**4.3. Long non-coding RNA molecules**

The success of this study led to other studies, especially in MM. However, different approaches lead to varying results. The main differences were type of samples (serum or plasma of PB),

Plasma of PB was reported to have lower levels of miR-92a in newly diagnosed MM patients in comparison to HD. Moreover, the level of miR-92a fluctuated based on progression of disease and treatment response, which would suggest a possible role of this miRNA as a predictive biomarker [68]. Another study showed increased expression of miR-148a, miR-181a, miR-20a, miR-221 and miR-88b in plasma of PB of MM patients in comparison with HD. Expression level of miR-20a and miR-148a was connected to shorter time to relapse of MM; this study suggested that circulating plasma miR-20a could be a marker of worse prognosis of MM [69]. On contrary, another study showed mostly decreased miRNA expression in MM patients compared to HD. MiR-483-5p and miR-20a were shown to have diagnostic and prognostic potential [70]. So far, most studies were performed using serum miRNA. Our own pilot study showed significantly increased levels of miR-29a, miR-660 and miR-142-5p in MM patients in comparison with HD. We showed that circulating serum miR-29a could be a biomarker for MM patients [71]. In our follow-up study, we showed dysregulation of five serum miRNA, miR-744, miR-130a, let-7d, let-7e and miR-34a in MGUS and MM patients in comparison with HD. Multivariate analysis showed that combination of miR-34a and let-7e distinguishes the patient cohorts with good sensitivity and specificity. Moreover, dynamics of serum miRNA with disease progression was shown [72].

In another study, increased expression of miR-181a/b, miR-221, miR-222 and miR-382 was found in relapsed MM patients and MM cell lines [51]. On contrary, lower expression of miR-15a and miR-16 was described; these miRNA have been described in chronic lymphocytic leukemia and seem to be part of pathogenesis of this disease. The genes for these miRNA are coded in the 13q14 locus; this locus is often deleted also in MM [73]. MiR-15a and miR-16 support apoptosis and decrease proliferation of MM cells by AKT and MAP kinase signaling [51]. In a study by Rocci et al., higher levels of miR-25, miR-16 and miR-30a in MM patients correlated with longer overall survival (OS) [74]. Another study showed that miR-19a and miR-4254 distinguish MM and HD. In addition, decreased level of serum miR-19a was positively correlated with international staging system (ISS) stage, presence of del(13q14) and gain 1q21 and shorter progression-free survival (PFS) and OS. Surprisingly, these patients responded to

Serum miRNA were also analyzed at CR after autologous stem cell transplantation (ASCT). MiR-16, miR-17, miR-19b, miR-20a and miR-660 were decreased in diagnostic samples in comparison with CR samples [76]. Patients with lower levels of miR-19b and miR-331 had shorter PFS after ASCT. Level of miR-19b was significantly lower in samples obtained at relapsed than at CR.

The most common clinical manifestation of MM is osteolytic lesions. Increased levels of serum miR-214 and miR-135b were found in MM patients with osteolytic lesions, and their expression correlated with severity of the symptoms [77]. Moreover, increased level of miR-

Using NGS, miRNA (let-7b a miR-18a) from exosomes isolated from serum of MM patients significantly correlated with PFS and OS in univariate analysis and with ISS and cytogenetic abnormalities in multivariate analysis [78]. Moreover, it was shown that levels of exosomal

bortezomib better [75].

74 Liquid Biopsy

214 associated with shorter PFS and OS.

design of experiments (patients vs. HD) and used methods and platforms.

LncRNA are an abundant class of RNA between 200 nt and 100 kb long [80, 81]. To date, approximately 16,000 lncRNA have been identified in the human genome (http://www.gencodegenes.org/). On the other hand, the functional characterization of most of them has not been determined yet.

Genes encoding for lncRNA are present in many types of organisms, including animals [82], plants [83], yeast [84], prokaryotic organisms [85] and viruses [86]. LncRNA do not possess protein-coding capacity due to the absence of open reading frames (ORFs) or because of insufficient length of ORFs [87–90]. They can be classified according to their genomic localization into three major groups: long intergenic non-coding RNA (lincRNA), long intronic RNA and long non-coding RNA transcribed from specific genomic regions.

The expression of lncRNA genes is developmental and tissue-specific, and they have been associated with a large spectrum of biological processes, for example, alternative splicing, modulation of protein activity, alternation of protein localization, epigenetic regulation and generally regulation of gene expression. These molecules can be precursors of small RNA and even tools for miRNA silencing [91–96]. LncRNA play an important role both in physiological and pathological processes. The deregulated expression levels of these molecules were identified in a large variety of tumor diseases: breast cancer [97], small-cell lung carcinoma [98] and also in MM [22]. It was shown that alterations in lncRNA can influence regulation of cancer progression [99]. Interestingly, lncRNA seem to have higher tissue specificity even in comparison with proteincoding mRNA and miRNA. Thus, they are even more interesting as new specific biomarkers [88].

Function of lncRNA can also be derived from their localization within the cell. These molecules can be found in the nucleus and in the cytoplasm. LncRNA are preferably located in the cell nucleus, deriving their significant effect on the DNA sequence [88].

The classification of lncRNA can be based on their influence on the DNA sequence. From this perspective, there are two classes of lncRNA: cis-lncRNA (cis-acting lncRNA) and translncRNA (trans-acting lncRNA). Cis-IncRNA can positively or negatively regulate expression of genes that are located in their genomic proximity [95]. On the other hand, trans-lncRNA regulate expression of distant genes [100]. Many lncRNA are transcribed by RNA polymerase II, just like protein-coding genes. If lncRNA are involved in the regulation of RNA polymerase II, they are transcribed by RNA polymerase III [87, 101–103]. High degree of evolutionary conservation, tissue-specific expression and stability of lncRNA point to significant functional role of these molecules [104].

and urine, but they can also be found within exosomes where they are protected against

Liquid Biopsies in Multiple Myeloma http://dx.doi.org/10.5772/intechopen.78630 77

Most studies of circulating lncRNA published so far were studies of solid tumors. In prostate cancer, PCA3 specificity was so high that a new test from urine has been approved for usage in Europe; it can be used together with currently used PSA test (prostate-specific antigen)

In urinary bladder cancer, increased level of UCA1 was detected not only in the tumor tissue but also in PB and urine of patients [79, 122]. It was shown that based on UCA1 expression, urinary bladder cancer may be distinguished from other urinary bladder diseases with high

Unfortunately, only very few studies were published about circulating lncRNA in MM. In a study of Isin et al. [123], five candidate lncRNA (TUG1, MALAT1, HOTAIR, GAS5, lincRNA-p21) were analyzed in plasma of PB of MM patients in comparison with CLL patients [103]. Plasma of PB of CLL patients contained significantly deregulated levels of lincRNA-p21. On the other hand, MM plasma contained deregulated levels of the other four lncRNA. When compared to HD, MM patients contained only TUG1 deregulated levels. There was a correlation of circulating lncRNA and clinical subgroups of MM, suggesting that TU1 could be a part of MM progression. Another study reported significantly higher levels of PCAT-1 in MM patients in comparison with HD. Its potential as a biomarker was proven by ROC analysis that showed sensitivity of 71.7% and specificity of 93.8%. A possible correlation with MM pathogenesis was suggested by a correlation with

While lncRNA molecules are generally described as being more tissue-specific than miRNA, not enough data have been published on circulating lncRNA in MM. Further studies that are

While not many studies have been published dealing with liquid biopsies of circulating molecules in multiple myeloma, they show a great promise. Liquid biopsies could be used as an adjunct to standard BM biopsy for disease monitoring to enable obtaining more complex results and easier follow-up of patients. While there are many candidate molecules that have been described in this review (cfDNA, miRNA and lncRNA), more studies are needed to

more comprehensive are needed to verify their claim as the more specific marker.

RNases [117].

[118–121].

specificity [28].

β2 microglobulin [123].

**5. Conclusion**

validate these findings.

**Acknowledgements**

This work was supported by grant AZV 15-29508A.

Dysregulation of lncRNA expression was observed in various human diseases, including cancer. LncRNA may be either oncogenes or tumor suppressors in development as well as progression of tumors [105, 106]. Changes of expression levels of several lncRNA have been reported in several malignancies; other lncRNA seem to be specific for a single tumor, suggesting that these molecules may be good biomarkers for tumor diagnostics as well as prognosis and prediction [107].

Moreover, it was shown that lncRNA are involved in regulation of hematopoiesis, including proliferation, differentiation and apoptosis of hematopoietic stem cells as well as progenitors and precursors of mature blood cells [108, 109]. Dysregulated expression of lncRNA was reported in lymphomas, leukemias and MM. It seems possible that expression profile of these lncRNA could have a potential clinical significance in diagnostics and prognostics of hematologic malignancies.

Current information about the role of lncRNA in pathogenesis of MM is very limited. So far, MALAT1 has been described as a marker of early progression [110]. Expression level of this lncRNA was increased in BM cells of newly diagnosed MM patients and changed during progression of the disease. Patients with lower levels of MALAT1 had a higher risk of early progression.

Handa et al. showed higher expression level of MALAT1 in MM patients in comparison to MGUS and HD [111]. These results are in correlation with another study of Ronchetti et al. who showed dysregulation of 31 lncRNA, including MALAT1, in MM patients [112]. Moreover, this lncRNA may be important in MM pathogenesis through activation of TGF-β, a factor important for osteolytic lesion formation [113].

An earlier study showed decreased expression of MEG3 in MM patients [114]. Decreased expression or loss of this lncRNA seems to be important in various types of human tumors [115]. In a study by Benetatos et al., MEG3 was reported to be lost in more than half of MM patients, and it seemed to have a prognostic significance for MM [116].

Our own study showed that UCA1 might be a marker of MM when HD, MGUS and MM plasma cells were compared (with sensitivity of 85.0% and specificity of 94.7%). UCA1 levels seemed to correlate with albumin and monoclonal immunoglobulin serum levels, cytogenetic aberrations, and survival of MM patients.

### *4.3.1. Circulating lncRNA*

Similar to circulating miRNA, even lncRNA may be detected in body fluids suggesting their possible role as biomarkers for diagnosis, prognosis and prediction. They were found in PB and urine, but they can also be found within exosomes where they are protected against RNases [117].

Most studies of circulating lncRNA published so far were studies of solid tumors. In prostate cancer, PCA3 specificity was so high that a new test from urine has been approved for usage in Europe; it can be used together with currently used PSA test (prostate-specific antigen) [118–121].

In urinary bladder cancer, increased level of UCA1 was detected not only in the tumor tissue but also in PB and urine of patients [79, 122]. It was shown that based on UCA1 expression, urinary bladder cancer may be distinguished from other urinary bladder diseases with high specificity [28].

Unfortunately, only very few studies were published about circulating lncRNA in MM. In a study of Isin et al. [123], five candidate lncRNA (TUG1, MALAT1, HOTAIR, GAS5, lincRNA-p21) were analyzed in plasma of PB of MM patients in comparison with CLL patients [103]. Plasma of PB of CLL patients contained significantly deregulated levels of lincRNA-p21. On the other hand, MM plasma contained deregulated levels of the other four lncRNA. When compared to HD, MM patients contained only TUG1 deregulated levels. There was a correlation of circulating lncRNA and clinical subgroups of MM, suggesting that TU1 could be a part of MM progression. Another study reported significantly higher levels of PCAT-1 in MM patients in comparison with HD. Its potential as a biomarker was proven by ROC analysis that showed sensitivity of 71.7% and specificity of 93.8%. A possible correlation with MM pathogenesis was suggested by a correlation with β2 microglobulin [123].

While lncRNA molecules are generally described as being more tissue-specific than miRNA, not enough data have been published on circulating lncRNA in MM. Further studies that are more comprehensive are needed to verify their claim as the more specific marker.
