P38 delta MAPK promotes breast cancer progression and lung metastasis by enhancing cell proliferation and cell detachment.

The protein p38 mitogen-activated protein kinase (MAPK) delta isoform (p38δ) is a poorly studied member of the MAPK family. Data analysis from The Cancer Genome Atlas database revealed that p38δ is highly expressed in all types of human breast cancers. Using a human breast cancer tissue array, we confirmed elevation in cancer tissue. The breast cancer mouse model, MMTV-PyMT (PyMT), developed breast tumors with lung metastasis; however, mice deleted in p38δ (PyMT/p38δ-/-) exhibited delayed primary tumor formation and highly reduced lung metastatic burden. At the cellular level, we demonstrate that targeting of p38δ in breast cancer cells, MCF-7 and MDA-MB-231 resulted in a reduced rate of cell proliferation. In addition, cells lacking p38δ also displayed an increased cell-matrix adhesion and reduced cell detachment. This effect on cell adhesion was molecularly supported by the regulation of the focal adhesion kinase by p38δ in the human breast cell lines. These studies define a previously unappreciated role for p38δ in breast cancer development and evolution by regulating tumor growth and altering metastatic properties. This study proposes MAPK p38δ protein as a key factor in breast cancer. Lack of p38δ resulted in reduced primary tumor size and blocked the metastatic potential to the lungs.

INTU is essential for oncogenic Hh signaling through regulating primary cilia formation in basal cell carcinoma.

Inturned (INTU), a cilia and planar polarity effector, performs prominent ciliogenic functions during morphogenesis, such as in the skin. INTU is expressed in adult tissues but its role in tissue maintenance is unknown. Here, we report that the expression of the INTU gene is aberrantly elevated in human basal cell carcinoma (BCC), coinciding with increased primary cilia formation and activated hedgehog (Hh) signaling. Disrupting Intu in an oncogenic mutant Smo (SmoM2)-driven BCC mouse model prevented the formation of BCC through suppressing primary cilia formation and Hh signaling, suggesting that Intu performs a permissive role during BCC formation. INTU is essential for intraflagellar transport A complex assembly during ciliogenesis. To further determine whether Intu is directly involved in the activation of Hh signaling downstream of ciliogenesis, we examined the Hh signaling pathway in mouse embryonic fibroblasts, which readily responds to the Hh pathway activation. Depleting Intu blocked Smo agonist-induced Hh pathway activation, whereas the expression of Gli2ΔN, a constitutively active Gli2, restored Hh pathway activation in Intu-deficient cells, suggesting that INTU functions upstream of Gli2 activation. In contrast, overexpressing Intu did not promote ciliogenesis or Hh signaling. Taken together, data obtained from this study suggest that INTU is indispensable during BCC tumorigenesis and that its aberrant upregulation is likely a prerequisite for primary cilia formation during Hh-dependent tumorigenesis.

Ron tyrosine kinase receptor synergises with EGFR to confer adverse features in head and neck squamous cell carcinoma.

BACKGROUND: Although EGFR inhibitors have shown some success in the treatment of head and neck squamous cell carcinomas (HNSCCs), the results are not dramatic. Additional molecular targets are urgently needed. We previously showed that the loss of Ron receptor activity significantly slowed squamous tumour growth and progression in a murine model. Based on these data, we hypothesised that Ron expression confers an aggressive phenotype in HNSCCs. We prospectively collected and evaluated 154 snap-frozen, primary HNSCCs for Ron and EGFR expression/phosphorylation. Biomarker correlation with clinical, pathological and outcome data was performed. The biological responses of HNSCC cell lines to Ron knockdown, its activation and the biochemical interaction between Ron and EGFR were examined. RESULTS: We discovered that 64.3% (99 out of 154) HNSCCs expressed Ron. The carcinomas expressed exclusively mature functional Ron, whereas the adjacent nonmalignant epithelium expressed predominantly nonfunctional Ron precursor. There was no significant association between Ron and sex, tumour differentiation, perineural/vascular invasion or staging. However, patients with Ron+HNSCC were significantly older and more likely to have oropharyngeal tumours. Ron+HNSCC also had significantly higher EGFR expression and correlated strongly with phosphorylated EGFR (pEGFR). Newly diagnosed HNSCC with either Ron/pEGFR or both had lower disease-free survival than those without Ron and pEGFR. Knocking down Ron in SCC9 cells significantly blunted their migratory response to not only the Ron ligand, MSP, but also EGF. Stimulation of Ron in SCC9 cells significantly augmented the growth effect of EGF; the synergistic effect of both growth factors in SCC9 cells was dependent on Ron expression. Activated Ron also interacted with and transactivated EGFR. CONCLUSION: Ron synergises with EGFR to confer certain adverse features in HNSCCs.

Is p16(INK4a) protein expression in oral ST lesions a reliable precancerous marker?

This study correlates the expression of p16(INK4a) and p53 with the detection of high risk human papillomavirus (HPV) in three clinical grades of smokeless tobacco keratosis (STK) as compared with patients without a history of smokeless tobacco use. Tissue samples, including squamous cell carcinoma (SCC) were evaluated for the expression of p16(INK4a) and p53 by indirect immunohistochemical methods using commercially obtained antibodies. HPV DNA analysis was performed using consensus sequence polymerase chain reaction (PCR). At least focal p16(INK4a) expression was detected in Grade I, II and III STK, SCC and control samples of alveolar ridge keratoses (ARK). p16(INK4a) expression in STK and in ARK was typically weak but was relatively strong in all SCC. Strong p53 nuclear staining was detected in STK, SCC and ARK. HPV DNA was detected in Grade I, II and III STK, SCC and ARK, but did not correlate with p16(INK4a) expression. p16(INK4a) distribution did not correlate with STK grade and does not appear to be related to the detection of HPV DNA by PCR in either STK or in SCC. There is an apparent relationship between the grade of STK and the presence of HPV. HPV was rarely detected in high-grade lesions.

Controlled aggregation of superparamagnetic iron oxide nanoparticles for the development of molecular magnetic resonance imaging probes.

A method for synthesizing superparamagnetic iron oxide (SPIO) multi-nanoparticle aggregates as molecular magnetic resonance imaging (MRI) contrast agents is described. The approach utilizes organic acid/base interactions in the colloid to induce highly controllable nanoparticle aggregation. Monodisperse aggregates with diameters as large as 100 nm are synthesized by manipulating the interfacial surface chemistry of the SPIO nanoparticles in tetrahydrofuran solvent. Subsequent phospholipid micelle encapsulation yields micellar multi-SPIO (mmSPIO) aggregates with enhanced T(2) relaxivity (368.0 s(-1) mmol(-1) Fe) as compared to micellar single particle SPIO (302.0 s(-1) mmol(-1) Fe). mmSPIO conjugated to anti-CA125 monoclonal antibodies were incubated with ovarian carcinoma cell lines to demonstrate targeted in vitro molecular MRI, resulting in a 66% shortening in T(2) time for CA125 positive NIH:OVCAR-3 cells and a less than 3% change in T(2) time for CA125 negative SK-OV-3 cells. The controllable aggregation of mmSPIO shows potential for the development of molecular MRI contrast agents with optimal sizes for specific diagnostic imaging applications.

Survivin expression in human osteosarcoma is a marker for survival.

AIMS: Osteosarcoma is the most frequent malignant bone tumor with a peak incidence in the second and third decade of life. Evaluation of prognosis of patients with osteosarcoma is limited to clinical parameters whereas molecular markers of tumor aggression have not yet been identified. Inhibition of apoptotic cell death could play a role in the development or progression of neoplasia. Survivin is a member of the inhibitor of apoptosis (IAP) protein gene family and is expressed both during normal fetal development and in human cancer. METHODS: The localization and distribution of survivin was investigated immunohistochemically in high-grade osteosarcomas by an indirect immunoperoxidase method. RESULTS: Survivin was detected in the cytoplasm in 23/40 and in the nucleus in 20/40 cases of osteosarcoma. Nuclear localization of survivin expression was significantly correlated with a prolonged survival (P=0.0347) but cytoplasmic staining showed no correlation with patient outcome. CONCLUSIONS: The results of this study indicates that the evaluation of survivin expression might be a useful prognostic marker in osteosarcoma. Patients with osteosarcoma exhibiting nuclear survivin expression could potentially benefit from stratification of neoadjuvant chemotherapy.

Expression of survivin, YB-1, and KI-67 in sporadic adenomas and dysplasia-associated lesions or masses in ulcerative colitis.

Sporadic adenomas are said to exhibit an orderly growth pattern with a reversal of proliferative and apoptotic cell distribution as compared with normal colonic crypts. Dysplastic polyps of patients with ulcerative colitis (UC) may represent dysplasia-associated lesions or masses (DALM) with a high associated cancer risk, or, alternatively, may represent sporadic adenomas. Histologic criteria to differentiate between sporadic adenomas and DALM have not focused on the balance between cell renewal and cell loss. The expression of the novel anti-apoptosis gene product, survivin, and the proliferation markers, Ki-67 and Y-box binding protein (YB-1), were investigated by immunohistochemical localization in sporadic adenomas and DALM lesions of patients with UC. In adenomas, KI-67 was expressed preponderantly at the luminal aspect of the polyp, whereas its expression was diffuse in DALM. Survivin was detected diffusely in both adenomas and DALM. YB-1 showed positive staining in the deep aspect of adenomatous glands but only to a minor degree at the surface, whereas both deep and diffuse expression patterns of YB-1 were seen in DALM. The authors conclude that DALM and sporadic adenomas exhibit different patterns of cellular proliferation and that molecular markers of cell proliferation, Ki-67 and YB-1, may be useful to distinguish sporadic adenomas from DALM. However, the similar expression of survivin suggests that the underlying mechanisms that regulate apoptotic cell death are uniform in these lesions.

Localization of telomerase hTERT protein and survivin in placenta: relation to placental development and hydatidiform mole.

OBJECTIVE: To find if a difference in telomerase or survivin expression exists between non-neoplastic tissues and hydatidiform moles, and explore expression of those proteins in normal placental development, post-term gestation, and preeclampsia. METHODS: Formalin-fixed placental tissues were selected from collections of the Department of Pathology at the University of Colorado. Five specimens of each trimester, five each of preeclamptic and post-term placentas, and 23 molar pregnancies were selected. The telomerase catalytic protein hTERT was localized in placental tissues using the catalyzed signal amplification system, and survivin was localized by conventional immunoperoxidase method. Staining was graded on a scale of zero to 4. RESULTS: hTERT staining was detected in sections of 42 of 48 specimens (23 of 23 hydatidiform moles, 19 of 25 non-neoplastic placental tissues). The intensity of staining for hTERT was higher in hydatidiform moles (mean 3.3, median 3) compared with levels in non-neoplastic placental tissues (mean 0.92, median 1) (P <.001). Survivin was detected in 39 of 48 specimens (22 of 23 hydatidiform moles, 17 of 25 non-neoplastic placental tissues). Compared with non-neoplastic tissues (mean 0.88, median 1), survivin levels were elevated in hydatidiform moles (mean 1.35, median 1) (P =.031). CONCLUSION: Survivin and telomerase were increased in hydatidiform moles, suggesting that regulation of apoptosis and stabilization of telomere length might be involved in neoplastic transformation of the placenta. The patterns of expression observed for survivin and telomerase in non-neoplastic placental tissues suggest that the control of apoptosis and stabilization of telomeric DNA might also be involved in normal gestational development.

Macrophage-rich reaction in lymph nodes as a mimic of metastatic renal cell carcinoma on fine needle aspiration. A case report.

BACKGROUND: Renal cell carcinomas have a high metastatic potential. Many of them are occult at initial presentation and mimic a primary neoplasm of the metastatic site. However, not all lymph node enlargements in a patient with a history of renal cell carcinoma are due to metastasis. Foamy macrophages can mimic metastatic renal cell carcinoma cells. CASE: A 60-year-old male with a known diagnosis of renal cell carcinoma of clear cell type developed enlarged neck nodes 44 months after the diagnosis. These were aspirated to yield cystic fluid that, on smears, showed numerous clear cells with low nuclear grade. Immunohistochemical stains revealed these cells to be foamy macrophages (CD68 immunoreactive) and not metastatic renal cell carcinoma, as had been suspected on initial examination of Diff-Quik and Papanicolaou-stained smears. CONCLUSION: Immunohistochemistry is a valuable adjunct in avoiding a false diagnosis of metastatic carcinoma in macrophage-rich nodal reactions in patients with a history of renal cell carcinoma.

Expression of survivin in normal, hyperplastic, and neoplastic colonic mucosa.

The regulation of apoptotic cell death may have a profound effect on the pathogenesis and progression of colon cancer. Survivin, a member of the inhibitor of apoptosis gene family, has been detected in fetal tissue and in a variety of human malignancies. In the current study, we investigated survivin expression by an immunohistochemical approach in benign, hyperplastic, premalignant, and malignant lesions of the colon. Survivin was detected in all cases of normal colonic mucosa (20/20), hyperplastic polyps (20/20), adenomatous polyps (20/20), and in both well differentiated and moderately differentiated colonic adenocarcinomas (20/20). In the normal colonic mucosa, survivin expression was mostly restricted to the base of the colonic crypts. All epithelial cells showed uniformly intense staining for survivin in hyperplastic polyps. By contrast, adenomas and adenocarcinomas showed a heterogeneous staining pattern with cell-to-cell, gland-to-gland, and regional variability in the intensity of survivin staining. In contrast to the basal preponderance of staining in normal colonic mucosa, numerous survivin positive cells were present at the luminal surface of hyperplastic polyps, adenomatous polyps, and adenocarcinomas. In conclusion, the expression of survivin is not a specific marker of adenocarcinoma of the colon but does show characteristic and reproducible patterns of expression in non-neoplastic proliferative lesions and in normal colonic mucosa.

Chromosomal aneuploidy precedes morphological changes and supports multifocality in head and neck lesions.

OBJECTIVE: To identify chromosome changes associated with the transformation of dysplastic lesions and to verify evidence for multifocality in synchronous premalignant lesions associated with head and neck squamous cell carcinoma (HNSCC). STUDY DESIGN: Chromosomal aneuploidy was evaluated in sections of formalin-fixed, paraffin-embedded tissues from 16 patients with HNSCC, including sites with normal squamous mucosa, dysplasia (low- and high-grade), and invasive tumor. METHODS: A panel of 6 centromeric probes (chromosomes 1, 3, 7, 8, 9, and 17) was analyzed in dual-color fluorescence in situ hybridization assays, using matched hematoxylin-eosin-stained sections for histologic correlation. RESULTS: Imbalances for most of the targets tested were found in 20 of 24 invasive carcinoma sites, mainly represented by gain in copy number per cell. However, cell populations with chromosome losses and gains in multimodal patterns were concomitantly observed in a number of tumors, indicating a high degree of chromosome instability. The detection of chromosomal aneuploidy precedes the malignant transformation as indicated by findings of monosomy and trisomy in normal squamous mucosa, and in low-grade and high-grade dysplasia sites. Loss of chromosomes 3 and 17 prevailed in low-grade dysplasias, and gain of chromosomes 7 and 8 were prevalent in high-grade dysplasias. Synchronous low-grade and high-grade dysplastic lesions displayed discordant molecular signatures, suggesting a multifocal origin. CONCLUSIONS: The interphase fluorescence in-situ hybridization (FISH) assay with centromeric may detect early changes in the progression of dyplastic epithelia to invasive carcinoma and supports the field cancerization theory of multifocality.

Localization of telomerase hTERT protein and hTR in benign mucosa, dysplasia, and squamous cell carcinoma of the cervix.

Telomerase has been detected by telomerase repeat amplification protocol (TRAP) assay in cervical dysplasia and squamous cell carcinoma but not in most normal cervical tissues. In the present study, the cellular localization of the protein catalytic subunit of telomerase (hTERT) and the RNA component (hTR) were investigated by a sensitive immunohistochemical technique and by in situ hybridization, respectively. hTERT protein was detected in all diagnostic categories of cervical specimens. hTERT was localized predominantly to the lower suprabasal levels of normal squamous mucosa but was detected throughout virtually all levels of the lesional epithelium in low-grade squamous intraepithelial lesions (LSILs), high-grade squamous intraepithelial lesions (HSILs), and squamous cell carcinoma (SCC). Telomerase expression correlated with hTERT detection in SCC and HSIL but was not detected by TRAP assay in most samples of normal mucosa or LSIL. The distribution of hTR correlated with the localization of hTERT in HSIL and SCC but was restricted to the basal and suprabasal cell layers in normal mucosa and LSIL.

Quantitative telomerase expression in glioblastomas shows regional variation and down-regulation with therapy but no correlation with patient outcome.

Despite the nearly ubiquitous expression of telomerase in almost all types of malignant human tumors, studies have shown widely varying positivity in the highest-grade glioma, the glioblastomas (GBMs), ranging from 26% to 100% of tumors analyzed. We have previously shown significant variability in positive versus negative telomerase expression from region to region within the same GBM. In this study, we hypothesized that application of new quantitative methodology would extend our previous observations and identify whether there is heterogeneity in levels of protein expression even within areas positive for telomerase in high-grade gliomas. Finally, we sought to correlate quantitative telomerase expression with patient outcome and therapeutic response. Quantitative analysis was achieved by polymerase chain-based TRAP assay with phosphorimager analysis and compared with clinical information obtained from 19 patients, most with primary, untreated GBMs. Results showed up to 3-fold variability in telomerase levels across multiple regional samples from the same patient, as well as between patients. In 5 of 6 patients with recurrent tumors who had received intervening radiation therapy or chemotherapy, telomerase was downregulated in the second, post-therapy sample. These data provide in vivo corroboration of recent in vitro experiments showing telomerase downregulation after radiation therapy or chemotherapy treatment of cell lines. Our finding of variability in levels of telomerase expression in GBMs parallels the known heterogeneity of these tumors for histologic features and cell growth-related factors. Statistical analysis showed no relationship between TRAP score and either time to clinical progression or time to death.

Immunohistochemical localization of telomerase hTERT protein and analysis of clonality in multifocal vulvar intraepithelial neoplasia.

Vulvar intraepithelial neoplasias (VINs) are potentially premalignant lesions of the squamous mucosa. The immunohistochemical distribution of the catalytic protein subunit of telomerase (hTERT) and the patterns of X chromosome inactivation were investigated as markers of neoplasia in samples from a patient with multifocal and diffuse VIN. hTERT nuclear staining in VIN correlated with squamous maturation and the degree of nuclear atypia. Normal mucosa revealed faint nuclear staining of parabasal cells and lower intermediate layer squamous cells. Monoclonal composition was demonstrated in 0 of 3 samples of VIN1, 2 of 3 samples of VIN2, and 13 of 13 samples of VIN3. The patterns of X chromosome inactivation indicated intramucosal extension and multifocal origin of individual lesions. Five samples of histologically normal vulvar squamous epithelium revealed a random pattern of X chromosome inactivation, consistent with polyclonal composition. All 19 samples from 9 lesions contained human papillomavirus (HPV)-16 sequences. Neither mutations in the p53 tumor suppressor gene or K-ras oncogenes nor loss of heterozygosity at 7 chromosomal loci were detected in any of the 19 samples of VIN. These results demonstrate that HPV-associated VIN may result from multifocal and diffuse 2-dimensional intraepithelial expansion of an immortalized monoclonal cell population.

Comparison of telomerase activity and GSTP1 promoter methylation in ejaculate as potential screening tests for prostate cancer.

New diagnostic tools are needed for the early detection of prostatic cancer. The molecular detection of prostate cancer cells in ejaculates was evaluated using complementary PCR-based methods. LNCaP cells, a cell line derived from prostatic carcinoma, were spiked into normal seminal ejaculates and the prostatic epithelial component of the specimens was isolated by immunomagnetic bead sorting, using a monoclonal antibody to prostate-specific membrane antigen (PSMA). Ejaculates from nine patients with a recent diagnosis of prostate cancer were processed in a similar fashion, using LNCaP-spiked aliquots as an internal positive control. Telomerase expression was evaluated by the telomeric repeat amplification protocol (TRAP) and glutathione S-transferase gene promoter (GSTP1) hypermethylation was evaluated by methylation-sensitive restriction endonuclease digestion and PCR amplification. Telomerase activity was detected in LNCaP cells recovered from normal seminal ejaculates but was not found in all nine samples from patients with prostate cancer. The sensitivity of GSTP1 analysis was similar to telomerase analysis for the detection of LNCaP cells from normal ejaculate samples but was positive in ejaculates from four out of nine patients with prostate cancer. GSTP1 DNA methylation status is more sensitive than telomerase analysis for the detection of malignant cells in seminal ejaculates from patients with prostate cancer.

Telomerase is a highly sensitive and specific molecular marker in fine-needle aspirates of breast lesions.

PURPOSE: Telomerase has been detected in a majority of human malignant tumors, making telomerase activity (TA) one key difference between mortal and immortal cells. In this study, we evaluated in blind-trial fashion the association of TA with cytologic and final clinical/pathologic diagnosis in fine-needle aspirates (FNAs) of breast lesions. MATERIALS AND METHODS: In 172 FNAs, including 80 samples that were cytologically malignant, 18 that were atypical but not diagnostic for malignancy, and 74 that were cytologically benign, TA was determined by a modified nonradioactive telomeric repeat amplification protocol (TRAP) assay. Final diagnosis was made by pathologic examination of follow-up surgical material available for all the cytologically malignant samples, a majority of the cytologically atypical samples, and a portion of the cytologically benign samples. RESULTS: TA was detected in 85 of 172 samples. Comparison of the cytologic and histologic diagnoses with TA showed that 80 of 87 samples from patients with breast cancer were telomerase-positive, resulting in a sensitivity of 92%. TA was found in four of five FNAs from carcinomas that were considered cytologically atypical but not diagnostic for malignancy. Eighty of 85 samples from patients with benign breast lesions were telomerase-negative, revealing a specificity of 94%. The five positive cases in this group were all fibroadenomas with low TA. Among the 18 cases with a cytologic diagnosis of atypia, there was a strong positive relationship between TRAP findings and histologic diagnosis. CONCLUSION: The detection of TA in FNAs of breast lesions is a highly sensitive and specific marker of malignancy and may be used as an adjunct in cases with an equivocal cytologic diagnosis.

Monoclonal endothelial cells in appetite suppressant-associated pulmonary hypertension.

Anorexigens such as aminorex fumarate and dexfenfluramine are associated with the development of severe pulmonary hypertension (PH), which clinically and histopathologically is considered indistinguishable from idiopathic or primary pulmonary hypertension (PPH). For the current study, we asked whether anorexigen-associated PH is characterized by monoclonal pulmonary endothelial cell proliferation (such as in PPH) or, alternatively, is associated with a polyclonal endothelial cell proliferation as found in secondary PH. Analysis of clonality by the human androgen receptor assay was performed in microdissected endothelial cells of plexiform lesions of two patients with anorexigen-associated PH. The four plexiform lesions of Patient 1 and the six of Patient 2 with anorexigen-associated PH exhibited a monoclonal expansion of pulmonary endothelial cells, with a mean clonality ratio of 0.03 +/- 0.01 SE. Our results indicate that appetite suppressant-associated PH is identical to PPH not only in clinical and histopathologic features but also, at a molecular level, in terms of the monoclonal nature of the endothelial cell proliferation. The anorexigens may accelerate the growth of pulmonary endothelial cells in patients with predisposition to develop PPH.

Carcinosarcoma of the breast: molecular-biological study for analysis of histogenesis.

The histogenesis of carcinosarcoma of the breast is controversial. In the current case, the demarcation between the carcinomatous and sarcomatous components was distinct in all microscopic fields. Immunohistochemical analysis was negative for epithelial membrane antigen (EMA) and keratin in the sarcomatous component and was negative for desmin in the carcinomatous component, suggesting that this tumor could be derived from the two different stem cells. To determine the histogenesis of this tumor, both carcinomatous and sarcomatous lesions were microdissected from formalin-fixed tissues and DNAs were prepared by proteinase K digestion. PCR amplification of the human androgen receptor (HUMARA) short tandem repeat (STR), after Hpa II digestion of the genomic DNA, indicated that the patterns of X-chromosome inactivation were identical in both components. Moreover, both components contained the identical TGT --> TTT transversion in codon 275 of the p53 gene. These observations strongly support the hypothesis that this tumor is derived from a single totipotent stem cell.

Amelogenin dosage compensation in carcinoma of colon, lung, liver and kidney, is not a marker of clonality in males.

The analysis of patterns of X-chromosome inactivation is becoming increasingly utilized as a marker of clonal composition of tissues from women. To date, however, no analogous system has been found for the study of clonality in tissue from men. In the current study, the methylation patterns for portions of the amelogenin genes are tested, which are encoded on both the X- and Y-chromosome (AMGX and AMGY). The polymerase chain reaction (PCR) was used to amplify portions of AMGX and AMGY from genomic DNA of carcinomas of the colon, lung, liver and kidney, as well as from matched normal somatic tissues. The amplification target included Alu I methylation sensitive restriction endonuclease sites as well as a 189 bp sequence which is present in AMGX but is absent in AMGY. Polymerase chain reaction amplification of AMGX and AMGY was successful using genomic DNA from both tumour and normal control tissue in 24 of the 26 cases. Pretreatment of genomic DNA with Alu I blocked amplification of AMGX in all cases from both normal tissue and tumour. This indicates that AMGX and AMGY undergo a non-random pattern of methylation in both normal tissues and in tumours, precluding their use as a marker of clonality. Methylation of Alu I sites in AMGY suggests that the amelogenin genes undergo dosage compensation, which raises the possibility that the expression of amelogenin is not restricted to the development of the tooth bud but may also play some other role in various tissues of the body.

Telomerase expression shows differences across multiple regions of oligodendroglioma versus high grade astrocytomas but shows correlation with Mib-1 labelling.

AIMS/BACKGROUND: Telomerase is an enzyme that is expressed in most human neoplasms and is associated with tumour immortality. Determination of the point in neoplastic transformation at which telomerase is expressed may aid the understanding of tumour pathogenesis and progression. Despite numerous reports on telomerase, few studies have investigated its expression in high grade glial tumours. These studies, performed on archival banked, single brain tumour specimens, have shown conflicting results for oligodendrogliomas and unexpectedly negative results for telomerase expression in high grade astrocytomas, with one third to one half of glioblastoma multiformes being negative. METHODS: 34 rapidly banked glioma specimens taken from patients undergoing gross total surgical resection of their tumours were studied. Telomerase expression was assessed across 3-8 sampled regions from each tumour by the telomeric repeat amplification protocol (TRAP) assay. Matched mirror image tissue samples were taken for histological analysis of tissue adequacy, statistical correlation of telomerase with tumour histological features, Mib-1 (a marker for cell cycling) labelling, and p53 immunohistochemistry. RESULTS: All five well differentiated oligodendrogliomas were homogeneously telomerase negative and two of three untreated anaplastic oligodendrogliomas were homogeneously positive. In contrast, 10 of 14 high grade astrocytomas showed heterogeneity for telomerase expression across the multiple regions sampled. All glioblastoma multiformes and two of three anaplastic astrocytomas showed at least one region positive for telomerase. When test samples were individually assessed in both oligodendrogliomas and high grade astrocytomas, telomerase expression was associated with Mib-1 labelling (p < 0.001). For the entire group, telomerase expression was associated with grade of tumour, age of patient, and vascular endothelial proliferation (all p < 0.001). CONCLUSIONS: This regional study clarifies that all glioblastoma multiformes are at least focally positive and that telomerase expression correlates with tumour grade in oligodendrogliomas. Homogeneity versus heterogeneity for telomerase expression across multiple regions of oligodendrogliomas versus high grade astrocytomas may provide important preclinical data on the use of antitelomerase agents in these adult glial tumours.