Exploratory open-label clinical study to determine the S-588410 cancer peptide vaccine-induced tumor-infiltrating lymphocytes and changes in the tumor microenvironment in esophageal cancer patients.

Cancer vaccines induce cancer-specific T-cells capable of eradicating cancer cells. The impact of cancer peptide vaccines (CPV) on the tumor microenvironment (TME) remains unclear. S-588410 is a CPV comprising five human leukocyte antigen (HLA)-A*24:02-restricted peptides derived from five cancer testis antigens, DEPDC1, MPHOSPH1, URLC10, CDCA1 and KOC1, which are overexpressed in esophageal cancer. This exploratory study investigated the immunologic mechanism of action of subcutaneous S-588410 emulsified with MONTANIDE ISA51VG adjuvant (median: 5 doses) by analyzing the expression of immune-related molecules, cytotoxic T-lymphocyte (CTL) response and T-lymphocytes bearing peptide-specific T-cell receptor (TCR) sequencing in tumor tissue or blood samples from 15 participants with HLA-A*24:02-positive esophageal cancer. Densities of CD8+, CD8+ Granzyme B+, CD8+ programmed death-1-positive (PD-1+) and programmed death-ligand 1-positive (PD-L1+) cells were higher in post- versus pre-vaccination tumor tissue. CTL response was induced in all patients for at least one of five peptides. The same sequences of peptide-specific TCRs were identified in post-vaccination T-lymphocytes derived from both tumor tissue and blood, suggesting that functional peptide-specific CTLs infiltrate tumor tissue after vaccination. Twelve (80%) participants had treatment-related adverse events (AEs). Injection site reaction was the most frequently reported AE (grade 1, n = 1; grade 2, n = 11). In conclusion, S-588410 induces a tumor immune response in esophageal cancer. Induction of CD8+ PD-1+ tumor-infiltrating lymphocytes and PD-L1 expression in the TME by vaccination suggests S-588410 in combination with anti-PD-(L)1 antibodies may offer a clinically useful therapy.Trial registration UMIN-CTR registration identifier: UMIN000023324.

Diagnosis of prostate cancer by detection of minichromosome maintenance 5 protein in urine sediments.

BACKGROUND: The accuracy of prostate-specific antigen (PSA) testing in prostate cancer detection is constrained by low sensitivity and specificity. Dysregulated expression of minichromosome maintenance (Mcm) 2-7 proteins is an early event in epithelial multistep carcinogenesis and thus MCM proteins represent powerful cancer diagnostic markers. In this study we investigate Mcm5 as a urinary biomarker for prostate cancer detection. METHODS: Urine was obtained from 88 men with prostate cancer and from two control groups negative for malignancy. A strictly normal cohort included 28 men with complete, normal investigations, no urinary calculi and serum PSA <2 ng ml(-1). An expanded control cohort comprised 331 men with a benign final diagnosis, regardless of PSA level. Urine was collected before and after prostate massage in the cancer patient cohort. An immunofluorometric assay was used to measure Mcm5 levels in urine sediments. RESULTS: The Mcm5 test detected prostate cancer with 82% sensitivity (confidence interval (CI)= 72-89%) and with a specificity ranging from 73 (CI=68-78%) to 93% (CI=76-99%). Prostate massage led to increased Mcm5 signals compared with pre-massage samples (median 3440 (interquartile range (IQR) 2280 to 5220) vs 2360 (IQR <1800 to 4360); P=0.009), and was associated with significantly increased diagnostic sensitivity (82 vs 60%; P=0.012). CONCLUSIONS: Urinary Mcm5 detection seems to be a simple, accurate and noninvasive method for identifying patients with prostate cancer. Large-scale prospective trials are now required to evaluate this test in diagnosis and screening.

Cell-cycle-phase progression analysis identifies unique phenotypes of major prognostic and predictive significance in breast cancer.

Multiparameter analysis of core regulatory proteins involved in G1-S and G2-M cell-cycle transitions provides a powerful biomarker readout for assessment of the cell-cycle state. We have applied this algorithm to breast cancer to investigate how the cell cycle impacts on disease progression. Protein expression profiles of key constituents of the DNA replication licensing pathway (Mcm2, geminin) and mitotic machinery (Plk1, Aurora A and the Aurora substrate histone H3S10ph) were generated for a cohort of 182 patients and linked to clinicopathological parameters. Arrested differentiation and genomic instability were associated with an increased engagement of cells into the cell division cycle (P<0.0001). Three unique cell-cycle phenotypes were identified: (1) well-differentiated tumours composed predominantly of Mcm2-negative cells, indicative of an out-of-cycle state (18% of cases); (2) high Mcm2-expressing tumours but with low geminin, Aurora A, Plk1 and H3S10ph levels (S-G2-M progression markers), indicative of a G1-delayed/arrested state (24% cases); and (3) high Mcm2-expressing tumours and also expressing high levels of the S-G2-M progression markers, indicative of accelerated cell-cycle progression (58% of cases). The active cell-cycle progression phenotype had a higher risk of relapse when compared with out-of-cycle and G1-delayed/arrested phenotypes (HR=3.90 (1.81-8.40, P<0.001)), and was associated with Her-2 and triple negative subtypes (P<0.001). It is of note that high-grade tumours with the G1-delayed/arrested phenotype showed an identical low risk of relapse compared with well-differentiated out-of-cycle tumours (HR=1.00 (0.22-4.46), P=0.99). Our biomarker algorithm provides novel insights into the cell-cycle state of dynamic tumour cell populations in vivo. This information is of major prognostic significance and may impact on individualised therapeutic decisions. Patients with an accelerated phenotype are more likely to derive benefit from S- and M-phase-directed chemotherapeutic agents.

Diagnosis of pancreaticobiliary malignancy by detection of minichromosome maintenance protein 5 in bile aspirates.

Biliary brush cytology is the standard method of sampling a biliary stricture but has a low sensitivity for the detection of malignancy. We have previously shown that minichromosome maintenance (MCM) replication proteins (Mcm2-7) are markers of dysplasia and have utilised these novel biomarkers of growth for the diagnosis of cervical and bladder cancer. We aimed to determine if MCM proteins are dysregulated in malignant pancreaticobiliary disease and if levels in bile are a sensitive marker of malignancy. In 30 tissue specimens from patients with malignant/benign biliary strictures, we studied Mcm2 and -5 expression by immunohistochemistry. Bile samples were also collected prospectively at endoscopic retrograde cholangiopancreatography from 102 consecutive patients with biliary strictures of established (n=42) or indeterminate aetiology (n=60). Patients with indeterminate strictures also underwent brush cytology as part of standard practice. Bile sediment Mcm5 levels were analysed using an automated immunofluorometric assay. In benign biliary strictures, Mcm2 and -5 protein expression was confined to the basal epithelial proliferative compartment - in contrast to malignant strictures where expression was seen in all tissue layers. The percentage of nuclei positive for Mcm2 was higher in malignant tissue (median 76.5%, range 42-92%) than in benign tissue (median 5%, range 0-33%) (P<0.0005), with similar results for Mcm5. Minichromosome maintenance protein 5 levels in bile were significantly more sensitive than brush cytology (66 vs 20%; P=0.004) for the detection of malignancy in patients with an indeterminate stricture, with a comparable positive predictive value (97 vs 100%; P=ns). In this study, we demonstrate that Mcm5 in bile detected by a simple automated test is a more sensitive indicator of pancreaticobiliary malignancy than routine brush cytology.

Microglial activation in white matter lesions and nonlesional white matter of ageing brains.

White matter lesions (WML), a common feature in brain ageing, are classified as periventricular (PVL) or deep subcortical (DSCL), depending on their anatomical location. Microglial activation is implicated in a number of neurodegenerative diseases, but the microglial response in WML is poorly characterized and its role in pathogenesis unknown. We have characterized the microglial response in WML and control white matter using immunohistochemistry to markers of microglial activation and of proliferation. WML of brains from an unbiased population-based autopsy cohort (Medical Research Council's Cognitive Function and Ageing Study) were identified by post mortem magnetic resonance imaging and sampled for histology. PVL contain significantly more activated microglia, expressing major histocompatibility complex (MHC) class II and the costimulatory molecules B7-2 and CD40, than either control white matter (WM) or DSCL. Furthermore, we show that significantly more microglia express the replication licensing protein minichromosome maintenance protein 2 within PVL, suggesting this is a more proliferation-permissive environment than DSCL. Although microglial activation occurs in both PVL and DSCL, our findings suggest a difference in pathogenesis between these lesion-types: the ramified, activated microglia associated with PVL may reflect immune activation resulting from disruption of the blood brain barrier, while the microglia within DSCL may reflect an innate, amoeboid phagocytic phenotype. We also show that microglia in control WM from lesional cases express significantly more MHC II than control WM from nonlesional ageing brain, suggesting that WML occur in a 'field-effect' of abnormal WM.

Mitogenic growth signalling, DNA replication licensing, and survival are linked in prostate cancer.

Activation of mitogen/extracellular-signal-regulated kinase kinase 5/extracellular signal-regulated kinase-5 (MEK5/ERK5) growth signalling is coupled to increased cell proliferation in prostate cancer (PCa). Dysregulation of the DNA replication licensing pathway, a critical step in growth control downstream of transduction signalling pathways, is associated with development of PCa. In this study we have investigated linkages between the MEK5/ERK5 pathway and DNA replication licensing during prostate carcinogenesis. The effects of increased MEK5/ERK5 signalling on the expression of replication licensing factors Mcm2 and geminin and the proliferation marker Ki67 were studied in an ecdysone-inducible system expressing a constitutively activated mutant of MEK5 in EcR293 cells and in stable ERK5 over-expressing PC3 clones. In parallel, expression of these biomarkers in PCa biopsy specimens (n=58) was studied and compared to clinicopathological parameters. In both in vitro systems induction of MEK5 expression resulted in increased levels of phosphorylated ERK5 and Mcm2, geminin and Ki67 proteins. In PCa specimens average Mcm2 expression was greater than Ki67 and geminin expression (median labelling index (LI) 36.7, 18.1, and 3.4% respectively), consistent with their differential expression according to growth status (P<0.0001). Mcm2, geminin and Ki67 expression were significantly associated with Gleason grade (P=0.0002, P=0.0003, P=0.004); however there was no link with T or M stage. There was a significant relationship between increasing ERK5 expression and increasing Mcm2 (P=0.003) and Ki67 (P=0.009) expression, with non-significant trends seen with increasing MEK5 expression. There were significant associations between Gleason grade and the number of cells traversing G1 phase (Ki67(LI)-geminin(LI); (P=0.001)), with high ERK5 levels associated with both an increase in replication licensed but non-cycling cells (Mcm2(LI)-Ki67(LI); (P=0.01)) and accelerated cell cycle progression (geminin(LI)/Ki67(LI); (P= 0.005)), all indicative of a shift towards increasing proliferative potential. While Mcm2 and Ki67 were both prognostic factors on univariate analysis, only Mcm2 remained an independent prognostic marker on multivariate analysis. Taken together, our data show that induction of MEK5/ERK5 signalling is linked to activation of the DNA replication licensing pathway in PCa, and that the strong prognostic value of MCM proteins may result from their function as relay stations coupling growth regulatory pathways to genome duplication.

Subtypes of oligodendroglioma defined by 1p,19q deletions, differ in the proportion of apoptotic cells but not in replication-licensed non-proliferating cells.

Oligodendrogliomas may be divided into those with deletion of chromosomes 1p and 19q (Del+), and those without (Del-). Del+ tumours show better survival and chemoresponsiveness but the reason for this difference is unknown. We have investigated whether these subgroups differ in (a) apoptotic index, (b) the proportion of cells licensed for DNA replication but not in-cycle, and (c) the relative length of G1-phase. Fluorescence in situ hybridisation with probes to 1p and 19q was used to determine the deletion status of 54 oligodendrogliomas, including WHO grades II and III. The apoptotic index was determined using counts of apoptotic bodies. Replication-licensed non-proliferating cells were determined from the Mcm2 minus Ki67 labelling index, whilst the geminin to Ki67 ratio was used as a measure of the relative length of G1. Del+ oligodendrogliomas showed a higher apoptotic index than Del- tumours (P=0.037); this was not accounted for by differences in tumour grade or in proliferation. There were no differences in the Mcm2-Ki67 index or in the geminin/Ki67 ratio between the subgroups, but grade III tumours showed a higher proportion of licensed non-proliferating cells than grade II tumours (P=0.001). An increased susceptibility to apoptosis in oligodendrogliomas with 1p+/-19q deletion may be important in their improved clinical outcome compared to Del- tumours.

Mcm2 labelling of balloon cells in focal cortical dysplasia.

Balloon cells (BC) are the prominent and defining cellular component of type IIB Focal Cortical Dysplasia (FCD), a common cause of focal epilepsy in patients undergoing surgical treatment. BC are considered immature cells of uncommitted cellular differentiation having immunophenotypical characteristics of both neurones and glia. They are often located in the lower cortical layers and white matter underlying the dysplastic cortex, suggesting migratory arrest during development. We investigated the proliferative potential of BC in 15 cases of FCD from patients with a wide range of ages using immunohistochemistry for Mcm2 (mini chromosome maintenance protein) and Ki67. In the majority of cases, BC showed Mcm2 nuclear positivity. In addition, cells with intermediate neuronal-glial characteristics were labelled whilst the dysmorphic or hypertrophic pyramidal neuronal components of FCD were not. Ki67 labelled only occasional BC. These findings support the view that BC cells represent a pool of less differentiated glial cells with proliferative capacity which may have potential for delayed neuronal differentiation. Furthermore, as Mcm2 specifically identifies BC populations, this marker may be of diagnostic value in the subtyping of FCD lesions in patients with epilepsy.

DNA replication licensing and cell cycle kinetics of normal and neoplastic breast.

Mcm2-7 (MCM) proteins are part of the origin licensing machinery that regulates initiation of DNA replication. Geminin is a licensing repressor and prevents reinitiation of DNA replication during S-G2-M phase by blocking reloading of Mcm2-7 at replication origins. Here, we have analysed these replication licensing factors (RLFs) to determine whether the pathway becomes deregulated during mammary carcinogenesis, and have assessed their potential value as prognostic markers. Protein expression profiles were generated for Ki67, Mcm2, geminin, HER-2, ER and PR in a series of reduction mammoplasty (n=18) and breast cancer specimens (n=120), and compared to clinicopathological parameters. A large proportion of epithelial cells of the terminal duct lobular unit reside in a primed 'replication licensed' but not proliferating state. This state is characterised by Mcm2 expression and absence of Ki67 and the S/G2/M marker geminin. In breast cancers, increasing tumour grade is associated with increased Ki67, Mcm2 and geminin expression. The Mcm2/Ki67 ratio decreases through the grades, indicating a shift from a predominantly licensed state to an actively proliferating state. This shift is associated with an increase in the geminin/Ki67 ratio, signifying a shortening of G1 phase in breast cancer cells. Ki67, Mcm2 and the Mcm2/Ki67 ratio are statistically significantly associated with the Nottingham Prognostic Index (NPI), but geminin and the geminin/Ki67 ratio are not. Ki67, Mcm2 and Mcm2/Ki67 are highly correlated with one another, with Mcm2 being the single most important predictor of NPI score (P<0.001). However, only 12% of variation in NPI is explained by Mcm2, as the labelling index for this marker is approaching 100% for many of the high-grade tumours. The origin licensing phenotypes of normal breast and breast cancers therefore relate to their cellular differentiation status, and high-level MCM expression in more poorly differentiated tumours severely constrains their use as prognostic markers in breast cancer.

Diagnosis of oesophageal cancer by detection of minichromosome maintenance 5 protein in gastric aspirates.

Symptomatic oesophageal cancer is usually advanced and the prognosis poor. Lethality of symptomatic oesophageal cancer has motivated screening for these diseases earlier in their evolution, but reliable methods for early diagnosis remain elusive. We have demonstrated that dysregulated expression of minichromosome maintenance (MCM) proteins 2-7 is characteristic of early epithelial carcinogenesis, and that these key DNA replication initiation factors can be used as diagnostic markers for cervical and genito-urinary tract cancer. In this study, we investigated whether minichromosome maintenance protein 5 (Mcm5) can be used to detect oesophageal cancer cells in gastric aspirates. Two monoclonal antibodies raised against His-tagged human Mcm5 were used in a time-resolved immunofluorometric assay to measure Mcm5 levels in cells isolated from gastric aspirates of 40 patients undergoing gastroscopy for suspected or known oesophageal carcinoma or symptoms of dyspepsia. The test discriminated with high specificity and sensitivity between patients with and without oesophageal cancer (85% sensitivity (95% confidence interval (CI)=62-97%), 85% specificity (CI=66-96%)), as demonstrated by the large area under the receiver operating characteristics curve (0.93 (95% CI=0.85-0.99)). Elevated levels of Mcm5 in gastric aspirates are highly predictive of oesophageal cancer. This simple test for oesophageal cancer is readily automated with potential applications in primary diagnosis, surveillance and screening.

DNA replication licensing and cell cycle kinetics of oligodendroglial tumours.

The convergence point of growth-signalling pathways that control cell proliferation is the initiation of genome replication, the core of which is the assembly of pre-replicative complexes (pre-RCs), resulting in chromatin being 'licensed' for DNA replication in the subsequent S phase. The Mcm2-7 complex is a core constituent of the pre-RC, whose recruitment to replication origins is dependent on the Cdt1 loading factor. Geminin is a potent inhibitor of the initiation of DNA replication by preventing Mcm2-7 assembly at origins via its interaction with Cdt1, ensuring genomic integrity through suppression of re-initiation events in S phase. Here we investigate the regulation of Ki67, Mcm2, p21, caspase 3 and Geminin in a series of 55 oligodendrogliomas to provide an integrated picture of how cellular proliferation and programmed cell death are dysregulated in these tumours. Geminin does not behave as an inhibitor of cell proliferation, its labelling index rising with increasing growth fraction as defined by Ki67 or Mcm2 expression. Geminin is expressed in a higher proportion of cells in higher grade tumours (P<0.001) and shows a strong correlation to proliferation and replication licensing (P<0.01), but not apoptosis. Increasing tumour anaplasia is not associated with loss of Geminin. Importantly, the G1 phase of the proliferative cell cycle, as assessed by the Geminin/Ki67 ratio, shortens with increasing anaplasia, providing new potential algorithms for prognostic assessment. Origin licensing proteins thus provide powerful novel tools for assessment of tumour cell cycle kinetics in routinely processed surgical biopsy material.

Aberrant expression of minichromosome maintenance proteins 2 and 5, and Ki-67 in dysplastic squamous oesophageal epithelium and Barrett's mucosa.

BACKGROUND: Minichromosome maintenance (Mcm) proteins are essential for eukaryotic DNA replication, and their expression implies potential for cell proliferation. Expression is dysregulated in dysplastic states but data for oesophageal squamous mucosa and Barrett's mucosa have not been published. AIM: To test the hypothesis that Mcm proteins are downregulated together with the proliferation marker Ki-67 in differentiating epithelial compartments of non-dysplastic squamous and Barrett's epithelium, and that this process does not occur in dysplastic mucosae. METHODS AND CASES: Forty five patients with Barrett's oesophagus included 20 with glandular dysplasia (10 low grade, eight high grade, two both, and four with invasive adenocarcinoma). Twenty five other patients included 12 with oesophageal squamous dysplasia (three low grade, six high grade, three both, and four with invasive squamous carcinoma). Formalin fixed paraffin embedded tissue sections from biopsy series and resections were immunostained using antibodies to Mcm2, Mcm5, and Ki-67. Percentage of nuclei positive for Mcm2, Mcm5, and Ki-67 was estimated and scored from 0 to 6 as: 0, none +; 1, <10%+; 2, 10-30%+; 3, 30-70%+; 4, 70-90%+; 5, >90%+; 6, all+. Four separate epithelial strata were scored: in squamous epithelium the basal layer and thirds to the surface, in Barrett's mucosa the luminal surface, upper and lower crypt, and deep glands. RESULTS: In non-dysplastic squamous epithelium and Barrett's mucosa, high level expression of Mcm2, Mcm5, and Ki-67 proteins was largely confined to the proliferative compartments and downregulated in differentiated compartments. Expression persisted up to the mucosal surface in dysplastic squamous epithelium and Barrett's mucosa. CONCLUSIONS: Persistent expression of Mcm2, Mcm5, and Ki-67 proteins in luminal compartments of dysplastic oesophageal squamous epithelium and dysplastic Barrett's mucosa may be diagnostic markers and imply disruption of cell cycle control and differentiation in these dysplastic epithelia.

Replicative Mcm2 protein as a novel proliferation marker in oligodendrogliomas and its relationship to Ki67 labelling index, histological grade and prognosis.

The grading and prognostic assessment of oligodendrogliomas is severely constrained and there remains a need for improved diagnosis. Recently, we have identified the minichromosome maintenance (MCM) family of proteins as a novel class of proliferation markers. Mcm2 is a protein which forms part of the prereplicative complex. It is necessary for this complex to be assembled at origins of future DNA replication during the G1 phase to allow genome replication in the subsequent S phase. Our aim was to determine whether analysis of Mcm2 protein expression in oligodendrogliomas is of diagnostic value. Immunohistochemical staining for Mcm2 was performed on an archival series of 32 oligodendrogliomas. These tumours have been previously characterized for Ki67, mitotic labelling index and outcome. Cells showing expression of Mcm2 were quantified as a percentage to provide an Mcm2 labelling index. We have demonstrated a good correlation between Mcm2 and Ki67 labelling indices (r = 0.76, P < 0.01) but immunohistochemistry for Mcm2 consistently identified a higher proportion of cells. Mcm2 labelling index was higher in grade III than grade II tumours (P < 0.001). Cases with a high Mcm2 labelling index showed a poorer prognosis than those with a low index (P = 0.497) in univariate analysis, but with wide variation in this small series. Demonstration of Mcm2 expression is of value to demonstrate the proliferative fraction of tumours and is likely to be of prognostic value. Its study in a larger series is therefore warranted.

Minichromosome maintenance protein 2 expression in prostate: characterization and association with outcome after therapy for cancer.

The minichromosome maintenance (MCM) proteins are highly conserved proteins essential for initiating and regulating eukaryotic DNA replication. Recent studies have demonstrated the potential use of MCM proteins as markers of proliferation. We characterized the pattern of Mcm 2 staining in benign and malignant prostate tissues and examined the role of Mcm 2 expression in disease-free survival after surgery in men with localized prostate cancer. Tumors from 92 patients who underwent radical prostatectomy for prostate cancer (median follow-up of 54 months) were examined for Mcm 2 expression by immunohistochemistry using a monoclonal antibody. Prostate tissue from five men without histopathological evidence of prostate cancer was also stained for Mcm 2. Mcm 2 expression was quantified by calculating a labeling index, and patients were grouped according to degree of staining. An analysis of the association between Mcm 2 expression with traditional clinicopathological characteristics of prostate cancer was carried out. A Cox proportional hazards analysis was performed to determine whether Mcm 2 staining was a significant independent predictor of disease-free survival. Mcm 2 expression is low (<2%) and limited to the basal cell layer in nonmalignant prostate glands. Mcm 2 expression is consistently increased in malignant glands and is significantly associated with disease-free survival in univariate (P = 0.002) and multivariate (P = 0.01) analyses. Patients with high Mcm 2 expression exhibited shorter disease-free survival. Mcm 2 expression was not associated with any traditional clinical or pathological factors and therefore is an independent predictor of survival in these patients with prostate cancer. These data support evidence that Mcm 2 may serve as a novel proliferation marker in the prostate. Mcm 2 expression is an independent predictor of disease-free survival after definitive local therapy and has potential as a molecular marker for clinical outcome in prostate cancer.

DNA replication licensing and human cell proliferation.

The convergence point of growth regulatory pathways that control cell proliferation is the initiation of genome replication, the core of which is the assembly of pre-replicative complexes resulting in chromatin being "licensed" for DNA replication in the subsequent S phase. We have analysed regulation of the pre-replicative complex proteins ORC, Cdc6, and MCM in cycling and non-proliferating quiescent, differentiated and replicative senescent human cells. Moreover, a human cell-free DNA replication system has been exploited to study the replicative capacity of nuclei and cytosolic extracts prepared from these cells. These studies demonstrate that downregulation of the Cdc6 and MCM constituents of the replication initiation pathway is a common downstream mechanism for loss of proliferative capacity in human cells. Furthermore, analysis of MCM protein expression in self-renewing, stable and permanent human tissues shows that the three classes of tissue have developed very different growth control strategies with respect to replication licensing. Notably, in breast tissue we found striking differences between the proportion of mammary acinar cells that express MCM proteins and those labelled with conventional proliferation markers, raising the intriguing possibility that progenitor cells of some tissues are held in a prolonged G1 phase or "in-cycle arrest". We conclude that biomarkers for replication-licensed cells detect, in addition to actively proliferating cells, cells with growth potential, a concept that has major implications for developmental and cancer biology.

Immunoassay for urothelial cancers that detects DNA replication protein Mcm5 in urine.

Cancer-screening tests for internal organs are severely constrained by low specificity or sensitivity, cost, and morbidity. We report a non-invasive immunofluorometric assay for detection of urothelial cancers based on ectopic expression of the DNA replication protein Mcm5.

Minichromosome maintenance proteins as biological markers of dysplasia and malignancy.

Dysplasia, an intermediate stage in the progression from normal tissue to neoplasia, is defined morphologically by a loss of normal orientation between epithelial cells, with changes in cellular and nuclear shape and size. However, little is known about the functional properties of dysplastic cells, including their replicative state, largely due to a lack of available biological markers. We have used novel antibodies against minichromosome maintenance (MCM) proteins to examine the proliferative status of a range of histological lesions and to characterize dysplastic cells in functional terms. Immunoperoxidase staining was used to localize the MCM proteins, components of the prereplicative complex that is essential for initiating eukaryotic DNA replication. These proteins are down-regulated in cells undergoing differentiation or quiescence and, thus, serve as specific markers for proliferating cells. In normal and some reactive tissues, MCM expression was present only in restricted proliferative compartments, consistent with our published findings in the uterine cervix. In dysplastic and malignant tissues, in contrast, MCM proteins were expressed in the majority of cells, extending to surface layers of dysplastic stratified epithelia. In carcinomas, the frequency of expression of MCM proteins showed an inverse correlation with the degree of tumor differentiation. Thus, we suggest that dysplastic cells may be characterized in functional terms as remaining in cell cycle, due to deregulation of normal controls over cell proliferation. Antibodies against MCM proteins have potential clinical applications, for example, in the assessment of tumor prognosis in histological sections and the identification of proliferating cells in clinical samples using biochemical or cytological assays.

Improved cervical smear assessment using antibodies against proteins that regulate DNA replication.

Carcinoma of the cervix is one of the most common malignancies. Papanicolaou (Pap) smear tests have reduced mortality by up to 70%. Nevertheless their interpretation is notoriously difficult with high false-negative rates and frequently fatal consequences. We have addressed this problem by using affinity-purified antibodies against human proteins that regulate DNA replication, namely Cdc6 and Mcm5. These antibodies were applied to sections and smears of normal and diseased uterine cervix by using immunoperoxidase or immunofluorescence to detect abnormal precursor malignant cells. Antibodies against Cdc6 and Mcm5 stain abnormal cells in cervical smears and sections with remarkably high specificity and sensitivity. Proliferation markers Ki-67 and proliferating cell nuclear antigen are much less effective. The majority of abnormal precursor malignant cells are stained in both low-grade and high-grade squamous intraepithelial lesions. Immunostaining of cervical smears can be combined with the conventional Pap stain so that all the morphological information from the conventional method is conserved. Thus antibodies against proteins that regulate DNA replication can reduce the high false-negative rate of the Pap smear test and may facilitate mass automated screening.

Cdc6 protein causes premature entry into S phase in a mammalian cell-free system.

We exploit an improved mammalian cell-free DNA replication system to analyse quiescence and Cdc6 function. Quiescent 3T3 nuclei cannot initiate replication in S phase cytosol from HeLa or 3T3 cells. Following release from quiescence, nuclei become competent to initiate semiconservative DNA replication in S phase cytosol, but not in G0 phase cytosol. Immunoblots show that quiescent cells lack Cdc6 and that minichromosome maintenance (MCM) proteins are not associated with chromatin. Competence of G1 phase nuclei to replicate in vitro coincides with maximum Cdc6 accumulation and MCM protein binding to chromatin in vivo. Addition of recombinant Cdc6 to permeabilized, but not intact, G1 nuclei causes up to 82% of the nuclei to initiate and accelerates G1 progression, making nuclei competent to replicate prematurely.