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.

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.

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.

MDS and AML show elevated fractions of CD34-positive blast cell populations with a high anti-apoptotic versus proliferation ratio.

This study investigates the intertwined processes of (anti-)apoptosis and cell proliferation in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). Utilizing antibodies to Bcl-2 and Ki-67, the CD34-positive blast cell compartments in bone marrow aspirates from 50 non-malignant cases, 25 MDS patients, and 25 AML patients were analyzed for their anti-apoptotic and proliferative cell fractions through ten-color flow cytometry. MDS patients exhibited a significantly increased anti-apoptotic (p=0.0014) and reduced proliferative cell fraction (p=0.0030) in their blast cell population as compared to non-malignant cases. AML patients showed an even more exacerbated trend than MDS patients. The resulting Bcl-2:Ki-67 cell fraction ratios in MDS and AML were significantly increased as compared to the non-malignant cases (p=0.0004 and p<0.0001, respectively). AML patients displayed, however, a high degree of variability in their anti-apoptotic and proliferation index, attributed to heterogeneity in maturation stage and severity of the disease at diagnosis. Using double-labeling for Bcl-2 and Ki-67 it could be shown that besides blast cells with a mutually exclusive Ki-67 and Bcl-2 expression, also blast cells concurrently exhibiting anti-apoptotic and proliferative marker expression were found. Integrating these two dynamic markers into MDS and AML diagnostic workups may enable informed conclusions about their biological behavior, facilitating individualized therapy decisions for patients.

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.

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.

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.

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.

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.

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.

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.

A new multiparameter assay to assess HPV 16/18, viral load and physical status together with gain of telomerase genes in HPV-related cancers.

Oncogenic human papillomavirus (HPV) is the most important risk factor for cancer of the uterine cervix and a subgroup of head and neck cancers. Viral load has been associated with persistence of infection, whereas integration of HPV into the host cell genome is associated with transition to invasive disease. Viral integration is frequently correlated with loss of viral E2 and gain of the telomerase-related genes TERC and TERT. The objective of this study was to develop a rapid and sensitive multiplex ligation-dependent probe amplification (MLPA) assay for the simultaneous analysis of viral load, integration and copy number gain of TERC and TERT in HPV16/18-associated lesions. The performance of the assay was tested for HPV vs. human gene copy number ratios ranging from 0.1 to 100 and for percentages of integration ranging from 0 to 100%. The model systems used include plasmid mixtures and the HPV-positive cell lines SiHa, HeLa and CaSki described to contain a range of 2-600 viral copies per cell. In samples with low-viral load, viral integration can be reliably determined when more than 30% of the virus is integrated. Gain of the telomerase-related genes in the cell lines as determined by our MLPA assay was in accordance with data reported in the literature. Our study demonstrates that within a single MLPA-reaction viral type, load, integration and gain of TERC and TERT can be reliably determined, which will improve risk assessment for patients suspected for HPV infection.

The two faces of cervical adenocarcinoma in situ.

In order of frequency, cervical intraepithelial neoplasia (CIN), combined adenocarcinoma in situ (AIS)/CIN lesions, and solitary AIS are the most prevalent premalignant lesions of the uterine cervix. As the morphologic distinction of these subtypes is not always straightforward, we performed an immunophenotyping analysis to establish distinguishing profiles for each of these squamous and glandular progenitor lesions of cervical carcinoma. A series of 26 premalignant cervical lesions, comprising 13 cases of AIS, of which 7 represented solitary AIS and 6 were combined with CIN (combined AIS/CIN), as well as 13 solitary high-grade CIN lesions, were immunophenotypically analyzed using antibodies directed against p16, p63, bcl-2, and cytokeratins (CK) 5, 7, 8, 13, 17, 18, and 19. Adjacent normal epithelia were also investigated. CIN lesions expressed the full panel of antibodies. Combined AIS/CIN lesions also expressed the full complement of markers in both the AIS and CIN compartments. However, the expression of p63, bcl-2, CK5, and CK17 was lower in AIS compared with CIN. The solitary AIS lesions could be subdivided into 2 subgroups, 1 expressing the full complement of markers and a second group in which no expression of p63, bcl-2, CK5, and a sporadically CK17 expression was observed. We conclude that 2 phenotypically distinct types of AIS can be identified, that is, AIS with a reserve cell marker phenotype and AIS with an endocervical glandular phenotype. These observations support the view that reserve cells are capable of bidirectional premalignant transformation, that is, into CIN and reserve cell-type AIS, as well as combined AIS/CIN. The endocervical type of AIS is probably a result of the unidirectional transformation of progenitor cells within the glandular cell compartment.

Genetic signatures of HPV-related and unrelated oropharyngeal carcinoma and their prognostic implications.

PURPOSE: Patients with human papillomavirus (HPV)-containing oropharyngeal squamous cell carcinomas (OSCC) have a better prognosis than patients with HPV-negative OSCC. This may be attributed to different genetic pathways promoting cancer. EXPERIMENTAL DESIGN: We used comparative genomic hybridization to identify critical genetic changes in 60 selected OSCC, 28 of which were associated with HPV-16 as determined by HPV-specific PCR and fluorescence in situ hybridization analysis and positive p16(INK4A) immunostaining. The results were correlated with HPV status and clinical data from patients. RESULTS: Two thirds of OSCC harbored gain at 3q26.3-qter irrespective of HPV status. In HPV-negative tumors this alteration was associated with advanced tumor stage (P=0.013). In comparison with HPV-related OSCC, the HPV-negative tumors harbored: (a) a higher number of chromosomal alterations and amplifications (P=0.03 and 0.039, respectively); (b) significantly more losses at 3p, 5q, 9p, 15q, and 18q, and gains/amplifications at 11q13 (P=0.002, 0.03; <0.001, 0.02, 0.004, and 0.001, respectively); and (c) less often 16q losses and Xp gains (P=0.02 and 0.03). Survival analysis revealed a significantly better disease-free survival for HPV-related OSCC (P=0.02), whereas chromosome amplification was an unfavorable prognostic indicator for disease-free and overall survival (P=0.01 and 0.05, respectively). Interestingly, 16q loss, predominantly identified in HPV-related OSCC, was a strong indicator of favorable outcome (overall survival, P=0.008; disease-free survival, P=0.01) and none of these patients had a tumor recurrence. CONCLUSIONS: Genetic signatures of HPV-related and HPV-unrelated OSCC are different and most likely underlie differences in tumor development and progression. In addition, distinct chromosomal alterations have prognostic significance.

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.

P21 Cip1/WAF1 expression is strongly associated with HPV-positive tonsillar carcinoma and a favorable prognosis.

Human papillomavirus is involved in the carcinogenesis of tonsillar squamous cell carcinomas. Here, we investigated the expression and the prognostic value of key cell cycle proteins in the pRb and p53 pathways in both human papillomavirus type 16-positive and -negative tonsillar squamous cell carcinomas. Using immunohistochemistry, 77 tonsillar squamous cell carcinomas with known human papillomavirus type 16 status and clinical outcome were analyzed for expression of Ki67, p16(INK4A,) cyclin D1, pRb, p14(ARF), MDM2, p53, p21(Cip1/WAF1), and p27(KIP1). Results were correlated with each other and with clinical and demographic patient data. A total of 35% of tonsillar carcinomas harbored integrated human papillomavirus type 16 DNA and p16(INK4A) overexpression, both being considered essential features for human papillomavirus association. These tumors also showed the overexpression of p14(ARF) (P<0.0001) and p21(Cip1/WAF1) (P=0.001), and downregulation of pRb (P<0.0001) and cyclin D1 (P=0.027) compared with the human papillomavirus-negative cases. Univariate Cox regression analyses revealed a favorable survival rate for non-smokers (P=0.006), as well as for patients with T1-2 tumors (P<0.0001) or tumors showing low expression of cyclin D1 (P=0.028), presence of human papillomavirus and overexpression of p16(INK4A) (P=0.01), p14(ARF) (P=0.02) or p21(Cip1/WAF1) (P=0.004). In multivariate regression analyses, smoking and tumor size, as well as expression of cyclin D1 and p21(Cip1/WAF1), were found to be independent prognostic markers. We conclude that human papillomavirus positivity in tonsillar squamous cell carcinomas strongly correlates with p21(Cip1/WAF1) and p14(ARF) overexpression and downregulation of pRb and cyclin D1. In particular p21(Cip1/WAF1) overexpression is an excellent favorable prognosticator in tonsillar squamous cell carcinomas.

Distribution pattern and marker profile show two subpopulations of reserve cells in the endocervical canal.

SUMMARY: A previous immunophenotyping study in the fetal uterine cervix provided evidence for the existence of 2 subpopulations of reserve cells, one giving rise to glandular epithelium and the other to squamous epithelium (5). In this study, we investigated whether the adult uterine cervix also harbors different populations of reserve cells on the basis of their marker profile and distribution pattern. Sagittal sections from 10 normal uteri, comprising the region from ectocervix to lower uterine cavity, were histologically examined and immunostained for p63, bcl-2 and cytokeratins (CKs) 5, 7, 8, and 17. The endocervical canal consists of three regions, that is, a part lined with squamous epithelium, a part lined with endocervical cells and a part lined with tubal type epithelial cells. Histologically, we found reserve cells in all 10 investigated cervices, with an abundancy in the area beneath the endocervical columnar epithelium close to the squamo-columnar junction, and high in the endocervical canal where the invaginations consist of tubal type epithelium. In between, an area lined with endocervical columnar cells without reserve cells was identified. No reserve cells were detected in the endometrial epithelium. We defined the end of the endocervix as the point where the surface of the cervical canal and the invaginations are completely lined with tubal type epithelium. From this point, reserve cells were no longer found. Reserve cells show strong expression for p63, CKs 5 and 7, and moderate expression for bcl-2. CK17 is strongly expressed in the reserve cells at the squamo-columnar junction and to a lesser extent in the reserve cells close to the endometrium. Endocervical columnar cells usually express CKs 7 and 8 and sporadically also p63 and CK5. CK17 was only found in endocervical cells in the vicinity of CK17-positive subcolumnar reserve cells. Tubal-type epithelium was present in all samples and contained bcl-2, along with CKs 5, 7, and 8. As a result, bcl-2 and CK5 expression distinguishes tubal epithelium from endocervical columnar cells. We conclude that reserve cells are present in all investigated cervices along the entire cervical canal. The concentration of subglandular reserve cells is highest close to the squamo-columnar junction and in the upper third of the cervix. The marker profile of reserve cells is the same in all parts of the cervix, except for CK17, which shows a decreasing gradient from distal to proximal, indicating a subpopulation of distal reserve cells as progenitor for squamous and columnar epithelium, and proximal reserve cells that can serve as progenitor cells for columnar epithelium.