Methylation and hydroxymethylation of cytosine moieties in CpG islands of specific genes are epig... more 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 paraffinembedded 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.
Standardization of the detection and quantification of leukocyte differentiation markers by the E... more 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.
This Data in Brief article presents a novel flow cytometric assay used to acquire and process the... more 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.
BACKGROUND Although flow cytometric detection of myelodysplastic syndrome (MDS) with the Ogata sc... more 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 proliferation marker Ki-67 is widely used within the field of diagnostic histopathology as a ... more 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 brightfield microscopical in-situ hybridization (ISH) technique was applied to semen samples of... more A brightfield microscopical in-situ hybridization (ISH) technique was applied to semen samples of two 47,XYY males, one 46,XY/47,XXY male with fertility problems, and two normal 46,XY men, who served as controls. The use of a standardized nuclear DNA decondensation method, together with double-target ISH and morphological staining, allowed an accurate study of the sex chromosomal content and morphology of spermatozoa. In the males carrying an extra sex chromosome, we detected X-and Y-bearing spermatozoa in a ratio which did not differ significantly from the .1:1 ratio found in normal males. Aneuploidy for the sex chromosomes was found in-15% of the spermatozoa of both XYY males and in 3% of the XXY male. The most striking finding was the relatively low percentage of spermatozoa in these patients, with an average of 65% in the XYY males and 84% in the XXY male. The other cells represented immature germ cells (IGC), including spermatogonia and spermatocytes arrested at various stages of spermatogenesis. Apparently, in XYY or XXY men, these IGC are shed into the semen to an increased extent as compared to normal, fertile men. The sex chromosome constitution of these IGC was heterogeneous. However, the finding that the majority of spermatozoa in semen of 47,XYY and 47,XXY males carried a single sex chromosome strengthens the hypothesis that a 46JCY germ cell line must be present, apparently with a proliferative advantage over the 47,XYY or 47,XXY cells.
Mercurated nucleic acid probes can be used for non-radioactive in situ hybridization. The princip... more Mercurated nucleic acid probes can be used for non-radioactive in situ hybridization. The principle of the method is based on the reaction of the mercurated pyrimidine residues of the in situ hybridized probe with the sulfhydryl group of a ligand which contains a hapten. Next, the hapten is immunocytochemically detected. Previous experiments showed that stable coupling of the sulfhydryl ligands could only be obtained when positively charged amino groups are present in the ligand. On basis of this finding, ligands were synthesized containing a sulfhydryl group, two lysyl residues and hapten groups such as trinitrophenyl, fluorescyl and biotinyl. The ligands, free or bound to mercurated nucleic acids, were immunochemically characterized in ELISAs. The method was shown to be specific and sensitive in the detection of target DNA in situ on microscopic preparations and in dot-blot hybridization reactions on nitrocellulose.
The mechanisms underlying a new hybridocytochemical method, which is based on mercurated nucleic ... more The mechanisms underlying a new hybridocytochemical method, which is based on mercurated nucleic acid probes and their binding to sulfhydryl-hapten ligands, have been studied. Furthermore we developed a simple procedure for the preparation of mercurated probes at a microgram scale. Nucleic acids immobilized on Sephadex beads have been immunochemically detected after hybridization with mercurated probes and binding of the sulfhydryl-hapten ligand trinitrophenyl-glutathione. In this system, the method proved to be specific and sensitive. However, the same procedure, when applied in situ, failed to give a positive result. ELISA experiments showed that these results cannot be attributed to a suboptimal immunochemical detection of the ligand. Chromatographic analysis of mercurated polynucleotide-ligand complexes revealed, however, an unexpected lability of the mercury-sulfhydryl bond. Under non-equilibrium conditions, as present during a cytochemical washing procedure, the mercury-sulfhydryl b ond was found to dissociate rapidly. On basis of these results the hypothesis was forwarded that the bond between mercurated nucleic acids immobilized on Sephadex and the ligand was stabilized by the positive charge of the Sephadex matrix. This charge was introduced during the cyanogen bromide activation and inactivation necessary for the covalent coupling of nucleic acids to Sephadex. In situ, however, no such positive charges are present. By reversing the charge of the ligand we expected to stabilize the mercury-sulfhydryl bond. In a subsequent paper data are presented that confirm this hypothesis.
An improved protocol for in situ hybridization (ISH) to routinely processed, paraffin-imbedded ti... more An improved protocol for in situ hybridization (ISH) to routinely processed, paraffin-imbedded tissue sections from transitional bladder carcinoma (TCC) is presented. The protocol to detect numerical chromosome aberrations involved treatment of sections with thiocyanate prior to proteolytic digestion, resulting in reproducible ISH reactions. It was used to explore the influence of nuclear truncation in the detection of numerical chromosome aberrations and the detection of tumor cells among stromal and inflammatory cells, to compare the flow cytometric DNA index with chromosome copy number, and to study chromosome heterogeneity within tumors. For this study, a DNA probe for the chromosome region 1q12 was used. Hybridization of model systems with known chromosome numbers, such as sections of paraffin-embedded lymph nodes, paraffin-embedded human peripheral lymphocytes, T24 and Molt-4 cells with two, three, and four chromosomes 1, respectively, showed in at least 50% of the cells the proper number of chromosome hybridization signals in standard 6-microns-thick sections. Depending on the size of the nucleus, a certain percentage of the cells showed lower copy numbers as a result of truncation. In four cases of normal urothelium in paraffin sections, the percentage of nuclei with more than two chromosome spots did not exceed 5%. Comparison of the number of ISH signals, as detected in ethanol-fixed single cell suspensions of 11 TCCs [five flow cytometric (FCM) diploid, three FCM aneuploid, and three FCM tetraploid], with ISH results obtained in paraffin sections of the same tumors showed that typical numerical chromosome aberrations, such as trisomy and tetrasomy up to nonasomy, could be detected. However, the real chromosome copy number is underestimated, especially in tumors with high copy numbers, as detected in the single cell suspensions of the same tumors. Hybridization of a TCC with extremely large nuclei (DNA index = 3.2) containing six to nine ISH signals as detected in the isolated tumor cells, showed that an indication of these real chromosome copy numbers could be obtained in 6-microns paraffin sections. The accuracy for the detection of the chromosome copy number was even higher in cases where hybridization signals were counted in the mitotic cells. Furthermore, chromosome heterogeneity was detected by ISH using centromeric probes for chromosomes 7, 9, and 18, even though nuclei are truncated in the section. The surplus value of ISH on paraffin sections, as compared with ISH on isolated tumor cells, can be summarized as follows. (a) The focal tumor cell areas with chromosome aberrations can be recognized in the sections and be correlated with the histologic appearance.(ABSTRACT TRUNCATED AT 400 WORDS)
In this study we tested the reliability of the new Agar Micro-biopsy technique through FNA, which... more In this study we tested the reliability of the new Agar Micro-biopsy technique through FNA, which were taken from 40 ex vivo kidneytumors. In 98% the new AM-biopsy technique was concordant with the classifying histological diagnosis of the kidneytumors.
The "TOPical Imiquimod treatment of high-grade Cervical intraepithelial neoplasia" (TOPIC) trial ... more The "TOPical Imiquimod treatment of high-grade Cervical intraepithelial neoplasia" (TOPIC) trial was stopped preliminary, due to lagging inclusions. This study aimed to evaluate the treatment efficacy and clinical applicability of imiquimod 5% cream in high-grade cervical intraepithelial neoplasia (CIN). The lagging inclusions were mainly due to a strong patient preference for either of the two treatment modalities. This prompted us to initiate a new study on the same subject, with a non-randomized, open-label design: the 'TOPical Imiquimod treatment of high-grade Cervical intraepithelial neoplasia (TOPIC)-3' study.
Background: Cervical intraepithelial neoplasia (CIN) is the premalignant condition of cervical ca... more Background: Cervical intraepithelial neoplasia (CIN) is the premalignant condition of cervical cancer. Whereas not all high grade CIN lesions progress to cervical cancer, the natural history and risk of progression of individual lesions remain unpredictable. Therefore, high-grade CIN is currently treated by surgical excision: large loop excision of the transformation zone (LLETZ). This procedure has potential complications, such as acute haemorrhage, prolonged bleeding, infection and preterm birth in subsequent pregnancies. These complications could be prevented by development of a non-invasive treatment modality, such as topical imiquimod treatment. The primary study objective is to investigate the efficacy of topical imiquimod 5 % cream for the treatment of high-grade CIN and to develop a biomarker profile to predict clinical response to imiquimod treatment. Secondary study objectives are to assess treatment side-effects, disease recurrence and quality of life during and after different treatment modalities. Methods/design: The study design is a randomized controlled trial. One hundred forty women with a histological diagnosis of high-grade CIN (CIN 2-3) will be randomized into two arms: imiquimod treatment during 16 weeks (experimental arm) or immediate LLETZ (standard care arm). Treatment efficacy will be evaluated by colposcopy with diagnostic biopsies at 20 weeks for the experimental arm. Successful imiquimod treatment is defined as regression to CIN 1 or less, successful LLETZ treatment is defined as PAP 1 after 6 months. Disease recurrence will be evaluated by cytology at 6, 12 and 24 months after treatment. Side-effects will be evaluated using a standardized report form. Quality of life will be evaluated using validated questionnaires at baseline, 20 weeks and 1 year after treatment. Biomarkers, reflecting both host and viral factors in the pathophysiology of CIN, will be tested at baseline with the aim of developing a predictive biomarker profile for the clinical response to imiquimod treatment. Discussion: Treatment of high-grade CIN lesions with imiquimod in a selected patient population may diminish complications as a result of surgical intervention. More knowledge on treatment efficacy, side effects and long-term recurrence rates after treatment is necessary.
FISH Oral premalignancies Leukoplakia Oral cancer Head and neck cancer s u m m a r y Objectives: ... more FISH Oral premalignancies Leukoplakia Oral cancer Head and neck cancer s u m m a r y Objectives: One of the main problems in reducing the incidence of oral squamous cell carcinoma (OSCC) is the inability to appropriately deal with leukoplakia. Accurately identifying lesions which will progress to malignancy is currently not possible. The present study aims to establish the value of chromosome instability (CI) detection by DNA image cytometry and FISH analysis for prognosis and monitoring of oral leukoplakia. Materials and methods: For this purpose, we included from our archives 102 oral leukoplakia cases, which had been diagnosed between 1991 and 2008. Patient follow-up data were collected and the histopathological diagnosis was revised. CI assessment was carried out on paraffin-embedded tissue sections using both DNA image cytometry (ICM) and dual target FISH for chromosomes 1 and 7. Results: 16 of 102 Patients developed carcinoma in situ or OSCC. Both detection methods were found to yield prognostic information independent of the histopathological diagnosis. CI was a strong individual marker of progression, with hazard ratios (HRs) of 7.2 and 6.8 for ICM and FISH respectively. Moreover, this approach seems suitable for monitoring lesions over time (especially ICM). Combining histopathology and CI enables subdivision of patients into three risk groups, with different probabilities of malignant progression. Conclusion: CI detection seems a reliable method for risk assessment of oral premalignancies and its application may contribute to a better risk-counselling and appropriate treatment regimen or watchfull-waiting approach of patients.
Methylation and hydroxymethylation of cytosine moieties in CpG islands of specific genes are epig... more 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 paraffinembedded 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.
Standardization of the detection and quantification of leukocyte differentiation markers by the E... more 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.
This Data in Brief article presents a novel flow cytometric assay used to acquire and process the... more 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.
BACKGROUND Although flow cytometric detection of myelodysplastic syndrome (MDS) with the Ogata sc... more 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 proliferation marker Ki-67 is widely used within the field of diagnostic histopathology as a ... more 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 brightfield microscopical in-situ hybridization (ISH) technique was applied to semen samples of... more A brightfield microscopical in-situ hybridization (ISH) technique was applied to semen samples of two 47,XYY males, one 46,XY/47,XXY male with fertility problems, and two normal 46,XY men, who served as controls. The use of a standardized nuclear DNA decondensation method, together with double-target ISH and morphological staining, allowed an accurate study of the sex chromosomal content and morphology of spermatozoa. In the males carrying an extra sex chromosome, we detected X-and Y-bearing spermatozoa in a ratio which did not differ significantly from the .1:1 ratio found in normal males. Aneuploidy for the sex chromosomes was found in-15% of the spermatozoa of both XYY males and in 3% of the XXY male. The most striking finding was the relatively low percentage of spermatozoa in these patients, with an average of 65% in the XYY males and 84% in the XXY male. The other cells represented immature germ cells (IGC), including spermatogonia and spermatocytes arrested at various stages of spermatogenesis. Apparently, in XYY or XXY men, these IGC are shed into the semen to an increased extent as compared to normal, fertile men. The sex chromosome constitution of these IGC was heterogeneous. However, the finding that the majority of spermatozoa in semen of 47,XYY and 47,XXY males carried a single sex chromosome strengthens the hypothesis that a 46JCY germ cell line must be present, apparently with a proliferative advantage over the 47,XYY or 47,XXY cells.
Mercurated nucleic acid probes can be used for non-radioactive in situ hybridization. The princip... more Mercurated nucleic acid probes can be used for non-radioactive in situ hybridization. The principle of the method is based on the reaction of the mercurated pyrimidine residues of the in situ hybridized probe with the sulfhydryl group of a ligand which contains a hapten. Next, the hapten is immunocytochemically detected. Previous experiments showed that stable coupling of the sulfhydryl ligands could only be obtained when positively charged amino groups are present in the ligand. On basis of this finding, ligands were synthesized containing a sulfhydryl group, two lysyl residues and hapten groups such as trinitrophenyl, fluorescyl and biotinyl. The ligands, free or bound to mercurated nucleic acids, were immunochemically characterized in ELISAs. The method was shown to be specific and sensitive in the detection of target DNA in situ on microscopic preparations and in dot-blot hybridization reactions on nitrocellulose.
The mechanisms underlying a new hybridocytochemical method, which is based on mercurated nucleic ... more The mechanisms underlying a new hybridocytochemical method, which is based on mercurated nucleic acid probes and their binding to sulfhydryl-hapten ligands, have been studied. Furthermore we developed a simple procedure for the preparation of mercurated probes at a microgram scale. Nucleic acids immobilized on Sephadex beads have been immunochemically detected after hybridization with mercurated probes and binding of the sulfhydryl-hapten ligand trinitrophenyl-glutathione. In this system, the method proved to be specific and sensitive. However, the same procedure, when applied in situ, failed to give a positive result. ELISA experiments showed that these results cannot be attributed to a suboptimal immunochemical detection of the ligand. Chromatographic analysis of mercurated polynucleotide-ligand complexes revealed, however, an unexpected lability of the mercury-sulfhydryl bond. Under non-equilibrium conditions, as present during a cytochemical washing procedure, the mercury-sulfhydryl b ond was found to dissociate rapidly. On basis of these results the hypothesis was forwarded that the bond between mercurated nucleic acids immobilized on Sephadex and the ligand was stabilized by the positive charge of the Sephadex matrix. This charge was introduced during the cyanogen bromide activation and inactivation necessary for the covalent coupling of nucleic acids to Sephadex. In situ, however, no such positive charges are present. By reversing the charge of the ligand we expected to stabilize the mercury-sulfhydryl bond. In a subsequent paper data are presented that confirm this hypothesis.
An improved protocol for in situ hybridization (ISH) to routinely processed, paraffin-imbedded ti... more An improved protocol for in situ hybridization (ISH) to routinely processed, paraffin-imbedded tissue sections from transitional bladder carcinoma (TCC) is presented. The protocol to detect numerical chromosome aberrations involved treatment of sections with thiocyanate prior to proteolytic digestion, resulting in reproducible ISH reactions. It was used to explore the influence of nuclear truncation in the detection of numerical chromosome aberrations and the detection of tumor cells among stromal and inflammatory cells, to compare the flow cytometric DNA index with chromosome copy number, and to study chromosome heterogeneity within tumors. For this study, a DNA probe for the chromosome region 1q12 was used. Hybridization of model systems with known chromosome numbers, such as sections of paraffin-embedded lymph nodes, paraffin-embedded human peripheral lymphocytes, T24 and Molt-4 cells with two, three, and four chromosomes 1, respectively, showed in at least 50% of the cells the proper number of chromosome hybridization signals in standard 6-microns-thick sections. Depending on the size of the nucleus, a certain percentage of the cells showed lower copy numbers as a result of truncation. In four cases of normal urothelium in paraffin sections, the percentage of nuclei with more than two chromosome spots did not exceed 5%. Comparison of the number of ISH signals, as detected in ethanol-fixed single cell suspensions of 11 TCCs [five flow cytometric (FCM) diploid, three FCM aneuploid, and three FCM tetraploid], with ISH results obtained in paraffin sections of the same tumors showed that typical numerical chromosome aberrations, such as trisomy and tetrasomy up to nonasomy, could be detected. However, the real chromosome copy number is underestimated, especially in tumors with high copy numbers, as detected in the single cell suspensions of the same tumors. Hybridization of a TCC with extremely large nuclei (DNA index = 3.2) containing six to nine ISH signals as detected in the isolated tumor cells, showed that an indication of these real chromosome copy numbers could be obtained in 6-microns paraffin sections. The accuracy for the detection of the chromosome copy number was even higher in cases where hybridization signals were counted in the mitotic cells. Furthermore, chromosome heterogeneity was detected by ISH using centromeric probes for chromosomes 7, 9, and 18, even though nuclei are truncated in the section. The surplus value of ISH on paraffin sections, as compared with ISH on isolated tumor cells, can be summarized as follows. (a) The focal tumor cell areas with chromosome aberrations can be recognized in the sections and be correlated with the histologic appearance.(ABSTRACT TRUNCATED AT 400 WORDS)
In this study we tested the reliability of the new Agar Micro-biopsy technique through FNA, which... more In this study we tested the reliability of the new Agar Micro-biopsy technique through FNA, which were taken from 40 ex vivo kidneytumors. In 98% the new AM-biopsy technique was concordant with the classifying histological diagnosis of the kidneytumors.
The "TOPical Imiquimod treatment of high-grade Cervical intraepithelial neoplasia" (TOPIC) trial ... more The "TOPical Imiquimod treatment of high-grade Cervical intraepithelial neoplasia" (TOPIC) trial was stopped preliminary, due to lagging inclusions. This study aimed to evaluate the treatment efficacy and clinical applicability of imiquimod 5% cream in high-grade cervical intraepithelial neoplasia (CIN). The lagging inclusions were mainly due to a strong patient preference for either of the two treatment modalities. This prompted us to initiate a new study on the same subject, with a non-randomized, open-label design: the 'TOPical Imiquimod treatment of high-grade Cervical intraepithelial neoplasia (TOPIC)-3' study.
Background: Cervical intraepithelial neoplasia (CIN) is the premalignant condition of cervical ca... more Background: Cervical intraepithelial neoplasia (CIN) is the premalignant condition of cervical cancer. Whereas not all high grade CIN lesions progress to cervical cancer, the natural history and risk of progression of individual lesions remain unpredictable. Therefore, high-grade CIN is currently treated by surgical excision: large loop excision of the transformation zone (LLETZ). This procedure has potential complications, such as acute haemorrhage, prolonged bleeding, infection and preterm birth in subsequent pregnancies. These complications could be prevented by development of a non-invasive treatment modality, such as topical imiquimod treatment. The primary study objective is to investigate the efficacy of topical imiquimod 5 % cream for the treatment of high-grade CIN and to develop a biomarker profile to predict clinical response to imiquimod treatment. Secondary study objectives are to assess treatment side-effects, disease recurrence and quality of life during and after different treatment modalities. Methods/design: The study design is a randomized controlled trial. One hundred forty women with a histological diagnosis of high-grade CIN (CIN 2-3) will be randomized into two arms: imiquimod treatment during 16 weeks (experimental arm) or immediate LLETZ (standard care arm). Treatment efficacy will be evaluated by colposcopy with diagnostic biopsies at 20 weeks for the experimental arm. Successful imiquimod treatment is defined as regression to CIN 1 or less, successful LLETZ treatment is defined as PAP 1 after 6 months. Disease recurrence will be evaluated by cytology at 6, 12 and 24 months after treatment. Side-effects will be evaluated using a standardized report form. Quality of life will be evaluated using validated questionnaires at baseline, 20 weeks and 1 year after treatment. Biomarkers, reflecting both host and viral factors in the pathophysiology of CIN, will be tested at baseline with the aim of developing a predictive biomarker profile for the clinical response to imiquimod treatment. Discussion: Treatment of high-grade CIN lesions with imiquimod in a selected patient population may diminish complications as a result of surgical intervention. More knowledge on treatment efficacy, side effects and long-term recurrence rates after treatment is necessary.
FISH Oral premalignancies Leukoplakia Oral cancer Head and neck cancer s u m m a r y Objectives: ... more FISH Oral premalignancies Leukoplakia Oral cancer Head and neck cancer s u m m a r y Objectives: One of the main problems in reducing the incidence of oral squamous cell carcinoma (OSCC) is the inability to appropriately deal with leukoplakia. Accurately identifying lesions which will progress to malignancy is currently not possible. The present study aims to establish the value of chromosome instability (CI) detection by DNA image cytometry and FISH analysis for prognosis and monitoring of oral leukoplakia. Materials and methods: For this purpose, we included from our archives 102 oral leukoplakia cases, which had been diagnosed between 1991 and 2008. Patient follow-up data were collected and the histopathological diagnosis was revised. CI assessment was carried out on paraffin-embedded tissue sections using both DNA image cytometry (ICM) and dual target FISH for chromosomes 1 and 7. Results: 16 of 102 Patients developed carcinoma in situ or OSCC. Both detection methods were found to yield prognostic information independent of the histopathological diagnosis. CI was a strong individual marker of progression, with hazard ratios (HRs) of 7.2 and 6.8 for ICM and FISH respectively. Moreover, this approach seems suitable for monitoring lesions over time (especially ICM). Combining histopathology and CI enables subdivision of patients into three risk groups, with different probabilities of malignant progression. Conclusion: CI detection seems a reliable method for risk assessment of oral premalignancies and its application may contribute to a better risk-counselling and appropriate treatment regimen or watchfull-waiting approach of patients.
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Papers by Anton Hopman