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Keywords:

  • microRNA;
  • diffuse large B-cell lymphoma;
  • lymphoma;
  • sera;
  • plasma

Summary

  1. Top of page
  2. Summary
  3. Methods
  4. Results and discussion
  5. Acknowledgements
  6. References
  7. Supporting Information

Circulating nucleic acids have been shown to have potential as non-invasive diagnostic markers in cancer. We therefore investigated whether microRNAs also have diagnostic utility by comparing levels of tumour-associated MIRN155 (miR-155), MIRN210 (miR-210) and MIRN21 (miR-21) in serum from diffuse large B-cell lymphoma (DLBCL) patients (n = 60) with healthy controls (n = 43). Levels were higher in patient than control sera (= 0·009, 0·02 and 0·04 respectively). Moreover, high MIRN21 expression was associated with relapse-free survival (= 0·05). This is the first description of circulating microRNAs and suggests that microRNAs have potential as non-invasive diagnostic markers for DLBCL and possibly other cancers.

MicroRNAs are a recently discovered class of naturally occurring short non-coding RNA molecules that negatively regulate eukaryotic gene expression through binding to complementary sequences in the 3′UTR of target mRNA. There are more than 400 known human microRNAs and more than 1000 predicted microRNA sequences await scientific confirmation (Griffiths-Jones et al, 2006). It is currently believed that between 10–30% of all human genes are targets for microRNA regulation. MicroRNAs play key regulatory roles in a diverse range of pathways, including haematopoiesis, and there is rapidly accumulating evidence to suggest that dysfunctional expression of microRNAs is a common feature in haematological malignancy (Lawrie, 2007).

The search for non-invasive tools for the diagnosis and management of cancer has long been a goal of cancer research that has led to great interest in the field of circulating nucleic acids in plasma and serum (Tsang & Lo, 2007). Many studies have shown that specific cancer characteristics, both genetic and epigenetic, are detectable in the plasma and serum of cancer patients and may be useful as a tool for early detection, diagnosis and follow-up of cancer patients (Tsang & Lo, 2007).

The current study investigated whether circulating microRNAs can be detected in serum and whether expression levels of specific microRNAs differ between cancer patients [diffuse large B-cell lymphoma (DLBCL)] and healthy individuals. As yet there is no detailed study of microRNA expression in DLBCL, therefore we investigated expression of MIRN155 (miR-155) and MIRN21 (miR-21), both found to be highly expressed in DLBCL (Lawrie et al, 2007) as well as MIRN210 (miR-210), which is associated with a more general malignant phenotype (Kulshreshtha et al, 2007).

Methods

  1. Top of page
  2. Summary
  3. Methods
  4. Results and discussion
  5. Acknowledgements
  6. References
  7. Supporting Information

We performed a retrospective study that analyzed serum samples from 60 patients diagnosed with de novo DLBCL, taken at the time of diagnosis (summarized in Table I). Further patient details, including treatment regimes and MIRN21 expression, can be found in Table SI. Forty-three serum samples from healthy individuals (27 males, 16 females, median age 57) were used as controls. Ethical permission and informed consent was obtained for the use of all samples.

Table I.   Summary of clinical details of DLBCL patients used for sera analysis (n = 60).
 n (%)
  1. *No survival data available for eight patients.

Sex 
 Men39 (65)
 Women21 (35)
Age range, years
 Mean (SD)63 (11)
Stage 
 120 (33)
 214 (23)
 318 (30)
 48 (13)
Extranodal involvement
 Yes33 (55)
 No27 (45)
International Prognostic Index score
 06 (10)
 111 (18)
 225 (42)
 312 (20)
 46 (10)
Follow-up, months (n = 52)*
 Mean [relapse-free (n = 38)] (SD)27 (16)

RNA was extracted from 2 ml of serum using Trizol LS reagent (Invitrogen, Paisley, UK) as described by the manufacturer. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was carried out using Taqman microRNA probes as described by the manufacturer (Applied Biosystems, Warrington, UK), using 10 ng of total RNA per reaction in a Roche LightCycler 480 machine (Roche, Sussex, UK). Triplicate samples were used throughout. As there is no current consensus on the use of house-keeping microRNAs for qRT-PCR analysis, and because we could not detect U6 or 5s in sera samples, based on previously published results (Mattie et al, 2006; Zhang et al, 2006) and as recommended by the manufacturer (Applied Biosystems), we used MIRN16 (miR-16) levels for normalization. In order to test variability of MIRN16 expression with other microRNAs that have previously been used for qRT-PCR normalization (Bandres et al, 2006; Lawrie et al, 2007), we compared sample MIRN16 Ct values with those of MIRNLET7A (let-7a) and MIRN24 (miR-24) and found a very good level of concordance (< 0·0001; r2 = 0·85 and 0·87 respectively; data not shown). There was no significant difference in MIRN16 Ct values, or any other microRNAs measured between control and lymphoma samples, suggesting there was no general over-representation of microRNA in lymphoma samples (data not shown). The ΔCt method was used for analysis [ΔCt =mean Ct (MIRN16) − mean Ct (microRNA of interest)].

MicroRNA expression levels were compared using Mann–Whitney independent t-test. Kaplan–Meier survival analysis was carried out on relapse-free survival times of DLBCL patients for whom follow-up details were available (n = 52) as a function of microRNA expression level, using the median value as a high/low cut-off. Relapse-free and overall survival times were calculated from the time of diagnosis to the date of relapse, death or last contact. Curves were compared by univariate (logrank) analysis. Statistical analyses were performed using GraphPad Prism version 4.00 (GraphPad, San Diego, CA, USA). P-values ≤ 0·05 were considered significant.

Results and discussion

  1. Top of page
  2. Summary
  3. Methods
  4. Results and discussion
  5. Acknowledgements
  6. References
  7. Supporting Information

The expression levels of three tumour-associated microRNAs (MIRN155, MIRN210 and MIRN21) were compared in serum samples from DLBCL patients (n = 60) with those of healthy controls (n = 43). We found levels of these microRNAs to be upregulated in patient sera (= 0·009, 0·02 and 0·04 respectively) (Fig 1A–C), corresponding to an average fold-change of 5·24, 4·15 and 2·56 respectively [fold change = 2(mean control ΔCt − mean patient ΔCt)].

image

Figure 1.  Sera levels of (A) MIRN155, (B) MIRN210 and (C) MIRN21 are higher in DLBCL patient sera (n = 60) than healthy controls (n = 43) and (D) sera levels of MIRN21 are associated with relapse-free survival in DLBCL patients. Values shown (ΔCt) are relative to levels of MIRN16. P-values were calculated by Mann–Whitney independent t-test. Kaplan–Meier survival curves were compared by univariate (logrank) analysis and MIRN21 expression levels in patients defined as high or low relative to the median.

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Moreover, high expression levels of MIRN21 in DLBCL patient sera were found to be associated with improved relapse-free survival times (= 0·05) (Fig 1D) but not overall survival (= 0·07; data not shown). These data are consistent with our previous findings in biopsy material from a different cohort of DLBCL patients, where high tumoural MIRN21 expression was also associated with a better prognostic outcome (Lawrie et al, 2007). We found no significant correlation between expression of either MIRN155 or MIRN210 and prognosis in this series of cases (= 0·22 and 0·86 respectively). We also investigated associations between microRNA levels and clinicopathological features of the DLBCL patients (i.e. sex, International Prognostic Index, stage and presence of extranodal disease) but found no significant correlations (data not shown).

Why high levels of MIRN21 should be associated with a more favourable clinical outcome for DLBCL patients remains to be determined. Although MIRN21 has been found to have an anti-proliferative effect in some cancers (Si et al, 2006), the opposite appears to be true for other cancers (Cheng et al, 2005). Presumably it is the targeting of oncogenes and/or anti-apoptotic molecules by MIRN21 that is important in determining clinical outcome in DLBCL patients. Amongst the 72 putative MIRN21 targets listed by two or more predictive algorithms (Table SII) there are several interesting candidate genes including PLAG1, E2F3, JAG1, SKI and STAT3 that should be explored further.

This is the first study, to our knowledge, that demonstrates the presence of microRNAs in biological fluids. Whilst it would be tempting to hypothesize that the detected extra-cellular microRNAs are derived from tumour cells, it is equally possible that these microRNAs arose from cells infiltrating the lymphomas or local organ or node involvement and may be part of an indirect response to cancer. For example MIRN155, in addition to being upregulated in certain lymphomas but not others (Kluiver et al, 2005), is necessary for normal immune function (Rodriguez et al, 2007). This is an area that clearly needs further research.

For this study we analysed serum samples but we have also successfully amplified microRNAs from plasma samples (data not shown). It would also be interesting to investigate microRNA expression in other body fluids, such as urine. Circulating microRNAs may have clinical utility for diagnosis, monitoring and follow-up of patients and this should be explored by conducting further studies with larger sample numbers and by recruiting patients prospectively within clinical trials so that serial measurements of the microRNA expression could be achieved before, during and after therapy.

In summary, we have shown that circulating microRNAs are clearly detectable in serum samples and that higher levels of specific microRNAs are associated with diagnosis and prognostic outcome in DLBCL patients. This data suggests that microRNAs have potential as clinically useful non-invasive biomarkers in cancer.

Acknowledgements

  1. Top of page
  2. Summary
  3. Methods
  4. Results and discussion
  5. Acknowledgements
  6. References
  7. Supporting Information

This study was supported by grants from the Leukaemia Research Fund (CHL, APL, KP, AHB, JB and JSW), Julian Starmer-Smith Memorial Fund (CHL), Cancer Research UK (FP, ALH.) and NTRAC (SG). We would especially like to thank everybody at the National Blood Service, Oxford for their help in collecting donor samples.

References

  1. Top of page
  2. Summary
  3. Methods
  4. Results and discussion
  5. Acknowledgements
  6. References
  7. Supporting Information
  • Bandres, E., Cubedo, E., Agirre, X., Malumbres, R., Zarate, R., Ramirez, N., Abajo, A., Navarro, A., Moreno, I., Monzo, M. & Garcia-Foncillas, J. (2006) Identification by real-time PCR of 13 mature microRNAs differentially expressed in colorectal cancer and non-tumoral tissues. Molecular Cancer, 5, 2939.
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  • Griffiths-Jones, S., Grocock, R.J., Van Dongen, S., Bateman, A. & Enright, A.J. (2006) miRBase: microRNA sequences, targets and gene nomenclature. Nucleic Acids Research, 34, D140D144.
  • Kluiver, J., Poppema, S., De Jong, D., Blokzijl, T., Harms, G., Jacobs, S., Kroesen, B.J. & Van Den Berg, A. (2005) BIC and miR-155 are highly expressed in Hodgkin, primary mediastinal and diffuse large B cell lymphomas. Journal of Pathology, 207, 243249.
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  • Lawrie, C.H. (2007) microRNAs and haematology: small molecules, big function. British Journal of Haematology, 136, 503512.
  • Lawrie, C.H., Soneji, S., Marafioti, T., Cooper, C.D.O., Palazzo, S., Paterson, J.C., Cattan, H., Enver, T., Mager, R., Boultwood, J., Wainscoat, J.S. & Hatton, C.S.R. (2007) MicroRNA expression distinguishes between germinal center B cell-like (GCB) and activated B cell-like (ABC) subtypes of diffuse large B cell lymphoma. International Journal of Cancer, 121, 11561161.
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  • Si, M.L., Zhu, S., Wu, H., Lu, Z., Wu, F. & Mo, Y.Y. (2006) miR-21-mediated tumor growth. Oncogene, 26, 27992803.
  • Tsang, J.C. & Lo, Y.M. (2007) Circulating nucleic acids in plasma/serum. Pathology, 39, 197207.
  • Zhang, L., Huang, J., Yang, N., Greshock, J., Megraw, M.S., Giannakakis, A., Liang, S., Naylor, T.L., Barchetti, A., Ward, M.R., Yao, G., Medina, A., O’Brien-Jenkins, A., Katsaros, D., Hatzigeorgiou, A., Gimotty, P.A., Weber, B.L. & Coukos, G. (2006) MicroRNAs exhibit high frequency genomic alterations in human cancer. Proceedings of the National Academy of Sciences of the United States of America, 103, 91369141.

Supporting Information

  1. Top of page
  2. Summary
  3. Methods
  4. Results and discussion
  5. Acknowledgements
  6. References
  7. Supporting Information

Table SI. Clinical details of DLBCL patients. *IPI group calculated according to N Engl J Med (1993) 329: 987–994. NA, not applicable because patient had no available follow-up data.

Table SII. Target genes predicted by two or more algorithms for miR-21 indicating cancer associations.

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Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.