A Combined Biomarker of Bright CD38 and MYC ≥55% Is Highly Predictive of Double-/Triple-Hit High-Grade B-Cell Lymphoma.

OBJECTIVES: Diagnosis of high-grade B-cell lymphoma with MYC and BCL2 or BCL6 rearrangements (double-/triple-hit lymphoma [DTHL]) appears to mandate fluorescence in situ hybridization (FISH) testing for all large B-cell lymphoma (LBCL). Given the low incidence of DTHL, we aimed to identify flow cytometry (FC) and immunohistochemistry (IHC) features of DTHL that could be used to develop an optimal screening strategy. This combined FC-IHC approach has not yet been studied. METHODS: We compared features of 40 cases of DTHL and 39 cases of diffuse LBCL (DLBCL) without MYC rearrangement. RESULTS: Bright CD38 expression (CD38bright) by FC, high MYC expression (≥55%), and double-expressor phenotype by IHC were significantly associated with DTHL. The biomarker combining FC and IHC, CD38bright and/or MYC ≥55%, was superior to FC and IHC markers alone in predicting DTHL. Restricting FISH testing to approximately 25% of LBCL based on CD38brightand/or MYC ≥55% would detect approximately 95% of DTHL-BCL2 and approximately 75% of DHL-BCL6. CONCLUSIONS: Our study demonstrated that the novel biomarker of CD38bright and/or MYC ≥55% is highly predictive of DTHL. Awareness of the advantages and limitations of this screening strategy would facilitate development of a rational diagnostic workflow to provide high-quality patient care.

Human Recombinant DNase I (Pulmozyme®) Inhibits Lung Metastases in Murine Metastatic B16 Melanoma Model That Correlates with Restoration of the DNase Activity and the Decrease SINE/LINE and c-Myc Fragments in Blood Cell-Free DNA.

Tumor-associated cell-free DNAs (cfDNA) play an important role in the promotion of metastases. Previous studies proved the high antimetastatic potential of bovine pancreatic DNase I and identified short interspersed nuclear elements (SINEs) and long interspersed nuclear elements (LINEs)and fragments of oncogenes in cfDNA as the main molecular targets of enzyme in the bloodstream. Here, recombinant human DNase I (commercial name Pulmozyme®), which is used for the treatment of cystic fibrosis in humans, was repurposed for the inhibition of lung metastases in the B16 melanoma model in mice. We found that Pulmozyme® strongly reduced migration and induced apoptosis of B16 cells in vitro and effectively inhibited metastases in lungs and liver in vivo. Pulmozyme® was shown to be two times more effective when administered intranasally (i.n.) than bovine DNase I, but intramuscular (i.m.) administration forced it to exhibit as high an antimetastatic activity as bovine DNase I. Both DNases administered to mice either i.m. or i.n. enhanced the DNase activity of blood serum to the level of healthy animals, significantly decreased cfDNA concentrations, efficiently degraded SINE and LINE repeats and c-Myc fragments in the bloodstream and induced apoptosis and disintegration of neutrophil extracellular traps in metastatic foci; as a result, this manifested as the inhibition of metastases spread. Thus, Pulmozyme®, which is already an approved drug, can be recommended for use in the treatment of lung metastases.

LncRNA ROR facilitates myocardial fibrosis in rats with viral myocarditis through regulating C-Myc expression.

OBJECTIVE: The aim of this study was to explore the effect of long non-coding ribonucleic acid (lncRNA) ROR on myocardial fibrosis in rats with viral myocarditis by regulating c-myc expression. MATERIALS AND METHODS: A total of 120 healthy male rats were randomly divided into three groups, including: blank control group (group N, healthy rats, n=40), model control group (group M, viral myocarditis, n=40), and group P (lncRNA ROR + viral myocarditis, n=40). At 5, 10, and 15 d after modeling, the rats were sacrificed. Meanwhile, the left ventricular mass index (LVMI), the left ventricular weight/tibial length (LVW/TL) and the heart mass index (HMI) were measured, respectively. The myocardial tissues of rats were observed via staining. The protein and mRNA expressions of the transforming growth factor-ß (TGF-ß) and c-myc in myocardial tissues were detected via Western blotting and quantitative Real Time-Polymerase Chain Reaction (qRT-PCR), respectively. Moreover, the level of the serum interleukin-6 (IL-6) was detected via enzyme-linked immunosorbent assay (ELISA). RESULTS: Compared with group N, HMI, LVMI, and LVW/TL in group M significantly increased at each time point (p<0.01). Meanwhile, they were remarkably elevated at each time point in group P when compared with those in group M (p<0.01). According to the results of hematoxylin-eosin staining (HE) staining, the myocardial fibers were normal and orderly arranged in group N. Meanwhile, no inflammatory cells were observed in group N. In group M, the proliferation of a large number of inflammatory cells was observed, and the myocardial cells were disorderly arranged and became enlarged. In group P, the myocardial fibrosis was significantly severer than that of the other two groups (p<0.05). The results of the Masson staining revealed that there was significantly less accumulation of collagen fibers in myocardium in group N. There was a little accumulation of collagen fibers in the myocardium and fibrosis declined in group M. The myocardial fibrosis in group P remarkably increased when compared with group M (p<0.05). According to the results of the Western blotting, the protein expressions of c-myc and TGF-ß were significantly up-regulated in group P, compared with those in group M and N (p<0.05). However, they were remarkably higher in group M than in group N (p<0.05). QRT-PCR showed that the relative messenger ribonucleic acid (mRNA) expressions of c-myc and TGF-ß were remarkably up-regulated in group P when compared with those in group M and N, showing statistically significant differences (p<0.05). Moreover, they were significantly higher in group M than those in group N (p<0.05). Besides, the expression level of serum IL-6 remained highest in group P (p<0.01). CONCLUSIONS: LncRNA ROR up-regulates the expression of c-myc and increases the level of serum IL-6, thereby facilitating the proliferation and differentiation of cardiac fibroblasts.

Plasma C-MYC level manifesting as an indicator in progression of breast cancer.

Aim: To investigate whether plasma C-MYC level could be an indicator in clinical progression of breast cancer. Materials & methods: Plasma level of C-MYC expression was detected by quantitative real time PCR and the level of c-myc protein in breast cancer tissues was detected by immunohistochemistry. The expression level of C-MYC mRNA in supernatant of cancer cells culture was measured compared with the nonbreast cancer cells. Results: Plasma C-MYC level was significantly higher in patients with breast cancer than that in the controls, which associated with clinical stages, lymph node status, etc. Receiver operating characteristic curve analysis showed the sensitivity and specificity of plasma C-MYC level for diagnosis of breast cancer were 63.6 and 81.8%, respectively. The expression of c-myc protein in breast cancer tissues was associated with plasma C-MYC level, even C-MYC level in supernatant of cancer cells was elevated. Conclusion: Plasma C-MYC level might be a potential indicator in progression of breast cancer.

Correlation of MACC1/c-Myc Expression in Endometrial Carcinoma with Clinical/Pathological Features or Prognosis.

BACKGROUND Endometrial carcinoma (EC) is a type of female reproductive malignant tumor, the incidence of which is generally 20~30%. Multiple factors and genes are involved in the regulation of EC occurrence and progression. This study aimed to measure the expressions of MACC1 and c-Myc in EC patients to analyze their correlation with pathological features of EC. MATERIAL AND METHODS A total of 60 EC patients were recruited in the experimental group, while another cohort of 30 people with endometrial inflammatory hyperplasia was enrolled in the control group. The levels of serum MACC1 and c-Myc were measured by ELISA, and the protein expressions in EC cancer tissues, tumor-adjacent tissues, and controlled endometrial tissues were detected by immunohistochemistry (IHC). The correlation between gene expression and clinical/pathological features was then determined. RESULTS Our data indicate that the level of serum MACC1 and c-Myc in the experimental group was 1.67±0.08 ng/ml and 1.78±0.07 ng/ml, respectively, both of which were significantly higher than that of the control group (p<0.05). However, no significant difference was found among levels of serum MACC1 or c-Myc at different TNM stages (p>0.05). In cancer tissues, the positive rate of MACC1 or c-Myc was 73.3% and 78.3%, respectively, which were significantly higher than that in adjacent or control tissues (p<0.05). MACC1/c-Myc expression was correlated with TNM stage, primary infiltration grade, lymph node metastasis, and distal metastasis (p<0.05). CONCLUSIONS MACC1 and c-Myc are highly expressed in serum and tumor tissues of EC patients. Both are correlated with TNM stage, primary infiltration, and lymph node or distal metastasis, which provides a scientific basis for the development of new biomarkers for the diagnosis of endometrial carcinoma.

Whole exome sequencing in Dandy-Walker variant with intellectual disability reveals an activating CIP2A mutation as novel genetic cause.

Dandy-Walker malformation (DWM) has been reported to have heterogeneous causes, including mutations in genes of fibroblast growth factors and in genes in the sonic hedgehog (Shh) signaling pathway. Here, we identified an activating cancerous inhibitor of protein phosphatase 2A (CIP2A) p.D269V mutation, located at the predicted protein-protein interaction groove, as a novel genetic cause of Dandy-Walker variant (DWV). CIP2A has been reported as an oncoprotein promoting tumor survival via inhibition of protein phosphatase 2A (PP2A). However, the impact of human germline CIP2A mutation is unknown. We report a novel heterozygous CIP2A p.D269V mutation via whole exome sequencing in two siblings with DWV and severe intellectual disability who were born to non-consanguineous parents. Only the older brother developed a slow-growing sacral leiomyoma in his teens. The CIP2A p.D269V mutation is associated with increased PP2A, mTOR, and c-Myc protein levels in peripheral blood mononuclear cells (PBMCs). The PP2A phosphatase activity, however, was not suppressed. Deep sequencing revealed that the father carries 16% of somatic CIP2A p.D269V mutation, suggesting potential inheritance from the mosaic sperm populations. Our study is the first to describe a pathogenic CIP2A mutation in humans, which might disrupt neuronal development via enhancing mTOR and c-Myc protein expressions, shedding light in mechanisms of DWV pathogenesis.

Methylparaben induces malformations and alterations on apoptosis, oxidant-antioxidant status, ccnd1 and myca expressions in zebrafish embryos.

Methylparabens (MP) are widely used as preservatives in cosmetics, pharmacy, and food industry. Although acute toxicity studies in animals indicated that parabens are not significantly toxic, the effects of chronic exposure under sublethal doses are still unknown and the number of related studies is limited. Our aim was to evaluate the effects of MP on the development of zebrafish embryos focusing on development, locomotor activity, oxidant-antioxidant status, apoptosis, and ccnd1 and myca expressions. The expressions of ccnd1 and myca were determined by RT-PCR. Lipid peroxidation (LPO), nitric oxide (NO), and glutathione-S-transferase (GST) activities were determined spectrophotometrically. Apoptosis was determined using acridine orange staining. Locomotor activity was measured using touch-evoked movement test. MP exposure increased malformations, LPO, apoptosis, ccnd1 and myca expressions, and decreased GST activities and NO levels compared with the control group. Our findings will lead to further understanding of the mechanism of MP toxicity, and merit further research.

MYC Mediates Large Oncosome-Induced Fibroblast Reprogramming in Prostate Cancer.

Communication between cancer cells and the tumor microenvironment results in the modulation of complex signaling networks that facilitate tumor progression. Here, we describe a new mechanism of intercellular communication originating from large oncosomes (LO), which are cancer cell-derived, atypically large (1-10 µm) extracellular vesicles (EV). We demonstrate that, in the context of prostate cancer, LO harbor sustained AKT1 kinase activity, nominating them as active signaling platforms. Active AKT1 was detected in circulating EV from the plasma of metastatic prostate cancer patients and was LO specific. LO internalization induced reprogramming of human normal prostate fibroblasts as reflected by high levels of α-SMA, IL6, and MMP9. In turn, LO-reprogrammed normal prostate fibroblasts stimulated endothelial tube formation in vitro and promoted tumor growth in mice. Activation of stromal MYC was critical for this reprogramming and for the sustained cellular responses elicited by LO, both in vitro and in vivo in an AKT1-dependent manner. Inhibition of LO internalization prevented activation of MYC and impaired the tumor-supporting properties of fibroblasts. Overall, our data show that prostate cancer-derived LO powerfully promote establishment of a tumor-supportive environment by inducing a novel reprogramming of the stroma. This mechanism offers potential alternative options for patient treatment. Cancer Res; 77(9); 2306-17. ©2017 AACR.

Identification of CCR2 and CD180 as Robust Pharmacodynamic Tumor and Blood Biomarkers for Clinical Use with BRD4/BET Inhibitors.

Purpose: AZD5153 is a novel BRD4/BET inhibitor with a distinctive bivalent bromodomain binding mode. To support its clinical development, we identified pharmacodynamic (PD) biomarkers for use in clinical trials to establish target engagement.Experimental Design: CCR2 and CD180 mRNAs, initially identified from whole transcriptome profiling, were further evaluated by quantitative PCR in hematologic cell lines, xenografts, and whole blood from rat, healthy volunteers, and patients with cancer. MYC and HEXIM1 mRNAs were also evaluated.Results: RNA-sequencing data showed consistent decreases in CCR2/CD180 expression across multiple hematologic cell lines upon AZD5153 treatment. Evaluation of dose dependence in MV4,11 cells confirmed activity at clinically relevant concentrations. In vivo downregulation of CCR2/CD180 mRNAs (>80%) was demonstrated in MV4,11 and KMS-11 xenograft tumors at efficacious AZD5153 doses. Consistent with in vitro rat blood data, an in vivo rat study confirmed greater inhibition of CCR2/CD180 mRNA in whole blood versus MYC at an efficacious dose. Finally, in vitro treatment of whole blood from healthy volunteers and patients with cancer demonstrated, in contrast to MYC, almost complete downregulation of CCR2/CD180 at predicted clinically achievable concentrations.Conclusions: Our data strongly support the use of CCR2 and CD180 mRNAs as whole blood PD biomarkers for BRD4 inhibitors, especially in situations where paired tumor biopsies are unavailable. In addition, they can be used as tumor-based PD biomarkers for hematologic tumors. MYC mRNA is useful as a hematologic tumor-based biomarker but suboptimal as a whole blood biomarker. Utility of HEXIM1 mRNA may be limited to higher concentrations. Clin Cancer Res; 23(4); 1025-35. ©2017 AACR.

Protein signatures as potential surrogate biomarkers for stratification and prediction of treatment response in chronic myeloid leukemia patients.

There is unmet need for prediction of treatment response for chronic myeloid leukemia (CML) patients. The present study aims to identify disease-specific/disease-associated protein biomarkers detectable in bone marrow and peripheral blood for objective prediction of individual's best treatment options and prognostic monitoring of CML patients. Bone marrow plasma (BMP) and peripheral blood plasma (PBP) samples from newly-diagnosed chronic-phase CML patients were subjected to expression-proteomics using quantitative two-dimensional gel electrophoresis (2-DE) and label-free liquid chromatography tandem mass spectrometry (LC-MS/MS). Analysis of 2-DE protein fingerprints preceding therapy commencement accurately predicts 13 individuals that achieved major molecular response (MMR) at 6 months from 12 subjects without MMR (No-MMR). Results were independently validated using LC-MS/MS analysis of BMP and PBP from patients that have more than 24 months followed-up. One hundred and sixty-four and 138 proteins with significant differential expression profiles were identified from PBP and BMP, respectively and only 54 proteins overlap between the two datasets. The protein panels also discriminates accurately patients that stay on imatinib treatment from patients ultimately needing alternative treatment. Among the identified proteins are TYRO3, a member of TAM family of receptor tyrosine kinases (RTKs), the S100A8, and MYC and all of which have been implicated in CML. Our findings indicate analyses of a panel of protein signatures is capable of objective prediction of molecular response and therapy choice for CML patients at diagnosis as 'personalized-medicine-model'.

Primary refractory diffuse large B cell lymphoma in the rituximab era.

PURPOSE OF REVIEW: The aim of this review is to describe the outcome of primary refractory diffuse large B cell lymphoma in the rituximab era and the different therapeutic options as well as new biological markers that could allow the pathologist to distinguish these cases at diagnosis. RECENT FINDINGS: Diffuse large B cell lymphoma outcome has been impressively improved since the introduction of rituximab in association with anthracycline-based chemotherapy; however, primary refractory patients still represent an unmet medical need. SUMMARY: If patients without relapse after 2 years from diagnosis have an outcome comparable to healthy individuals, primary refractory patients still represent 20% of the cases with a very poor overall survival. These cases are usually described as progressive patients during first line or patients reaching a nonadequate partial response or those relapsing within a year after reaching a response.

Rho kinase inhibition drives megakaryocyte polyploidization and proplatelet formation through MYC and NFE2 downregulation.

The processes of megakaryocyte polyploidization and demarcation membrane system (DMS) formation are crucial for platelet production, but the mechanisms controlling these processes are not fully determined. Inhibition of Rho kinase (ROCK) signalling leads to increased polyploidization in umbilical cord blood-derived megakaryocytes. To extend these findings we determined the effect of ROCK inhibition on development of the DMS and on proplatelet formation. The underlying mechanisms were explored by analysing the effect of ROCK inhibition on the expression of MYC and NFE2, which encode two transcription factors critical for megakaryocyte development. ROCK inhibition promoted DMS formation, and increased proplatelet formation and platelet release. Rho kinase inhibition also downregulated MYC and NFE2 expression in mature megakaryocytes, and this down-regulation correlated with increased proplatelet formation. Our findings suggest a model whereby ROCK inhibition drives polyploidization, DMS growth and proplatelet formation late in megakaryocyte maturation through downregulation of MYC and NFE2 expression.

[Expression of P53, NPM1, Kras, c-Myc, p14(ARF) genes in blood cells of cancer patients before and after radiation therapy].

The mRNA levels of P53gene, as well as NPM1, Kras, c-Myc, p14(ARF) genes, which, according to the published data, code for the proteins regulating the p53 activity, were studied using RT-PCR method in blood cells of patients with different localization of tumor process (prostate cancer, breast cancer and head and neck cancer) before and after application of radiation therapy. Changes in gene expression of cancer patients were compared with the control group of healthy donors. We have established that all patients had a decreased level of the Kras gene expression even before radiotherapy; moreover, the group of patients with prostate cancer had a low content of mRNA in NPM1 and p14(ARF), and the group of patients with head and neck cancerhad a reliably reduced mRNA in P53, NPM1 and p14(ARF). The radiation therapy did not cause essential changes in the expression of these genes of cancer patients, ecpect for the Kras gene, whose the mRNA level in the group of patients with head and neck cancer was reliably lower than the mRNA level prior to beginning of radiation therapy. The correlations of P53, NPM1, Kras, p14(ARF) gene expression were studied. We have shown that p14(ARF) mRNA level negatively correlates with Kras mRNA (R = -0.6, p = 0.002) and P53 mRNA levels (R = -0.49, p = 0.013) in the control group of healthy donors. A positive correlation was observed between P53 mRNA and NPM1 mRNA (R = 0.54, p = 0.006). Similar correlations between mRNA levels of these genes in blood cells were absent in the cancer patients before radiotherapy. After radiotherapy in patients with prostate cancer, p14(ARF) mRNA level positively correlated with NPM1 mRNA (R = 0.7, p = 0.001) and negatively with Kras mRNA (R = - 0.5, p = 0.03). Our results provide evidence that expression P53, NPM1, Kras and p14(ARF) genes may be coordinated in blood cells of healthy donors. The low expression levels of the studied genes in patients can contribute to the increase in the mutation changes in blood cells of the examined subjects after the action of genotoxic factors.

Combined serum and tissue proteomic study applied to a c-Myc transgenic mouse model of hepatocellular carcinoma identified novel disease regulated proteins suitable for diagnosis and therapeutic intervention strategies.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer death in the U.S. Notably, most HCCs display c-Myc hyperactivity but this transcription factor participates in the regulation of as many as 15-20% of genes of the human genome. To better understand its oncogenic activity, a mass spectrometry-based proteomic approach was employed to search for disease-regulated proteins in liver tissue and serum of c-Myc transgenic mice that specifically developed HCC. Overall, a total of 90 differentially expressed proteins were identified with retinol binding protein 4, transthyretin, major urinary protein family, apolipoprotein E, and glutathione peroxidase being regulated in common in tissue and serum of HCC mice. Importantly, this study identified n = 22 novel tumor tissue-regulated proteins to function in cell cycle and proliferation, nucleotide and ribosomal biogenesis, oxidative stress, and GSH metabolism, while bioinformatics revealed the coding sequences of regulated proteins to enharbour c-Myc binding sites. Translation of the findings to human disease was achieved by Western immunoblotting of serum proteins and by immunohistochemistry of human HCC. Taken collectively, our study helps to define a c-Myc proteome suitable for diagnostic and possible therapeutic intervention strategies.

Evaluating markers for treatment selection based on survival time.

For many medical conditions, several treatment options may be available for treating patients. We consider evaluating markers based on a simple treatment selection policy that incorporates information on the patient's marker value. For example, colon cancer patients may be treated by surgery alone or surgery plus chemotherapy. The c-myc gene expression level may be used as a biomarker for treatment selection. Although traditional regression methods may assess the effect of the marker and treatment on outcomes, it is more appealing to quantify directly the potential impact on the population of using the marker to select treatment. A useful tool is the selection impact (SI) curve proposed by Song and Pepe for binary outcomes (Biometrics 2004; 60:874-883). However, the current SI method does not deal with continuous outcomes, nor does it allow to adjust for other covariates that are important for treatment selection. In this paper, we extend the SI curve for general outcomes, with a specific focus on survival time. We further propose the covariate-specific SI curve to incorporate covariate information in treatment selection. Nonparametric and semiparametric estimators are developed accordingly. We show that the proposed estimators are consistent and asymptotically normal. The performance is assessed by simulation studies and illustrated through an application to data from a cancer clinical trial.

[IL-6, p53 and proto-oncogene c-myc play different roles as biological markers of plasma cell dyscrasias]. / IL-6, p53 e il proto-oncogene c-myc esercitano ruoli diversi come marcatori biologici di discrasie plasmacellulari.

PURPOSE: To determine the role of serum levels of IL-6 and p53 mutant protein as well as of c-myc proto-oncogene alterations: a) in discriminating between benign (MGUS) and malignant Plasma cell dyscrasias (Multiple and Microsecreting Myeloma, Plasmocytoma); b) in monitoring the clinical course of malignant forms of this disease. PATIENTS AND METHODS: Eighty-eight patients affected by Plasma cell dyscrasias (58 MGUS, 24 MM and 6 PLC) entered this study. Using commercially available ELISA kits, serum levels of IL-6 and p53 have been determined in all the patients. In addition, a selected group of patients (n = 30) was also analyzed for structural c-myc gene alterations by Southern blot technique. RESULTS: The results show that, conversely from p53 protein, IL-6 and c-myc gene may represent useful diagnostic markers for discriminating benign from malignant forms of Plasma cell dyscrasia. On the contrary, preliminary findings of the same work indicate a potential role for the mutant p53 protein in monitoring the response to chemotherapy of patients affected by MM or PLM. CONCLUSIONS: Overall, these data suggest that the combined use of IL-6, p53 and c-myc may provide a new approach for a more rational management of Plasma cell dyscrasia patients.

Infrequent presence of anti-c-Myc antibodies and absence of c-Myc oncoprotein in sera from lung cancer patients.

To clarify the host immune response and explore a new serological marker of lung cancer, we examined serum c-Myc antigens and auto-antibodies against c-Myc in 68 lung cancer patients and 30 healthy volunteers using bacterially synthesized glutathione S-transferase c-Myc fusion proteins and immunoblotting. The detection rate of anti-c-Myc antibodies was 13.2% (9/68) in lung cancer patients and 3.3% (1/30) in healthy volunteers. These anti-c-Myc antibodies were directed toward exon 2 alone (4/68), exon 3 alone (1/68), and both exon 2 and exon 3 (4/68) of c-Myc. Circulating c-Myc antigen was not detected in any individuals with lung cancer and normal controls. Age, sex, performance status, histology, stage, smoking history, and prior treatment of the patients with and without anti-c-Myc antibodies were not significantly different. The low incidence of anti-c-Myc antibodies and c-Myc antigens in peripheral blood suggests that these examinations are not useful in the serological diagnosis of lung cancer.

Molecular markers of carcinogenesis.

The protein products of oncogenes and tumor suppressor genes play critical roles in the development of many cancers. The expression of a number of these proteins can be detected in extracellular fluids such as blood. This article reviews the literature on the application of methods for the detection of the proteins of oncogenes and tumor suppressor genes in the blood of humans with cancer or at risk for the development of cancer. The detection of these proteins in blood may be useful molecular markers of carcinogenesis that could play an important part in cancer diagnosis, prognosis, and prevention.

Differential expression of c-myc protein in B and T lymphocytes.

Overexpression of c-myc may play a role in the multistep pathogenesis of B- and T-cell malignancies. To determine whether this expression is inappropriate requires information on the normal cellular counterparts. There is no agreement in the literature on the levels of expression of c-myc mRNA and protein in normal peripheral blood lymphocytes and there are no reports on the differential expression in different lymphocyte populations. The aim of this study was to assess the state of c-myc expression in normal peripheral blood lymphocytes at the single cell level by immunocytochemistry and flow cytometry. Two monoclonal antibodies against c-myc and specific peptide inhibition controls were tested in mononuclear cells from nine healthy volunteers and the HL60 cell line. The expression of c-myc in B- and T-lymphocyte subsets was studied by two-colour immunocytochemistry and flow cytometry. Using calibrated reference standards, we quantified the c-myc protein and results were referred as molecules of equivalent soluble fluorochrome. Almost all lymphocytes express c-myc by both techniques. Two patterns of nuclear staining (weak and strong) were found by immunocytochemistry and this was confirmed by two peaks of fluorescence intensity by flow cytometry. Double immunostaining showed that the stronger pattern of c-myc staining corresponds to B lymphocytes and the weak one to T cells. Quantification confirmed these results which demonstrated a statistically significant difference in the expression of c-myc in these two lymphocyte populations (p < 0.005). Our results demonstrate for the first time that normal circulating B cells express higher levels of c-myc protein than T lymphocytes.