**2. Circulating plasma cells**

MM is the second most common hematological malignancy [4]. It is caused by malignant transformation of PC, which infiltrate the bone marrow (BM) disrupting normal hematopoiesis. Moreover, they produce M-Ig that is found in serum and/or urine of MM patients. MM is characterized by a set of clinical features known as CRAB features (hypercalcemia, renal

MM represents about 13% of all hematological and about 1% of all malignancies. The incidence in the Czech Republic has been reported at 4.8/100000 per year [7], while in Europe it is slightly higher, 6/100000 per year [8]. Interestingly, MM is quite common in North America,

In 2003, the International Myeloma Working Group (IMWG) published diagnostic criteria for MM: infiltration of BM by malignant PC >10%, CRAB features and presence of M-Ig in serum and/or urine [2]. Thus, MM was treated only when CRAB feature(s) were fully developed. At that time, most treatment options were quite toxic, which is why treatment was postponed [10]. The past 10 years, however, have brought unprecedented new treatment options (immunomodulatory drugs, proteasome inhibitors, monoclonal antibodies) that improved survival rates as well as quality of life of MM patients. This led to revision of diagnostic criteria in 2014 from diagnostics based on clinical symptoms to diagnostics based on biomarkers allowing

Since the new drugs have been introduced into clinics, detection of minimal residual disease (MRD) has become even more important as MRD negativity is a prognostic factor in MM. Moreover, sensitivity of the detection method is becoming an issue since new drugs induce deep response, and MRD needs to be measured with sensitivity up to 10−6. The two most common methods used for MRD detection are multiparametric flow cytometry and ASO-PCR (allele-specific PCR) [11–13]. ASO-PCR is based on the detection of patient-specific V(D)J rearrangement in bone marrow plasma cells (BMPC) [14]. Nowadays, next-generation sequencing (NGS) is gaining more attention as a more precise and modern method of detection. However,

NGS needs to be standardized and more accessible before it can be used more broadly.

The genome of MM cells is highly unstable and harbors various cytogenetic abnormalities, including translocations, deletions or duplications. From the cytogenetic point of view, MM may be divided into two groups: hyperdiploid and non-hyperdiploid. Hyperdiploid genome is mostly characterized by trisomies of odd chromosomes (3,5,7,9,11,15,19,21) and is connected to better prognosis [5], while non-hyperdiploid genome is characterized by monosomies of chromosomes 8,13,14,16,17 and 22 and recurrent chromosomal translocations involving the immunoglobulin heavy chain (IgH) locus at 14q32. In MM, the most frequent chromosomal translocations are t(11;14)(q13;q32) (15–20% of MM patients) and t(4;14)(p16;q32) (12–15% of MM patients). Other translocations are less frequent, found only in less than 5% of patients (t(14;16)(q32; q23), t(14;20)(q32;q11) and t(6;14) (p21;q32)). In MM cells with t(11;14)(q13;q32), cyclin D1 gene is translocated under the control of immunoglobulin heavy chain enhancer. Similarly, cyclin D3 gene at 6p21 is overexpressed in MM cells carrying t(6;14)(p21;q32) [14]. In addition to MM being a genetically heterogeneous disease, it is also characterized by multifocal tumor deposits throughout the BM and focal lesions elsewhere. Malignant PC in these lesions carry various cytogenetic aberrations with varying level of prognostic value [15].

Europe and Australia, while it is rare in the Middle East and Asia [9].

failure, anemia and bone lesions) [5, 6].

68 Liquid Biopsy

earlier treatment of the disease [6].

In some cases, PC may migrate out of the BM into peripheral blood (PB), then they are called circulating PC (cPC) [23, 24]. While the reason for the migration is unclear, it is clear that these cPC lose (in some cases only temporarily) their dependence on the BM microenvironment due to the loss of adhesion molecules, increased proliferation, increased number of chromosomal aberrations and increased angiogenesis [25, 26]. The presence of cPC in newly diagnosed MM patients has been associated with shorter survival of patients, and it is an independent negative prognostic factor [24]. It is also possible that it is the first feature of extramedullary relapse, which is characterized by infiltration of PC into soft tissues and bad prognosis for patients [27]. In case that the number of cPC increases to over 20% in PB, it may turn into progression to secondary plasma cell leukemia [28].

cPC can be detected in a small fraction of newly diagnosed MM patients (15%) by conventional morphology [29]. However, this frequency increases up to 50–70% once more sensitive techniques, such as flow cytometry, are used [30]. Interestingly, the presence of cPC has been associated with an increased risk of malignant transformation to symptomatic MM in MGUS patients as well as with an inferior survival among symptomatic newly diagnosed and relapse/ refractory MM [31]. In a study by Paiva et al., cPC were analyzed by multiparametric flow cytometry, FISH and cell cycle analysis; cPC were compared to paired PC samples from the BM. Their results showed that cPC are a unique subpopulation of malignant PC in MM; cPC are characterized by decreased expression of integrins (CD11a/CD11c/CD29/CD49d/CD49e) and adhesion molecules (CD33/CD56/CD117/CD138). cPC were also mostly quiescent with higher clonogenic potential than BMPC [31].

Mishima et al. investigated genomic characterization of MM patients using cPC and wondered if mutational profile of cPC is in concordance with mutational profile of PC from the BM. This study showed that both populations have similar mutations. Interestingly, 100% of clonal mutations found in PC from BM were also detected in cPC. Moreover, 99% of clonal mutations of cPC were also found in BMPC. Whole genome sequencing did not find any major differences between these two groups of MM cells, suggesting that the change in biological behavior of cPC could be based on the changes of expression of ncRNA molecules [32].
