Inhibition of cytosine 5-hydroxymethylation during progression of cancer precursor lesions in the uterine cervix.

Methylation and hydroxymethylation of cytosine moieties in CpG islands of specific genes are epigenetic processes shown to be involved in the development of cervical (pre)neoplastic lesions. We studied global (hydroxy)methylation during the subsequent steps in the carcinogenic process of the uterine cervix by using immunohistochemical protocols for the detection of 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) in paraffin-embedded tissues of the normal epithelia and (pre)malignant lesions. This approach allowed obtaining spatially resolved information of (epi)genetic alterations for individual cell populations in morphologically heterogeneous tissue samples. The normal ectocervical squamous epithelium showed a high degree of heterogeneity for both modifications, with a major positivity for 5-mC in the basal and parabasal layers in the ectocervical region, while 5-hmC immunostaining was even more restricted to the cells in the basal layer. Immature squamous metaplasia, characterized by expression of SOX17, surprisingly showed a decrease of 5-hmC in the basal compartments and an increase in the more superficial layers of the epithelium. The normal endocervical glandular epithelium showed a strong immunostaining reactivity for both modifications. At the squamocolumnar junctions, a specific 5-hmC pattern was observed in the squamous epithelium, resembling that of metaplasia, with the typical weak to negative reaction for 5-hmC in the basal cell compartment. The reserve cells underlying the glandular epithelium were also largely negative for 5-hmC but showed immunostaining for 5-mC. While the overall methylation status remained relatively constant, about 20% of the high-grade squamous lesions showed a very low immunostaining reactivity for 5-hmC. The (pre)malignant glandular lesions, including adenocarcinoma in situ (AIS) and adenocarcinoma showed a progressive decrease of hydroxymethylation with advancement of the lesion, resulting in cases with regions that were negative for 5-hmC immunostaining. These data indicate that inhibition of demethylation, which normally follows cytosine hydroxymethylation, is an important epigenetic switch in the development of cervical cancer.

Impact of the gating strategy for Ki-67 and Bcl-2 on the determination of proliferation and anti-apoptosis data by flow cytometry in non-malignant bone marrow aspirates and aspirates from patients with myeloid malignancies.

This Data in Brief article displays a flow cytometric assay that was used for the acquisition and analyses of proliferative and anti-apoptotic activity in hematopoietic cells. This dataset includes analyses of the Ki-67 positive fraction (Ki-67 proliferation index) and Bcl-2 positive fraction (Bcl-2 anti-apoptotic index) of the different myeloid bone marrow (BM) cell populations in non-malignant BM, and in BM disorders, i.e. myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). The present dataset comprises 1) the percentage of the CD34 positive blast cells, erythroid cells, myeloid cells and monocytic cells, and 2) the determined Ki-67 positive fraction and Bcl-2 positive fraction of these cell populations in tabular form. This allows the comparison and reproduction of the data when these analyses are repeated in a different setting. Because gating the Ki-67 positive and Bcl-2 positive cells is a critical step in this assay, different gating approaches were compared to determine the most sensitive and specific approach. BM cells from aspirates of 50 non-malignant, 25 MDS and 27 AML cases were stained with 7 different antibody panels and subjected to flow cytometry for determination of the Ki-67 positive cells and Bcl-2 positive cells of the different myeloid cell populations. The Ki-67 or Bcl-2 positive cells were then divided by the total number of cells of the respective cell population to generate the Ki-67 positive fraction (Ki-67 proliferation index) or the Bcl-2 positive fraction (Bcl-2 anti-apoptotic index). The presented data may facilitate the establishment and standardization of flow cytometric analyses of the Ki-67 proliferation index and Bcl-2 anti-apoptotic index of the different myeloid cell populations in non-malignant BM as well as MDS and AML patients in other laboratories. Directions for proper gating of the Ki-67 positive and Bcl-2 positive fraction are crucial for achieving standardization among different laboratories. In addition, the data and the presented assay allows application of Ki-67 and Bcl-2 in a research and clinical setting and this approach can serve as the basis for optimization of the gating strategy and subsequent investigation of other cell biological processes besides proliferation and anti-apoptosis. These data can also promote future research into the role of these parameters in diagnosis of myeloid malignancies, prognosis of myeloid malignancies and therapeutic resistance against anti-cancer therapies in these malignancies. As specific populations were identified based on cell biological characteristics, these data can be useful for evaluating gating algorithms in flow cytometry in general by confirming the outcome (e.g. MDS or AML diagnosis) with the respective proliferation and anti-apoptotic profile of these malignancies. The Ki-67 proliferation index and Bcl-2 anti-apoptotic index may potentially be used for classification of MDS and AML based on supervised machine learning algorithms, while unsupervised machine learning can be deployed at the level of single cells to potentially distinguish non-malignant from malignant cells in the identification of minimal residual disease. Therefore, the present dataset may be of interest for internist-hematologists, immunologists with affinity for hemato-oncology, clinical chemists with sub-specialization of hematology and researchers in the field of hemato-oncology.

Impact of antigen retrieval protocols on the immunohistochemical detection of epigenetic DNA modifications.

This study compares three different pretreatment protocols for the immunohistochemical detection of 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) in nuclear DNA. The human biological samples analyzed included formalin-fixed and paraffin-embedded (FFPE) normal squamous epithelium, ethanol-fixed cultured cells, and metaphase chromosomes. The antigen retrieval methods included low pH Citrate and high pH Tris-ethylenediaminetetraacetic acid (EDTA) protocols, as well as a method using Pepsin pretreatment combined with HCl for DNA denaturation. A gradual increase in the detection levels of 5-mC and 5-hmC was observed when going from Citrate via Tris/EDTA to Pepsin/HCl retrieval. While the Citrate retrieval protocol was the least efficient for the detection of 5-mC and 5-hmC, it did preserve nuclear morphology and enabled visualization of differences in intra- and internuclear distribution patterns in tissue and cell culture samples by single- and double-fluorescence detection. Quantification of (hydroxy)methylation levels in FFPE material demonstrated a significant heterogeneity and differences in 5-mC and 5-hmC levels within and between nuclei in the different compartments of normal squamous epithelium. It was concluded that immunohistochemical detection of 5-mC and 5-hmC enables the correlation of these DNA modifications with histomorphological features in heterogeneous tissues, but this is influenced by different pretreatment protocols that must be carefully chosen to allow an appropriate interpretation of these epigenetic switches.

SOX2 expression in the pathogenesis of premalignant lesions of the uterine cervix: its histo-topographical distribution distinguishes between low- and high-grade CIN.

SOX2 expression in high-grade cervical intraepithelial neoplasia (CIN3) and cervical squamous cell carcinoma is increased compared to that in the normal cervical epithelium. However, data on the expression and histological distribution of SOX2 in squamous epithelium during progression of CIN are largely lacking. We studied SOX2 expression throughout the epithelium in 53 cases of CIN1, 2, and 3. In general, SOX2 expression increased and expanded from basal/parabasal to the intermediate/superficial compartment during early stages of progression of CIN. An unexpected, specific expression pattern was found in areas classified as CIN2 and CIN3. This pattern was characterized by the absence or low expression of SOX2 in the basal/parabasal compartment and variable levels in the intermediate and superficial compartments. It was significantly associated with CIN3 (p = 0.009), not found in CIN1 and only seen in part of the CIN2 lesions. When the different patterns were correlated with the genetic make-up and presence of HPV, the CIN3-related pattern contained HPV-positive cells in the basal/parabasal cell compartment that were disomic. This is in contrast to the areas exhibiting the CIN1 and CIN2 related patterns, which frequently exhibited aneusomic cells. Based on their SOX2 localisation pattern, CIN1 and CIN2 could be delineated from CIN3. These data shed new light on the pathogenesis and dynamics of progression in premalignant cervical lesions, as well as on the target cells in the epithelium for HPV infection.

Proliferative and anti-apoptotic fractions in maturing hematopoietic cell lineages and their role in homeostasis of normal bone marrow.

Recent developments in clinical flow cytometry allow the simultaneous assessment of proliferative and anti-apoptotic activity in the different hematopoietic cell lineages and during their maturation process. This can further advance the flow cytometric diagnosis of myeloid malignancies. In this study we established indicative reference values for the Ki-67 proliferation index and Bcl-2 anti-apoptotic index in blast cells, as well as maturing erythroid, myeloid, and monocytic cells from normal bone marrow (BM). Furthermore, the cell fractions co-expressing both proliferation and anti-apoptotic markers were quantified. Fifty BM aspirates from femoral heads of patients undergoing hip replacement were included in this study. Ten-color/twelve-parameter flow cytometry in combination with a software-based maturation tool was used for immunophenotypic analysis of Ki-67 and Bcl-2 positive fractions during the erythro-, myelo-, and monopoiesis. Indicative reference values for the Ki-67 and Bcl-2 positive fractions were established for different relevant hematopoietic cell populations in healthy BM. Ki-67 and Bcl-2 were equally expressed in the total CD34 positive blast cell compartment and 30% of Ki-67 positive blast cells also showed Bcl-2 positivity. The Ki-67 and Bcl-2 positive fractions were highest in the more immature erythroid, myeloid and monocytic cells. Both fractions then gradually declined during the subsequent maturation phases of these cell lineages. We present a novel application of an earlier developed assay that allows the simultaneous determination of the Ki-67 proliferative and Bcl-2 anti-apoptotic indices in maturing hematopoietic cell populations of the BM. Their differential expression levels during the maturation process were in accordance with the demand and lifespan of these cell populations. The indicative reference values established in this study can act as a baseline for further cell biological and biomedical studies involving hematological malignancies.

Optimized gating strategy and supporting flow cytometry data for the determination of the Ki-67 proliferation index in the diagnosis of myelodysplastic syndrome.

This Data in Brief article presents a novel flow cytometric assay used to acquire and process the data presented and discussed in the research paper by Mestrum et al., co-submitted to Leukemia Research, entitled: "Integration of the Ki-67 proliferation index into the Ogata score improves its diagnostic sensitivity for low-grade myelodysplastic syndromes." [1]. The dataset includes the gated fractions of the different myeloid populations in bone marrow (BM) aspirates (total BM cells, CD34 positive blast cells, erythroid cells, granulocytes and monocytes. The raw data is hosted in FlowRepository, while the analyzed data of 1) the fractions of the different myeloid cell populations and 2) the Ki-67 proliferation indices of these myeloid cell populations are provided in tabular form to allow comparison and reproduction of the data when such analyses are performed in a different setting. BM cells from aspirates of 50 myelodysplastic syndrome (MDS) patients and 20 non-clonal cytopenic controls were stained using specific antibody panels and proper fixation and permeabilization to determine the Ki-67 proliferation indices of the different myeloid cell populations. Data was acquired with the three laser, 10-color Navios™ Flow cytometer (Beckman Coulter, Marseille, France) with a blue diode Argon laser (488 nm, 22 mW), red diode Helium/Neon laser (638 nm, 25 mW) and violet air-cooled solid-state diode laser laser (405 nm, 50 mW). A minimum of 100,000 relevant events were acquired per sample, while we aimed at acquiring 500,000 events per sample. Gating was performed with the Infinicyt v2.0 software package (Cytognos SL, Salamanca, Spain). These data may guide the development and standardization of the flow cytometric analysis of the Ki-67 proliferation index (and other markers for cell behavior) for differentiation between non-clonal cytopenic patients and MDS patients. In addition, this assay may be used in myeloid malignancies for research and clinical purposes in other laboratories. This data can be used to encourage future research regarding stem-/progenitor cell resistance against anti-cancer therapies for myeloid malignancies, diagnostics of myeloid malignancies and prognosis of myeloid malignancies. Therefore, these data are of relevance to internist-hematologists, clinical chemists with sub-specialization of hematology and hemato-oncology oriented researchers.

Integration of the Ki-67 proliferation index into the Ogata score improves its diagnostic sensitivity for low-grade myelodysplastic syndromes.

BACKGROUND: Although flow cytometric detection of myelodysplastic syndrome (MDS) with the Ogata score has a high specificity, its sensitivity for low-grade MDS is low. Additional markers are needed to improve its diagnostic reliability. Therefore, we investigated the diagnostic performance of the Ki-67 proliferation index in bone marrow (BM) cell populations for detection of MDS. METHODS: BM aspirates from 50 MDS patients and 20 non-clonal cytopenic controls were analyzed with flow cytometry to determine the Ogata score and the Ki-67 proliferation indices in different cell populations. RESULTS: Ki-67 proliferation indices alone could be used to detect MDS with a sensitivity of up to 80 % and specificity of up to 70 %. Combining the Ogata score with the Ki-67 proliferation index of erythroid cells significantly improved its sensitivity for detection of MDS from 66 % to 90 %, while maintaining a specificity of 100 %. Particularly, the sensitivity for detection of low-grade MDS improved from 56 % to 91 %. CONCLUSIONS: This is the first study using Ki-67 proliferation indices to detect MDS and shows their particularly high diagnostic sensitivity for detection of low-grade MDS. Integration of the Ki-67 proliferation index of erythroid cells into the Ogata score significantly improved its sensitivity without loss of the high specificity.

The potential of proliferative and apoptotic parameters in clinical flow cytometry of myeloid malignancies.

Standardization of the detection and quantification of leukocyte differentiation markers by the EuroFlow Consortium has led to a major step forward in the integration of flow cytometry into classification of leukemia and lymphoma. In our opinion, this now enables introduction of markers for more dynamic parameters, such as proliferative and (anti)apoptotic markers, which have proven their value in the field of histopathology in the diagnostic process of solid tumors and lymphoma. Although use of proliferative and (anti)apoptotic markers as objective parameters in the diagnostic process of myeloid malignancies was studied in the past decades, this did not result in the incorporation of these biomarkers into clinical diagnosis. This review addresses the potential of these markers for implementation in the current, state-of-the-art multiparameter analysis of myeloid malignancies. The reviewed studies clearly recognize the importance of proliferation and apoptotic mechanisms in the pathogenesis of bone marrow (BM) malignancies. The literature is, however, contradictory on the role of these processes in myelodysplastic syndrome (MDS), MDS/myeloproliferative neoplasms, and acute myeloid leukemia. Furthermore, several studies underline the need for the analysis of the proliferative and apoptotic rates in subsets of hematopoietic BM cell lineages and argue that these results can have diagnostic and prognostic value in patients with myeloid malignancies. Recent developments in multiparameter flow cytometry now allow quantification of proliferative and (anti)apoptotic indicators in myeloid cells during their different maturation stages of separate hematopoietic cell lineages. This will lead to a better understanding of the biology and pathogenesis of these malignancies.

Proliferative activity is disturbed in myeloproliferative neoplasms (MPN), myelodysplastic syndrome (MDS), and MDS/MPN diseases. Differences between MDS and MDS/MPN.

The proliferation marker Ki-67 is widely used within the field of diagnostic histopathology as a prognostic marker for solid cancers. However, Ki-67 is hardly used for prognostic and diagnostic purposes in flow cytometric analyses of hematologic neoplasms. In the present study, we investigated to what extent the proliferative activity, as determined by Ki-67 expression, is disturbed in myeloproliferative neoplasms (MPN), myelodysplastic syndrome (MDS), and MDS/MPN diseases. Bone marrow aspirates from 74 patients suffering from MPN, MDS, or MDS/MPN, and aspirates from 50 non-malignant cases were analyzed by flow cytometry for Ki-67 expression in the erythro-, myelo-, and monopoiesis. Ki-67 expression was used to investigate the proliferative activity during the various maturation steps within these hematopoietic cell lineages. In the MPN patient cohort, the proliferative activity of all cell lineages is significantly higher during almost all maturation stages compared to those of the benign control cohort. In the MDS and MDS/MPN cohort, a significantly lower proliferative activity is observed in the early maturation stages. In the MDS/MPN patient cohort, increased proliferative activity is seen in the later stages of the maturation. MDS and MDS/MPN display a distinct pattern in the proliferating fraction of maturing hematopoietic cells. This could become of added value in order to classify these malignancies based on their biological background and behavior, as well as in gaining a better understanding into the pathobiology of these malignancies.

Molecular characterization, prevalence and clinical relevance of Phodopus sungorus papillomavirus type 1 (PsuPV1) naturally infecting Siberian hamsters (Phodopus sungorus).

Phodopus sungorus papillomavirus type 1 (PsuPV1), naturally infecting Siberian hamsters (Phodopus sungorus) and clustering in the genus Pipapillomavirus (Pi-PV), is only the second PV type isolated from the subfamily of hamsters. In silico analysis of three independent complete viral genomes obtained from cervical adenocarcinoma, oral squamous cell carcinoma and normal oral mucosa revealed that PsuPV1 encodes characteristic viral proteins (E1, E2, E4, E6, E7, L1 and L2) with conserved functional domains and a highly conserved non-coding region. The overall high prevalence (102/114; 89.5 %) of PsuPV1 infection in normal oral and anogenital mucosa suggests that asymptomatic infection with PsuPV1 is very frequent in healthy Siberian hamsters from an early age onward, and that the virus is often transmitted between both anatomical sites. Using type-specific real-time PCR and chromogenic in situ hybridization, the presence of PsuPV1 was additionally detected in several investigated tumours (cervical adenocarcinoma, cervical adenomyoma, vaginal carcinoma in situ, ovarian granulosa cell tumour, mammary ductal carcinoma, oral fibrosarcoma, hibernoma and squamous cell papilloma) and normal tissues of adult animals. In the tissue sample of the oral squamous cell carcinoma individual, punctuated PsuPV1-specific in situ hybridization spots were detected within the nuclei of infected animal cells, suggesting viral integration into the host genome and a potential etiological association of PsuPV1 with sporadic cases of this neoplasm.

Chromosome instability predicts progression of premalignant lesions of the larynx.

The histopathology of premalignant laryngeal lesions does not provide reliable information on the risk of malignant transformation, hence we examined new molecular markers which can easily be implemented in clinical practice. Dual-target fluorescence in situ hybridisation (FISH) for chromosome 1 and 7 centromeres was performed on tissue sections of laryngeal premalignancies in 69 patients. Chromosome instability was indicated by numerical imbalances and/or polysomy for chromosomes 1 and 7. Additionally, immunostainings for p53, Cyclin D1 and (p)FADD expression were evaluated. Malignant progression was recorded. Eighteen patients with carcinoma in situ (CIS) were treated after diagnosis and excluded from follow-up. Chromosome instability was strongly associated with a high risk of malignant transformation, especially in lower grade lesions (hyperplasia, mild and moderate dysplasia; odds ratio = 8.4, p = 0.004). Patients with lesions containing chromosome instability showed a significantly worse 5-year progression-free survival than those with premalignancies without chromosome instability (p = 0.002). Neither histopathology nor the protein markers predicted progression in univariate analysis, although histopathological diagnosis, p53 and FADD contributed positively to chromosome instability in multivariate analysis. Chromosome instability is associated with malignant progression of laryngeal premalignancies, especially in lower grade lesions. These results may contribute to better risk counselling, provided that they can be validated in a larger patient set.

The majority of metachronous CIN1 and CIN3 lesions are caused by different human papillomavirus genotypes, indicating that the presence of CIN1 seems not to determine the risk for subsequent detection of CIN3.

Cervical intraepithelial neoplasia (CIN) is historically viewed as a progressive biologic continuum leading to cervical cancer. However, it has been questioned whether CIN1 lesions ever progress. To this end, we evaluated the number of patients with a CIN3 and a previous CIN1 diagnosis. Subsequently, metachronous CIN1 and CIN3 lesions were reviewed and human papillomavirus (HPV) genotyping was performed to evaluate whether CIN1 lesions do progress. The medical records of 1819 patients diagnosed with a CIN3 lesion were retrieved from the archives, and prior Pap smear surveillance was available for 1474 patients. Forty-four CIN3 patients (3.0%) had a previous CIN1 lesion, and review of the biopsies confirmed 43 out of 44 CIN3 lesions and 37 out of 44 CIN1 lesions (78%). Three cases were not available for analysis, and in another three cases the quality of the isolated DNA was insufficient for further analysis. Out of the 30 remaining patients, 19 patients had different HPV genotypes in their CIN1 and CIN3 lesion. The cytological diagnosis leading to the CIN1 biopsy showed high-grade squamous intraepithelial lesion in 11 out of 19 patients with a different HPV genotype in the metachronous CIN1 and CIN3 lesions. High-grade squamous intraepithelial lesion was detected in 7 out of 11 patients with the same HPV genotype. Our results show that CIN3 lesions are rarely preceded by a CIN1 lesion. The majority of metachronous CIN1 and CIN3 lesions are caused by different HPV genotypes, indicating that the presence of CIN1 seems not to determine the risk for subsequent detection of CIN3.

Mesenchymal stem cells induce T-cell tolerance and protect the preterm brain after global hypoxia-ischemia.

Hypoxic-ischemic encephalopathy (HIE) in preterm infants is a severe disease for which no curative treatment is available. Cerebral inflammation and invasion of activated peripheral immune cells have been shown to play a pivotal role in the etiology of white matter injury, which is the clinical hallmark of HIE in preterm infants. The objective of this study was to assess the neuroprotective and anti-inflammatory effects of intravenously delivered mesenchymal stem cells (MSC) in an ovine model of HIE. In this translational animal model, global hypoxia-ischemia (HI) was induced in instrumented preterm sheep by transient umbilical cord occlusion, which closely mimics the clinical insult. Intravenous administration of 2 x 10(6) MSC/kg reduced microglial proliferation, diminished loss of oligodendrocytes and reduced demyelination, as determined by histology and Diffusion Tensor Imaging (DTI), in the preterm brain after global HI. These anti-inflammatory and neuroprotective effects of MSC were paralleled by reduced electrographic seizure activity in the ischemic preterm brain. Furthermore, we showed that MSC induced persistent peripheral T-cell tolerance in vivo and reduced invasion of T-cells into the preterm brain following global HI. These findings show in a preclinical animal model that intravenously administered MSC reduced cerebral inflammation, protected against white matter injury and established functional improvement in the preterm brain following global HI. Moreover, we provide evidence that induction of T-cell tolerance by MSC might play an important role in the neuroprotective effects of MSC in HIE. This is the first study to describe a marked neuroprotective effect of MSC in a translational animal model of HIE.

Human papillomavirus multiplex ligation-dependent probe amplification assay for the assessment of viral load, integration, and gain of telomerase-related genes in cervical malignancies.

We evaluated the reliability of a novel multiplex ligation-dependent probe amplification (MLPA) assay in detecting integration of human papillomavirus (HPV) based on the viral E2/E6 copy number ratio in formalin-fixed and paraffin-embedded cervical lesions. The MLPA results were compared with those of amplification of papillomavirus oncogene transcripts for RNA, detection of integrated papillomavirus sequences for DNA, and HPV fluorescence in situ hybridization (FISH). DNA was isolated from 41 formalin-fixed and paraffin-embedded HPV-positive cervical lesions (cervical intraepithelial neoplasia grade 3 lesions, squamous cell carcinomas, and adenocarcinomas) for MLPA analysis. From 13 matching frozen samples, DNA and RNA were isolated for the detection of integrated papillomavirus sequences and/or the amplification of papillomavirus oncogene transcripts, respectively. Integrated HPV16, HPV18, or both were identified. The MLPA assay detected viral integration in 12 of these 13 cases, and episomal copies also were detected in 7 cases. In 20 of the 24 cases with exclusive viral integration or episomal viral copies as detected by FISH, MLPA confirmed the physical status of the virus. In the cases classified as mixed by FISH, the presence of excess episomal copies complicated the recognition of viral integration by MLPA. Furthermore, the feasibility of detecting gain of the telomerase genes with the HPV MLPA assay was evaluated. The MLPA confirmed the FISH data in 12 of 13 cases in which the status of copy number gain for telomerase RNA component was known. In conclusion, the HPV MLPA assay can be performed on routinely processed cervical lesions for the detection of viral load and HPV integration.

Use of the HPV MLPA assay in cervical cytology for the prediction of high grade lesions.

Current screening methods for uterine cervical cancer such as Papanicolaou smears and/or high risk human Papillomavirus (HR-HPV) detection have a high negative predictive value but a low positive predictive value for the presence of high grade cervical lesions. Therefore, new parameters are needed to reduce the rate of unnecessary referrals for colposcopy. The predictive value of the HPV multiplex ligation-dependent probe amplification (MLPA) assay, which can assess simultaneously HPV16/18 viral load and viral integration, was evaluated. The assay was applied to 170 cervical cytological samples, and the results were correlated with the matching histological follow-up. The GP5+/6+ assay and qPCR were used as a control for HR-HPV typing. The MLPA assay classified a higher percentage of cases as high-risk (high-viral load and/or viral integration) with higher grades of dysplasia. There was a high correlation between the HPV MLPA assay and qPCR for viral load and HPV genotyping, and between the MLPA assay and the GP5+/6+ assay for HPV genotyping. The sensitivity and specificity of the HPV MLPA assay for the detection of high-grade lesions were 44% and 93%, respectively. This study demonstrates that the HPV MLPA assay can reliably detect HPV 16/18, viral load, and viral integration in cytological samples. Also, high-risk classification correlated well with the presence of high-grade dysplasia. However, for the implementation of the MLPA assay into clinical practice, additional HR-HPV types need to be included to increase the sensitivity of the assay, and thereby increase its negative predictive value.

Molecular biomarkers in cervical cancer diagnosis: a critical appraisal.

INTRODUCTION: It is expected that in the near future high-risk human papillomavirus (hr-HPV) testing will be implemented as the primary cervical cancer screening method in some countries. However, only a fraction of hr-HPV positive women will have a clinically relevant lesion. As a result, there is an urgent need for additional biomarkers that can detect these lesions and that can at the same time be applied to cytological specimens. This overview evaluates the most promising cytological biomarkers. AREAS COVERED: Cytological biomarkers that can be used are being discussed in view of their molecular background. The most promising biomarkers are p16(INK4a)/Ki-67 dual immunostaining; methylation of the promoter region of the cell adhesion molecule 1 (CADM1) gene and the T-lymphocyte maturation associated protein (MAL) gene and viral integration. Their sensitivity, specificity and limitations are discussed in detail and their diagnostic accuracy is evaluated. EXPERT OPINION: The most promising cytological biomarkers for cervical cancer screening are p16(INK4a)/Ki-67 dual immunostaining, methylation of CADM1 and MAL and viral integration. Although some of the biomarkers are very promising for this purpose, no studies have evaluated how accurately these biomarkers classify or predict the outcome. Additional clinical trials are needed to determine the true clinical value of these promising cytological biomarkers.

Chromosome stability in tonsillar squamous cell carcinoma is associated with HPV16 integration and indicates a favorable prognosis.

Tonsillar squamous cell carcinoma (TSCC) is frequently associated with human papillomavirus (HPV) and chromosome instability. Data from cellular model systems are, however, controversial concerning a relation between HPV and chromosome instability development. Here we studied this association in 77 primary TSCC with known clinical outcome and cell cycle protein expression profiles. Thirty-two tumors (42%) showed HPV16-integration. All 77 cases were analyzed by fluorescence in situ hybridization using chromosome 1- and 7-specific centromere DNA probes to detect chromosome instability, indicated by the presence of chromosome imbalances and/or polyploidization for these chromosomes. In addition, eight HPV-positive dysplasias, seven of which were adjacent to a carcinoma, were analyzed. Disomy for chromosome 1 and 7 was present in 29 out of 77 TSCC (38%), of which 19 were HPV16-positive (p = 0.002). Aneusomy was observed in the remaining 48 TSCC, of which 13 were HPV-positive. Aneusomies correlated significantly with tobacco- and alcohol consumption (p = 0.001 and p = 0.016, respectively) and a higher T-stage (p = 0.018). Both HPV-positivity and chromosome disomy were significantly associated with a favorable disease-free survival (p = 0.001 and p = 0.025, respectively). Particularly in the HPV16-positive group chromosome instability is a very strong indicator for an unfavorable prognosis (p = 0.032). In the dysplasias an identical HPV and chromosome copy number status was identified as in the adjacent tumors. We conclude that HPV-positive TSCC and their precursor lesions are more often genetically stable than HPV-negative lesions and that these tumors are associated with a favorable prognosis. Chromosome instability is an indicator for unfavorable prognosis, particularly in the HPV-positive patient group.

Increase in viral load, viral integration, and gain of telomerase genes during uterine cervical carcinogenesis can be simultaneously assessed by the HPV 16/18 MLPA-assay.

Oncogenic human papillomavirus (HPV) infection is the most important risk factor in cervical carcinogenesis cases; high viral loads, viral integration into the host genome, and gain of the telomerase-related genes, TERT and TERC, are all factors associated with progression to cancer. A recently developed multiparameter HPV 16/18 multiplex ligation-dependent probe amplification (MLPA) assay, which allows the simultaneous assessment of these factors, was applied to a series of 67 normal and (pre)malignant frozen uterine cervical samples, as well as to 91 cytological preparations, to test the ability of the MLPA assay to identify high-risk lesions on the basis of these factors. Validation was performed using quantitative PCR, the PapilloCheck and fluorescence in situ hybridization. Only 5 out of 37 normal tissue samples or low-grade cervical lesions (ie, CIN1 and condyloma) showed either an HPV16 viral load higher than 25 copies per cell, viral integration, and/or gain of one of the telomerase-related genes, whereas for the high-grade cervical lesions, one or more of these risk factors was found in 25 of 30 cases. The HPV MLPA assay showed a sensitivity of 83% and a specificity of 86% in frozen cervical specimens. Furthermore, the feasibility of the MLPA assay was shown for cytological samples, where in 57% of high-grade squamous intraepithelial lesion cases, the high-risk factors were detected using this assay.

Diagnostic value of processing cytologic aspirates of renal tumors in agar cell (tissue) blocks.

OBJECTIVE: To adapt a method enabling utilization of most of the harvest from a fine needle aspirate in an effort to improve diagnostic accuracy in the assessment of a renal tumor in a single histologic slide. STUDY DESIGN: In a series of 43 renal tumors, 2 fine needle aspirations were performed, 4 smears were prepared after each aspiration for conventional cytology and the remaining aspirate was processed for the improved agar microbiopsy (AM) method. Conventional cytology slides, AM slides and surgical specimens were diagnosed separately, after which the diagnoses were compared. Immunohistochemistry was performed as required on the AM sections. Surgical specimens served as the gold standard. RESULTS: In 53% of conventional cytologic smears, the cellular yield was sufficient to render a correct diagnosis. In 12% the diagnosis was incorrect, in 21% only a differential diagnosis could be formulated, and in 14% too few diagnostic cells were present in the conventional smears for a cytologic diagnosis. It was, however, possible to correctly diagnose histologic sections from 97% of AM tissue blocks. In 11 cases this was facilitated with immunohistochemistry. In only 1 case did the AM tissue block contain too few cells to formulate a diagnosis; the conventional cytologic sample in this case contained enough diagnostic cells. In all cases the AM diagnosis was confirmed in the definitive surgical specimen. CONCLUSION: Our AM technique for processing fine needle aspirates from renal tumors results in a major enhancement of diagnostic accuracy of such aspirates and should be valuable in the preoperative diagnosis of large, as well as small, renal tumors.