Diagnostic significance of dysregulated miRNAs in T-cell malignancies and their metabolic roles

T-cell malignancy is a broad term used for a diverse group of disease subtypes representing dysfunctional malignant T cells transformed at various stages of their clonal evolution. Despite having similar clinical manifestations, these disease groups have different disease progressions and diagnostic parameters. The effective diagnosis and prognosis of such a diverse disease group demands testing of molecular entities that capture footprints of the disease physiology in its entirety. MicroRNAs (miRNAs) are a group of noncoding RNA molecules that regulate the expression of genes and, while doing so, leave behind specific miRNA signatures corresponding to cellular expression status in an altered stage of a disease. Using miRNAs as a diagnostic tool is justified, as they can effectively distinguish expressional diversity between various tumors and within subtypes of T-cell malignancies. As global attention for cancer diagnosis shifts toward liquid biopsy, diagnosis using miRNAs is more relevant in blood cancers than in solid tumors. We also lay forward the diagnostic significance of miRNAs that are indicative of subtype, progression, severity, therapy response, and relapse. This review discusses the potential use and the role of miRNAs, miRNA signatures, or classifiers in the diagnosis of major groups of T-cell malignancies like T-cell acute lymphoblastic lymphoma (T-ALL), peripheral T-cell lymphoma (PTCL), extranodal NK/T-cell lymphoma (ENKTCL), and cutaneous T-cell lymphoma (CTCL). The review also briefly discusses major diagnostic miRNAs having prominent metabolic roles in these malignancies to highlight their importance among other dysregulated miRNAs.


Introduction
Cancer research has made remarkable progress in recent years, bringing many potent drugs to the fore for its treatment and management. However, the effectiveness of these cancer therapies largely depends on accurate and timely diagnosis. In most cases, delayed or inaccurate diagnosis leads to faulty clinical judgment, which incurs toxic responses in patients and renders therapeutic endeavors less effective (1). This disadvantage of diagnostic delays and inaccuracy can be overcome by investigating circulating fluids that may be home to disease-specific markers, especially in cases of lymphoma more than in solid tumors (2). Although the incidence of various T-cell lymphomas may be statistically rare, it is considered very aggressive and is often associated with poorer disease outcomes (3). Patients harboring such malignancies can also face treatment failures, as they develop resistance to established chemotherapeutic regimens faster than solid tumors (4). To diagnose such aggressive malignancies, conventional methods may not be sufficient to identify these diseases as early as possible. However, the growing prospects of liquid biopsy can be exploited to diagnose these lymphomas (5). One such biomarker is microRNAs (miRNAs), whose global expression can be investigated not only in patient tissues but also in their circulating fluids, providing scope for liquid biopsy (6). However, miRNA profiling, as a diagnostic option is still limited within the research domain; its applicability in routine clinical practice has not yet materialized as it requires high-throughput techniques. The present review describes the diagnostic and prognostic potential of miRNAs that are relevant for the efficient discriminatory diagnosis of a particular T-cell lymphoma from a wide spectrum of T-cell malignant lymphomas (TCLs). miRNAs were first discovered in Caenorhabditis elegans as lin-4, a noncoding RNA reported to downregulate the expression of another gene, lin-14 (7). miRNAs are short (usually 21-23 nucleotides), noncoding (nc), ssRNA molecules produced endogenously in animal and plant cells that function posttranscriptionally, consequently regulating gene expression. Most often, miRNAs repress gene expression by binding to the 3′untranslated regions (3′UTRs) of their respective mRNA targets, thus cleaving them to suppress translation (8). These miRNAs can co-express and co-regulate one or more cellular pathways and result in a phenotypic consequence of oncogenic or onco-suppressive functions. miRNAs and their repressive roles have been widely studied using both in vitro and in silico models, in addition to their gene-inducing capabilities (7)(8)(9).
Cancers have overwhelming involvement in multiple pathways, for which miRNAs certainly create a miRNA signature while regulating those pathways and may indicate the cell's physiological, metabolic, pathologic, and tumor-progression status (10). It is also noteworthy that T-cell malignant formations shed circulating miRNAs in circulating body fluids, which can be quantified to design promising diagnostic applications and monitor therapy response during treatment (10). In this review, we discuss different miRNAs with important diagnostic and prognostic applications in various types of T-cell malignancies. For this review, the diversity of T-cell malignancies is based on various pathological studies and the WHO classification (11). However, a clear classification is yet to be explored and is beyond the scope of this review (12). Hence, we discuss major T-cell malignancies and their dysregulated miRNAs, contributing to aspects of diagnostic or prognostic values in adult Tlymphoblastic leukemia/lymphoma (T-ALL), peripheral T-cell lymphoma (PTCL) and its types, extranodal T-cell lymphoma (ENKTCL), and cutaneous T-cell lymphoma (CTCL) (Figure 1). We also discuss how diagnostic miRNAs have a significant role in reprogramming cancer metabolism, as we underline emerging evidence suggesting a crucial role of miRNAs in regulating metabolic processes such as insulin sensitivity, glucose and lipid metabolism, and energy homeostasis. As metabolic miRNAs are being used for diagnostic and prognostic applications in metabolic disorders (13), they can have similar diagnostic and prognostic utilities in cancers as well, which undergo metabolic reprogramming as a key hallmark toward transformation. A recent computational biology study using machine learning has identified seven key miRNAs associated with mRNAs involved in lipid metabolism that are differentially expressed between prostate cancer and its benign counterpart. The differential expression of this lipid metabolism-related signature was also validated by PCR and in publicly available datasets (14). In addition to forming a molecular signature, miRNA expression profiles can identify distinct metabolic phenotypes within a disease spectrum. By identifying patients who may respond differently to a particular therapy or need specialized care, sub-phenotyping patients based on miRNA signatures can help precision medicine efforts (15,16). Therefore, miRNAs along with their downstream metabolite signatures, can also potentially distinguish high-risk patient groups from others (17). The diagnostic and prognostic potential of miRNA profiling can be harnessed in clinical settings if it can be narrowed down to a few miRNAs defining a particular cancer subtype. The diagnostic focus required to distinguish a spectrum of disease manifestations can also be achieved by quantifying key miRNAs and their metabolic targets. Moreover, finding out how dysregulated miRNAs affect functional outcomes can reveal their role in metabolic processes verified through in vitro and in vivo research, which can also prove the miRNAs' potential as therapeutic targets.

T-lymphoblastic leukemia/ lymphoma
According to the International Lymphoma study group and the WHO, T-lymphoplastic leukemia/lymphoma comprises two kinds of malignancies namely T-cell lymphoblastic lymphoma (T-LBL) and T-ALL (18,19). Both are malignancies of immature thymocytes that share large similarities in morphology and immunophenotypes (20,21). Both malignancies have considerable infiltration in the mediastinum, differing in their proportion of bone marrow infiltration. T-ALL has more (>25%) bone marrow infiltration, whereas T-LBL has considerably high infiltration in the lymph nodes along with low infiltration in the bone marrow (<25%). Malignant thymocytes in T-LBL and T-ALL also infiltrate peripheral blood, bone marrow, and cerebrospinal fluid (22). As far as genetic and epigenetic aberrations in both T-LBL and T-ALL are concerned, their molecular features largely overlap with some differential mutations in each. The early T-cell precursor LBL/ALL (ETP-LBL or ETP-ALL) is another aggressive subtype of immature T-cell malignancy whose mutational and transcriptional profiles closely resemble those of myeloid progenitors and hematopoietic stem cells (21,22). However, there exist other immunophenotypic subgroups, such as Near-ETP-ALL and Pro-T-ALL, whose distinct phenotypic and clinical presentations are not clear enough (23).

miRNAs having diagnostic significance in T-ALL
When T-ALL microRNA expression was compared with that of healthy T cells, higher expression of miR-128a and miR-181b was observed alongside significant underexpression of miR-100 and let-7e (50). A study noted a general downregulation of miR-708 among all T-cell malignant patient groups, whereas miR-196b was found to be upregulated significantly only in patients with T-ALL immunophenotypes (51). A separate study evaluated the increased expression of seven miRNAs (miR-92, miR-20a, miR-19a, miR-19b, miR-18a, miR-17-3p, and miR-17-5p) in the miRNA cluster (miR-17-92) and reported their involvement in chromosomal rearrangement activity, including translocation (27). Among these miRNAs, significant overexpression of miR-19b and miR-19a has been reported in patients with T-ALL. An miRNA panel that co-expresses miR-26a and miR-92, along with miR-19a and miR-19b, is strongly associated with T-ALL patients (52). Moreover, among miRNA species that are underexpressed, miR-150 has a significant role in increasing hematopoietic progenitor growth and cell proliferation in T-ALL (53). The underexpressing miRNAs, miR-30 and miR-451, are believed to be tumor suppressive in nature, inhibiting NOTCH1 and NOTCH2 expression. These aforementioned miRNAs play a significant role in regulating the signaling pathways involved in the progression of T-ALL ( Figure 2) (54).
In a study, several miRNAs were reported to have stage-and subtype-specific expression statuses. For instance, upregulated miR-196b in T-ALL corresponds with a more immature immunophenotype of malignant T cells, whereas upregulated miR-363 and miR-19a correspond with a more mature malignant T-cell immunophenotype (44,55). Another miRNA couple, miR-221 and miR-222, have high Major types of T-cell malignancies. Malignant transformation at various stages of T-cell development gives rise to a spectrum of malignancies defined by a variety of clinical manifestations. expression in T-ALL cases but more significantly in its ETP-ALL subtype, as high as eight-and fivefold, respectively (56). Moreover, an independent study used microRNA profiling and noted the underexpression of miR-124a, miR-146b-5p, miR-150, miR-193b-3p, miR-326, miR-451, miR-30, and miR-708 in T-ALL. Among them, lower expression of miR-146b-5p was correlated with the TAL1+ T-ALL patient group (35). Lower expression of a different miRNA, miR-193b-3p, was associated with the TAL-rearranged T-ALL subtype and the NOTCH-induced T-ALL subgroup. MicroRNAs can also differentially associate with adult and pediatric T-ALL disease biology; for instance, in children with T-ALL, miR-221 was found to overexpress significantly in their peripheral blood mononuclear cells (PBMNCs). Interestingly, miR-221 is also associated with the refractory stage of T-ALL, signifying its prognostic role in both adult and pediatric cases. Similarly, a different miRNA (miR-664) was found to be overexpressed in pediatric T-ALL, underlying a possible association with miR-221 ( Figure 2). Additionally, miR-664 has been reported to confer malignant features such as cell growth promotion, cell migration, and apoptotic inhibition of cells in T-ALL (51) ( Table 1).

miRNAs and prognosis of T-ALL disease
Dysregulated miRNAs can also indicate survival outcomes in patients; for instance, a miRNA signature of overexpressed miR-221 and miR-222 in T-ALL, more so in ETP-ALL, is reported to be associated with the lowest 5-year survival scores and is also known to induce chemoresistance (44). Overexpression of miR-221 was found to be associated with poorer response to induction therapy, risk typing, and blood cell counts, making it a reliable marker for the diagnosis and prognosis of T-ALL (57). A different pair of miR-99a and miR-125b is associated with the TLK3 T-ALL subtype that exhibits poorer patient survival (35). Patients diagnosed with T-ALL have lower expression of miR-30 and miR-100, and while the role of miR-30 is still under investigation, miR-100 underexpression correlates with poor overall and disease-free survival rates, making it a high-risk prognostic biomarker (27, 37). As high expression of miR-30 in breast cancer is associated with better survival and prognosis (58), its low expression in T-ALL may possibly resemble poor survival, which requires further investigation. Lower expression of miR-326, along with high expression of BAALC (brain and acute lymphoblastic leukemia cytoplasmic protein), is associated with poor survival, especially in children, along with leukemogenesis, shorter survival, resistance to therapy, and MRD (minimal residual disease) (46). There are other miRNAs, for instance, miR-16, which, despite having a nonsignificant overexpression in T-ALL, gave better 1-year survival rates as high as 50% in patients having comparatively higher levels of miR-16 (28). Another, miR-142-3p, is overexpressed in T-ALL cells and is involved in the downregulation of the cAMP/PKA pathway, causing enhanced cell migration. On the one hand, this miRNA causes increased sensitivity to steroid therapy; on the other hand, its inhibition causes increased sensitivity to dexamethasone in patients with prednisone resistance (27). A poorer response to prednisone is also associated with lower expression of miR-708, a miRNA associated with childhood T-ALL. Higher levels of miR-708 can be of diagnostic significance and can signify moderate survival, whereas lower expression of miR-708 is associated with higher risk and higher chances of relapse (59) (Figure 2) ( Table 1).

Metabolic roles of dysregulated miRNAs in T-ALL
T-ALL finds significant overexpression and underexpression of many miRNAs, amongst which we discuss prominently dysregulated miRNAs having metabolic roles, either reported in T-cell malignancies or in other cancers. In T-ALL, an upregulated miR-16, positively influencing patient survival (28), is reported to reprogram glycolysis and chemoresistance (29) in cervical cancer by activating PKD4, and also increase the radiosensitivity of prostate cancer cells (60). A downregulated let-7e in T-ALL was reported to regulate metabolic rewiring by inhibiting oxidative phosphorylation and adipogenesis and increasing lactate accumulation in a breast cancer study (61). A negative prognostic factor of underexpressing miR-17-5p in T-ALL leads to tumor metabolic reprogramming via its cluster miR-17-92 in B-lymphoma cells (31). Similarly, miR-99a and miR-125b, resembling poor prognosis and patient survival, were reported to be involved in glycolytic downregulation (34,36) by facilitating decreased glucose uptake and oxygen consumption (32,33). Moreover, an upregulated miR-100 attributed to poor overall and disease-free survival in T-ALL is involved in reduced lipid accumulation in metabolic syndromes (62) and probably involved in inositol phosphate metabolism in multiple myeloma (38). Similarly, miR-124a and miR-128b were found to be downregulated in T-ALL, and are associated with poor prognosis, and have a role in the inhibition of the Warburg effect and suppression of glucose metabolism, respectively (40, 42). Furthermore, the enhanced miR-221 and miR-222 in T-ALL are not yet known to have any metabolic reprogramming function in cancer, but they are reported to have roles in adipogenesis in obese conditions (45). An underexpressed miR-326 is found to impact key glucose metabolism regulators (47). The significance of miR-708, which is found underexpressed in T-ALL and correlates with poor survival, has not yet been investigated for its metabolic role in cancer, but it has been reported to affect glucose metabolism by influencing insulin secretion (49) and also influence prostaglandin E2 production, affecting tumorigenesis (48). Thus, these miRNAs with diagnostic and prognostic significance expectedly mold the metabolic status of cancer cells by prominently affecting glucose metabolism to support malignant growth ( Figure 2).

Peripheral T-cell lymphomas
Unlike T-ALL, which originates from immature thymocytes, PTCL is a malignancy of mature T cells. It is a heterogeneous class of nodal and some extranodal tumors, corresponding to approximately 10%-15% of all lymphoid malignancies. PTCL can be subdivided into three major types: angioimmunoblastic T-cell Various miRNAs and their significance in patient survival outcomes, prognosis, and subtyping, highlighting miRNAs involved in metabolic reprogramming of cancers. lymphoma (AITL), anaplastic large-cell lymphoma (ALCL), and peripheral T-cell lymphoma-not otherwise specified (PTCL-NOS) (63). As far as the diagnosis of PTCLs is concerned, conventional diagnostic methodologies have not performed efficiently, and therefore, more than 30% of all PTCL cases remain unclassified and remain at the intersection of various subtypes. Although at the molecular level, these subgroups, despite sharing an overwhelming number of commonly altered pathways and transcriptional signatures, have distinct expression levels revealed by gene expression profiling of patients (64). MicroRNA profiling of PTCL cases revealed specific miRNA signatures based on differential expression of various miRNAs when compared to normal cells. Many miRNA profiling studies, however, have reported differentially expressed miRNAs within various subtypes (65), but their diagnostic accuracies will depend on their practical ability to discriminate the subtypes at the molecular level. A notable miRNA, miR-187 overexpression, is associated with peripheral Tcell lymphoma progression, which is also related to sensitivity to bortezomib (66) and is associated with better survival in other cancers (67). However, its clinical role in PTCL patient outcomes and prognosis remains unclear.

Angioimmunoblastic T-cell lymphoma
AITL is a rare but the second most frequently reported malignant lymphoma of peripheral T cells that originates in mature T lymphocytes. It also accounts for around 1%-2% of all non-Hodgkin lymphoma (NHL) cases. Clinically, AITL cases manifest symptoms such as lymphadenopathy, hepatosplenomegaly, hypergammaglobulinemia, and anemia. Less common symptoms include arthritis, ascites, and some autoimmune characteristics, such as circulating rheumatoid antibodies, antismooth muscle antibodies, and hemolytic anemia (68). Generally, death in AITL patients occurs due to overwhelming immunodeficiency and not due to a serious tumor load (69). AITL pathogenesis and progression are also associated with EBV; in some cases, its onset closely mimics an EBV infection confirmed by EBV-specific antibodies in AITLpositive patient serology (70). miRNA profiling of 30 patients with AITL revealed suppressed expression of four miRNAs, miR-140-3p, let-7g, miR-30b, and miR-664, whereas three miRNAs, miR-146a, miR-193b, and miR-34a, were significantly upregulated (71). Among these, miR-146a and miR-34a influence the expression of the EBVassociated protein LMP1 in an NF-kB-dependent manner, thus helping in tumorigenesis; however, these miRNAs are also overexpressed in B-cell lymphomas (72), indicating their lower specificity for AITLs.
Genome-wide miRNA expression profiling of AITL biopsy samples when compared with their polarized CD4+ T-cell counterparts, produced by polarizing them and grouping them based on their cytokine signatures, revealed many overlapping and distinct miRNA signatures. In general, AITL was associated with many differentially expressed miRNAs compared with their normal counterparts or other subtypes of PTCL-NOS, such as the PTCL-GATA3 and PTCL-TBX21 subtypes. These 13 differentially expressed miRNAs (Table 2) signify a distinct miRNA signature that distinguishes AITL from other T-cell malignancies. Among these miRNAs, miR-126-3p is important as it regulates angiogenesis (85), maintains endothelial vasculature (86), and has roles in T-cell activation (87) and differentiation (88), all of which are interesting roles in the context of AITL pathobiology. Moreover, two miRNAs, miR-126-3p and miR-145-5p, are important because they show marginal and significant associations with AITL overall survival (OS) (Figure 3). Ectopic expression of miR-126-3p in cell line models showed repression of sphingosine-1 phosphate receptor-2 (S1PR2), which has been reported to activate RhoA signaling and is associated with poor prognosis, whereas ectopic expression of miR-145-5p represses ROCK1, which is a downstream target of RhoA. Hence, these miRNAs, along with their association with poor prognosis, are also responsible for regulating Rho-GTPase and inhibiting T-cell migration, as observed in AITL pathobiology (77). These enriched miRNA signatures, when fed into an in silico computational annotation tool (89) for pathway analysis, showed the involvement of two major pathways. These enriched miRNAs activated T-cell receptor signaling along with the activation of PI3K-Akt signaling (77). Therefore, these overexpressing miRNAs and their involvement in peculiar AITL pathobiology hold promising potential and call for more focused investigations.

Anaplastic large cell lymphoma
ALCL is a malignancy of mature T lymphocytes characterized by anaplastic characteristics, such as horseshoe-shaped nuclei with large pleomorphic lymphoid cells. Molecularly, ALCL is further subdivided into two groups, ALK + ALCL and ALK− ALCL, based on rearrangement (translocation) of the anaplastic lymphoma kinase (ALK) gene (90). Diseases in which this gene is rearranged give rise to the expression of chimeric nucleophosmin (NPM) protein, which causes constitutive ALK expression (91). This translocation and eventual formation of the fusion protein NPM-ALK is the definitive molecular feature of ALK+ ALCL, whereas the other subgroup, that is, ALK− ALCL, is coarsely defined as cases devoid of expressing the fusion protein NPM-ALK. This NPM-ALK fusion protein activates many growth-promoting and antiapoptotic pathways, such as cJun, cMyc, Jak/STAT, and PI3K-Akt/mTOR (92). In a major study in which miRNA profiling of ALCL cases was performed and compared to miRNA profiles of other PTCL cases, such as AITL and PTCL-NOS, diagnostic miRNA signatures were identified after nullifying miRNAs that were also expressed in stromal cells. This diagnostic miRNA signature differentiates ALCL from other PTCL cases and comprises 13 upregulated miRNAs (Table 2), led by the highest expression of miR-149 along with others, and 15 downregulated miRNAs (Table 2), led by miR-155. These miRNA signatures do hold promising diagnostic potential owing to their discriminatory nature regarding various molecular and clinical manifestations of PTCLs (81).

ALK+ and ALK− ALCL
ALK+ ALCL has positive expression of the oncogene fusion protein NPM-ALK, which promotes miR-135b expression by activating signal transducer and activator of transcription 3 (STAT3). This miR-135b also targeted FOXO1 and inhibited its growth suppression activities (82). As STAT3 activation is essential for ALK-mediated transformation, its knockdown in ALK cells and forced expression of a certain miRNA cluster (miR-17-92) result in STAT3 rescue, suggesting a positive role of this cluster in STAT3 and ALK-mediated transformation (93). Similar to most ALCL phenotypes that overexpress anti-apoptotic genes like myeloid cell leukemia-1 (MCL1), it has been reported that this overexpression is done by the NPM-ALK fusion oncoprotein by downmodulating another miRNA called miR-29a (83). However, the role of miR-29a in determining clinical outcomes and patient survival remains unclear.
To differentiate ALCL subtypes from other PTCL subtypes, research suggests unique miRNA signatures associated with both ALK+ and ALK− subtypes. ALK+ ALCL has a significant association with seven miRNAs, of which the expression of five miRNAs was elevated (miR-512-3p, miR-886-5p, miR-886-3p, miR-708, and miR-135b) and the expression of two miRNAs (miR-146a and miR-155) was repressed (Table 2) (81). In contrast, ALK− ALCL was found to be associated with 11 miRNAs, of which four were elevated (led by miR-191) and seven had lower expression (led by miR-22) (Figure 3) (81). Therefore, these miRNAs can be used as classifiers to diagnose and differentiate ALK subtypes of ALCL from other PTCL subtypes.

PTCL-NOS (not otherwise specified)
PTCL-NOS consists of a group of malignancies that are not similar to the other main PTCL subtypes. Generally, PTCL-NOS is molecularly characterized by an aberrant T-cell immunophenotype, usually with the loss of CD5 and CD7 (94). Similar to many other PTCLs, PTCL-NOS shows molecular expression similarity to activated CD4+ and CD8+ T lymphocytes in in vitro studies. In PTCL-NOS, overexpression of miR-187 has been reported to be associated with tumor progression and a poor prognosis (66). In this regard, many studies have attempted to classify PTCL-NOS cases based on their miRNA expression patterns. A study by Laginestra et al. found 158 miRNAs that significantly affect the PTCL-NOS transcriptome compared to normal activated CD8+ and CD4+ T lymphocytes. Of these miRNAs, there was a significant underexpression of miR-132-3p, a miRNA found to be downregulated in many other solid tumors, but in PTCL-NOS cases, it showed differential expression compared to normally activated CD4+ and CD8+ T cells (63). In addition to the above study, another miRNA profiling study reported a miRNA signature of 20 miRNAs (led by kshv-miR-K12-12-5p) in PTCL-NOS that can be used for diagnostic purposes while narrowing down five miRNAs, namely hsa-miR-224-3p, hsa-miR-614, hsa-miR-3196, hsa-miR-1290, and hsa-miR-4485, (Table 2), which related to common cancer progression pathways (84). They reported a significant increase in expression of miR-1290 and miR-4485 in PTCL-NOS among all other subtypes (84).
Although miRNA expression analysis has shown a large number of miRNAs expressed in various subtypes of PTCLs, their diagnostic efficacy has not been assessed in previous studies. Laginestra et al. (63) not only studied differentially expressed miRNAs in PTCL subtypes but also narrowed down the huge number of miRNAs to a set of few miRNA classifiers and devised a practical tool for their diagnostic efficiency. According to this study, PTCL-NOS can be differentiated from ALCL/ALK− using a miRNA classifier comprising five miRNAs (led by has-miR-155) (Table 2). Similarly, PTCL-NOS can be distinguished from AITL using another miRNA classifier with eight miRNAs (led by has-miR-324-5p) and a four-miRNA classifier (led by hsa-miR-124) ( Table 2) to differentiate ALK+ and ALK− ALCLs (63). However, a few other studies have subdivided PTCL-NOS into two groups, namely PTCL-NOS-GATA3 and PTCL-NOS-TBX21, based on patient gene expression profiling (GEP). The PTCL-NOS-GATA3 subtype is associated with PI3KP and mTOR upregulation, whereas PTCL-NOS-TBX21 activates the NF-kB pathway. As far as the PTCL-NOS-GATA3 subtype is concerned, it showed nine distinct miRNAs (led by miR-18a-5p), like a signature peculiar to PTCL-NOS-GATA3 (77). PTCL-NOS-TBX21 also showed five distinct miRNA expressions (led by let-7f-5p) when compared against AITL or other PTCL-NOS-GATA3 cases (Figure 3) (77). As the incidence of PTCL-NOS is the highest among all PTCLs, an increasing number of clinical studies validating these miRNA classifiers will help to understand and validate their diagnostic accuracies and their effect on survival outcomes.

Metabolic roles of dysregulated miRNAs in PTCLs
As PTCL has several subtypes, upregulation of microRNA miR-187 reported across PTCL subtypes is known to decrease glucosestimulated insulin secretion (GSIS) in metabolic syndromes; however, its role in cancer metabolic reprogramming is unknown (73). In AITL, the overexpression of miR-146a has a global implication on cancer metabolic reprogramming, starting from glycolysis, oxidative phosphorylation, and TCA, along with amino acid and lipid metabolism (74). The miR-34a, which is also overexpressed in AITL, has a negative role in regulating glucose metabolism by lowering the expression of lactate dehydrogenase A (LDHA) (95), whereas the downregulated miR-30b has a negative role in regulating lipid metabolism as reported in hepatocellular carcinoma (76). Similarly, an overexpressed miR-126-3p, suggesting poor prognosis in AITL, expectedly is a global regulator of angiogenesis, having a role in neovascularization and glycometabolism in cardiovascular diseases (78). This miR-126 is also suggested to have been enriched in microvesicles (96), indicating its prospective role as a noninvasive circulating biomarker potentially useful for clinical management. Another miRNA, miR-145, associated with poor survival in AITL, finds significance in negatively regulating aerobic glycolysis and has also been found to inhibit mitochondrial metabolism in cervical cancer and ovarian cancer (79,80).
In ALCL, few miRNAs among the 13 found to have diagnostic specificity against other PTCLs have metabolic roles. Among them are the highly upregulated hypoxia-responsive miR-210 and miR-517, which are implicated in facilitating aerobic glycolysis (75,97), whereas miR-708 may have a role in facilitating cellular metabolism (48). For ALK+ ALCL, the upregulated diagnostic miRNA signature, including miR-512 and miR-135b, is reported to have an inhibitory effect on glycolysis (98,99), whereas a repressed miR-155 may signify reduced glucose metabolism (100). Another downregulated miR-146a in ALK+ ALCL may signify an increase in the Warburg effect and lactate export (101) to support cancer growth. Like ALK+ ALCL, miR-210 is also upregulated in ALK− ALCL, which has a well-known role in facilitating the Warburg effect by decreasing tri-carboxylic acid (TCA) cycle activity and increasing lactate production (102). Furthermore, in PTCL-NOS-TBX21, miRNAs let-7f and miR-26a were found to be most upregulated and have well-documented roles in modulating metabolism in cancers (103,104), whereas an upregulated miR-30b is known to negatively regulate lipid metabolism (76). In PTCL-NOS-TBX21, an upregulated miR-106b is one of the global regulators of cancer metabolism (74). In this TBX21 subtype, an upregulated miR-181a is found to reprogram metabolism to influence glucose metabolism (105), lactate production (106), and lipid metabolism (107), and can inhibit IDH1/2 and TCA cycle enzymes (108). Moreover, an upregulated miR-20 is reported to have been involved in increasing fatty acid synthesis in separate studies (109) on metastatic cancers; therefore, its levels in aggressive forms of PTCL-NOS can be of clinical and diagnostic importance ( Figure 3).

Extranodal NK/T-cell lymphomas
Another subtype of PTCL, extranodal NK/T-cell lymphomas, should be discussed separately because of their peculiar nature of affecting non-nodal regions. Almost 80% of the reported cases occur around the nose and the region associated with the nasopharynx and oropharynx (110). As far as the incidence of ENKTCL is concerned, it is more commonly diagnosed in men than in women, along with an ethnic bias where it is reported more in Asian, South American, and Central American populations than in Western countries (111). Apart from its apparent clinical course associated with extensive necrosis, ENKTCL is also associated with Epstein-Barr virus (EBV) infection, a peculiar and defining feature of this malignancy. This malignancy is sustained by upregulation of pro-proliferation pathways like JAK/ STAT3-related pathways, NF-kB pathways, RUNX3, platelet-derived growth factor (PDGF) pathway, NOTCH1, and aurora kinase pathway, among others (112).
When considering the role of miRNAs in ENKTCL malignancies, earlier studies have shown the diagnostic significance of circulating miR-221, whose upregulation in plasma is so enriched that it can be quantified without total mRNA extraction (113). miR-221 is also upregulated in T-ALL and associated with shorter survival (56). Earlier studies also found downregulation of miR-26a, miR-26b, miR-28-5p, miR-101, and miR-363 in nasal-type ENKTCL when compared to normal NK cells (114). As more than 90% of ENKTCL cases have EBV infection, EBV-encoded miRNAs also play a significant role in driving the transformation of NK/T cells and evading scrutiny by cytotoxic T cells. Among these EBV-encoded miRNAs, BART9 is directly involved in modulating latent membrane protein 1 (LMP1) expressed in almost all EBV-associated lymphomas, providing ENKTCL and its relationship with BART9 with a discriminatory edge compared to other non-EBV-associated lymphomas (115). Other EBV-encoded miRNAs, such as miR-BART20-5p and miR-BART8, are upregulated and involved in inhibiting the IFN-g STAT1 pathway (116) and evading immune scrutiny, whereas miR-BART9 was found to directly inhibit transcription factor T-bet, resulting in repressed expression of IFN-g (117). In non-EBV-associated lymphomas, two miRNAs (miR-205 and miR-142-3p) were found to support tumor progression by upregulating the expression of BCL6 and interleukin-A. Interestingly, these two miRNAs were found to be repressed in EBVassociated lymphomas (Table 3) (118), indicating their diagnostic role in discriminating between EBV and non-EBV-linked lymphomas. Various other miRNAs such as miR-21, miR-155, miR-221, miR-223, and miR-4943p are overexpressed, and several others such as miR-15a, miR-16, miR-143, miR-146a, miR-150, and miR-205 are found to be underexpressed in independent studies but have less diagnostic potential because of their association with other malignancies (119).

Cutaneous T-cell lymphomas
Cutaneous T-cell lymphoma is a heterogeneous group of malignancies with different clinical, histological, and prognostic features that are grouped according to their apparent effects on skin and cutaneous tissues. A general feature of all CTCLs is the presence of malignant T cells proliferating in a chronic inflammatory environment that expands to the skin, like the sezary syndrome (SS) and mycosis fungicides (MF), which are aggressive variants of CTCLs and constitute the majority of the cases (120). Past research has successfully identified miRNA signatures that differentiate CTCLs from other similar diseases/malignancies, for example, benign inflammatory dermatosis (121). These specific miRNA signatures may be potent in not only identifying CTCLs from other tumors/lymphomas but can also differentiate between various subtypes of CTCLs such as MF, erythrodermic mycosis fungoides (eMF), SS and others (122,123).

Mycosis fungoides
One of the most prevalent types of CTCL is the indolent form known as MF, which accounts for almost 50% of all CTCL cases (120). An early-stage MF resembles small patches and plaques on the skin that advance to form large multifocal itchy lesions and cutaneous tumors in later stages, which may start disseminating malignant T cells to the surrounding nodes, peripheral blood, and vital organs (136). MFs can also be characterized in many groups based on their different clinical and pathological presentations (for example, folliculotropic MF, erythrodermic MF, unilesional MF, hyperpigmented MF, pagetoid MF, granulomatous MF, granulomatous slack skin MF, and vasculature atropicans MF) (137, 138).
In MF, several miRNAs have been implicated to not only have diagnostic potential but also act as oncogenes and drive the pathogenesis of MF. One of them is miR-155, whose upregulation has been consistently observed in various studies concerning MFs. Considering MF's resemblance to inflammatory dermatoses, differential expression of microRNAs in both disease types is reportedly apparent (121, 139). A study observed significantly higher upregulation of miR-155, miR-92a, miR-92b, and miR-93 in MFs than in inflammatory dermatoses, among which miR-93 showed the highest upregulation. Many miRNAs, including miR-93, miR-146a, miR-181, miR-203, miR-205, miR-16, miR-342-3p, and miR-146b-5p, can diagnose MFs early and efficiently (121) (140). These miRNAs can also be used for differential diagnosis of MFs. Among these, miR-93 is important, as its levels are suggestive of specific stages of MFs. In advanced MFs, miR-93 is upregulated when compared with inflammatory dermatosis, whereas another study suggested its downregulation in early MFs when compared with normal and eczema cases (128).

Large cell transformation of mycosis fungoides diagnosis
Another advanced form of MF, called large cell transformation of mycosis fungoides (LCT-MF), is associated with rapid progression of the disease and poor survival outcomes (142). LCT-MFs are characterized by the presence of large T cells that are almost four times bigger than normal T cells and have been reported as an independent prognostic factor for poor survival in MF and SS (143, 144). These malignant transformed T cells (LCT) are often found in advanced stages of MF or SS and very rarely in early stages (145). A study showed significant upregulation of miR-34a, miR-93-5p, miR-181a, miR-181b, and miR-326 in LCT-MF when compared to control cases (130). Another study focusing on the miRNA profile of patients with LCT-MF found the highest upregulation of miRNAs like miR-21-3p, miR-146a-3p, miR136-5p, Major cutaneous T-cell lymphoma (CTCL) subtypes and miRNAs of diagnostic and prognostic significance. Illustrated here are the miRNAs involved in mycosis fungiode (MF) diagnosis in a stage-and lesion-specific diagnosis.

Folliculotropic mycosis fungoides
An aggressive subtype of MF known as folliculotropic MF (FMF), which involves deep infiltration of malignant T cells in and around hair follicles of the patient's body, has a quite similar miRNA profile to LCT-MF with upregulation of miR-34a, miR-93-5p, and miR-181a (130). Both LCT-MF and FMF cases can be distinguished by upregulated miR-181b and miR-326 in LCT-MF and FMF (130). There have been no discriminatory miRNAs discovered to date to diagnose FMF cases positively.

Sezary syndrome
SS is a rare form of CTCL that can be distinguished from other types of lymphomas molecularly by the expression of CD4+ and morphologically by the cerebriform appearance of the nucleus, commonly known as sezary cells. These sezary cells can be found in the peripheral blood, skin, and lymph nodes of the suffering patient. Among all the miRNAs, miR-21 specifically regulates the oncogenic progression in SS by activating the STAT3 signaling pathway. This activation of the signaling pathway is also strongly regulated by IL-21 cytokine. Hence, targeting the miR-21 can provide a new therapeutic approach to SS management (146).

Diagnostic miRNAs of CTCLs and their metabolic significance
In CTCLs, miR-155 is found upregulated irrespective of its subtype but is interestingly found downregulated in PTCLs, where it has a positive role in regulating and driving glucose metabolism (100,148,149). Its increased expression in CTCL subtypes may signify a significant role of glucose metabolism in the pathogenesis of CTCLs. The miR-326, which is upregulated in CTCLs in general more so in MFs, along with its FMF variant, is underexpressed in T-ALL. It is known to modulate glucose metabolism by influencing many key regulators (47). Moreover, upregulated miR-146a in all CTCL subtypes and AITL are well known as global regulators of cancer metabolism, influencing glycolysis and TCA cycle activity (74). Similarly, miR-181 is found to regulate glucose and lipid metabolism is upregulated in PTCL-NOS-TBX21 and CTCL subtypes more prominently in mycosis fungiodes and also in T-ALL (77, 105-107, 121). This indicates a similar contribution of miRNA regulators of glucose metabolism to the metabolic landscape of these T-cell malignancies and, hence, can suggest interesting future studies in the discovery of more accurate diagnostic miRNAs.
6 Applicability of miRNA-based diagnosis and new avenues miRNAs for diagnosis have been of research interest in cancers for a long time now. Its application in the diagnosis of T-cell malignancies is, however, of more importance given the wide range of pathobiological, clinical, and cytogenetic variations in its malignant subtypes (150). To avoid inaccurate clinical judgment (151), it is essential to devise new methods of diagnosis that cannot only capture specific tumor markers but also quantify molecular variations among overlapping clinical features. miRNA profiling can be employed to look for miRNA classifiers for specific patterns in T-cell malignancy subtypes (152). For instance, in AITL biology, miR-146a and miR-34a are associated with EBV-linked AITL cases; therefore, their upregulation can be used to establish EBVassociated AITL cases and rule out non-EBV cases. Similarly, miR-30b is significantly associated with angiogenesis and can be used for the differential diagnosis of AITL from others. Moreover, subtypes like extranodal NK/T-cell lymphomas (ENKTCL) overexpresses EBV-associated miRNAs like miR-BART8, miR-BART9, and miR-BART20-5p, along with non-EBV-associated miRNAs found to be over/underexpressed (116). These miRNAs, in combination, can be effectively used to differentially diagnose and discriminate ENKTCL lymphomas from other subtypes.
Circulating miRNAs have attracted a great deal of interest as potential diagnostic biomarkers because of their stability and detectability. miRNAs derived from extracellular vesicles (EVs) (54) and exosomes (153) may reflect a miRNA signature that may resemble a very early disease condition (154). For instance, miR-126 overexpressed in AITL signifies poor prognosis, and its enrichment in the exosome is found to distinguish early and advanced nonsmall cell lung cancer (NSCLC), while it is downregulated for advanced NSCLC (155). This miR-126 can be investigated in AITL for its possible correlation with the stage and aggressiveness of AITL and whether its exosomal enrichment in body fluids can be used as a noninvasive tool to monitor disease and suggest better therapies. Similarly, in ALCL, an upregulated miR-223 can have a potential role in inducing chemoresistance, whose expression levels can be quantified in exosomes to potentially diagnose and use for disease surveillance (156). Studies also indicate that an upregulated miR-197 in ALK− ALCL can be investigated for its enrichment in exosomes as it has been implicated in metastatic promotion in lung cancer (157). The above insights project miRNAs and their profiling as powerful tools to be potentially used for differential diagnosis of malignancies with overlapping clinical manifestations.
More recently, liquid biopsies, which include examining circulating miRNAs in bodily fluids, have presented a simple, noninvasive method for early and efficient diagnosis along with disease tracking and surveillance capacities (158). However, assessing tumor-specific miRNA expression by liquid biopsy or miRNA enrichment in EVs or exosomes requires many clinicalbased studies to establish their clinical applicability. Currently, the applicability of liquid biopsies in clinical settings is debatable (159). On the one hand, the method provides a noninvasive and easily accessible option that incurs less discomfort and risk to the patient than standard tissue biopsies, evaluation of biomarkers derived from it, remains unstandardized (160). The task of standardizing miRNA profiling data analysis is ongoing as platforms and procedures used by various laboratories differ, causing results to fluctuate and making it difficult to reproduce and compare findings (160). A liquid biopsy also gives clinicians and scientists an upper hand in the early detection of tumors, along with its promising potential to record the molecular heterogeneity of tumors, thus providing scope for precise diagnosis and personalized treatment strategies. However, molecular and genetic complexities of T-cell malignancies can influence miRNA profiles depending on the tumor microenvironment, disease stage, individual patient characteristics, or the presence of any additional disease (161). Moreover, circulating miRNA levels can be affected by a number of preanalytical factors, including sample type, storage conditions, and RNA separation techniques. The processing of samples and the selection of the sample type (serum, plasma, or other biofluids) can affect the stability and production of miRNA (162). For trustworthy diagnostic outcomes and to reduce technical variability, these preanalytical factors must be standardized. The clinical relevance, sensitivity, specificity, and positive predictive value of diagnostic tests must be demonstrated through thorough validation and clinical investigations, just like with any other diagnostic test (163). Additionally, in order to authorize miRNAbased diagnostic tests for clinical use, regulatory authorities demand strong proof to avoid faulty clinical judgments while in use (164).
Tumor-associated miRNAs can be detected and quantified in body fluids such as serum (165) or plasma (166) using techniques such as reverse transcriptase PCR (167), microarray (124), and next-generation sequencing (NGS) that were traditionally used for miRNA profiling studies. Quantitative PCR and microarray analysis are two miRNA detection methods that have limitations in terms of sensitivity and specificity. Results that are falsely positive or falsely negative can be recorded, which can reduce the precision of miRNA-based diagnosis. More reliable and powerful detection techniques such as droplet digital PCR (ddPCR) promise increased sensitivity, absolute quantification and precision, multiplexing capabilities, and resistance to PCR inhibitors, and their ability to detect rare variants, which can lead to betterstandardized results than traditionally used methods (168).
In malignancies or otherwise, miRNAs were never singularly used for diagnosis, as there are overlapping miRNAs in various malignant disease phenotypes. Therefore, miRNA classifiers and panels encompassing a wide range of over-and underexpressing miRNAs are fit to be used for diagnostic applications. In this regard, many researchers have already devised miRNA classifiers whose diagnostic applicability has been validated in clinical settings (140,169,170). However, research is still underway to identify and validate particular miRNAs linked to better prognosis and accurate diagnosis. In this regard, miRNAs having a metabolic impact on tumors may be investigated for their promising diagnostic potential. Many studies have identified miRNA species that have regulatory roles in reprogramming cell metabolism to support or inhibit cancer progression. Such studies often develop an overexpression system using many strategies, such as transfecting cells with miRNA-viral construct or miRNA mimics to transiently overexpress or inhibit a particular miRNA in order to study their functional roles. A brief summary of the methodological strategies and outcomes of such studies is featured in Supplementary Table 1. Future investigations exploring such metabolic miRNAs for their diagnostic potential along with the metabolic status of patients could be potentially utilized for more accurate subtyping and subgrouping of diseases and leading precision medicine efforts.

Conclusion
Various malignancies of the T-cell display overlapping physical symptoms despite each one having different molecular and disease progression profiles. Even though imaging techniques such as PET and CT scans can efficiently diagnose the location, stage, and spread of cancer, they cannot suggest valuable information regarding the best therapeutic choice with minimum toxicity and maximum survival. However, investigating disease-specific molecular biomarkers in tumor tissues or in circulating fluids is a promising avenue of molecular diagnostics and can reduce cancer mortality by suggesting the best therapeutic choices for better survival outcomes. Studies capturing miRNA signatures and unique miRNAs in miRNAome need more variety in terms of patient diversity in addition to the range of miRNAs used for screening. miRNAs involved in regulating diverse signaling pathways can be included in the panel of miRNAs for better diagnosis and categorization of malignant T-cell subtypes. Therefore, more such studies identifying and evaluating tissue or serum miRNAs derived from EVs or exosomes will help identify specific signature miRNAs for various other clinical outcomes and may lead to early and efficient diagnosis and therapy of this diverse disease group. Future studies can explore miRNAs along with their metabolic impact to devise more precise miRNA panels for differential diagnostic purposes.

Author contributions
Corresponding and lead authors from Guru Ghasidas Vishwavidyalaya wrote and edited the manuscript. All authors contributed intellectual contributions to designing, editing, and proofreading the manuscript. All authors contributed to the article and approved the submitted version.