particular kind of meat. We suggest that some constituent of animal flesh predisposes to replication of HPV7 in keratinized epithelium.

Detection of human papilloma virus type 16 DNA in oral squames from normal young adults.

We have employed the polymerase chain reaction (PCR) to detect Human Papillomavirus (HPV) type 16 in oral squames and mononuclear cells from 62 healthy young adult volunteers. Two groups were screened for the presence of this virus, but in not all cases was DNA obtained from the scrapes. In the first (n = 30), the results show that 43% of normal individuals harbour HPV 16 (a genital type) in their buccal mucosa, epithelium of dorsum of tongue and hard palate. In the second group (n = 18), 44% of individuals were positive for HPV 16 in their oral epithelial scrapes, while only 6% were positive for the same virus in mononuclear cells. Interestingly, in 2 cases, peripheral blood lymphocyte DNA gave a positive reaction with the HPV 16 primers. To investigate possible HPV infection of lymphocytes, a further 42 lymphocyte samples, taken from the same age group as the epithelial study group, were analysed. None of these lymphocytes were positive for the presence of HPV 16 DNA.