Disorder	Disease Definition	References
Monoclonal gammopathy of undetermined significance (MGUS)	All three criteria must be met: • Serum M protein < 3 g/dL • Clonal bone marrow plasma cells <10%, and • Absence of end organ damage such as hypercalcemia, renal insufficiency, anemia, and bone lesions (CRAB) that can be attributed to the plasma cell proliferative disorder; or in the case of immunoglobulin M (IgM) MGUS no evidence of anemia, constitutional symptoms, hyperviscosity, lymphadenopathy, or hepatosplenomegaly that can be attributed to the underlying lymphoproliferative disorder	International Myeloma Working Group³¹⁶
Smoldering multiple myeloma (also referred to as asymptomatic multiple myeloma)	Both criteria must be met: • Serum M protein (IgG or IgA) ≥ 3 g/dL and/or clonal bone marrow plasma cells ≥ 10%, and • Absence of end organ damage such as lytic bone lesions, anemia, hypercalcemia, or renal failure that can be attributed to a plasma cell proliferative disorder	International Myeloma Working Group³¹⁶
Multiple myeloma	All three criteria must be met except as noted: • Clonal bone marrow plasma cells ≥ 10% • Presence of serum and/or urinary M protein (except in patients with true nonsecretory multiple myeloma), and • Evidence of end organ damage that can be attributed to the underlying plasma cell proliferative disorder, specifically: • Hypercalcemia: serum calcium ≥ 11.5 mg/dL or • Renal insufficiency: serum creatinine > 1.73 mmol/L (or > 2 mg/dL) or estimated creatinine clearance < 40 mL/min • Anemia: normochromic, normocytic with a hemoglobin value of > 2 g/dL below the lower limit of normal or a hemoglobin value < 10 g/dL • Bone lesions: lytic lesions, severe osteopenia or pathological fractures	International Myeloma Working Group³¹⁶; Rajkumar and Kyle³¹⁷
IgM monoclonal gammopathy of undetermined significance (IgM MGUS)	All three criteria must be met: • Serum IgM M protein < 3 g/dL • Bone marrow lymphoplasmacytic infiltration < 10%, and • No evidence of anemia, constitutional symptoms, hyperviscosity, lymphadenopathy, or hepatosplenomegaly that can be attributed to the underlying lymphoproliferative disorder	Owen et al²³⁷; Baldini et al²³⁸; Gobbi et al²³⁹; Kyle et al^240,³¹⁸
Smoldering Waldenström macroglobulinemia (also referred to as indolent or asymptomatic Waldenström macroglobulinemia)	Both criteria must be met: • Serum IgM M protein ≥ 3 g/dL and/or bone marrow lymphoplasmacytic infiltration ≥ 10%, and • No evidence of anemia, constitutional symptoms, hyperviscosity, lymphadenopathy, or hepatosplenomegaly that can be attributed to the underlying lymphoproliferative disorder	Owen et al²³⁷; Baldini et al²³⁸; Gobbi et al²³⁹; Kyle et al^240,241,318
Waldenström macroglobulinemia	All criteria must be met: • IgM monoclonal gammopathy (regardless of the size of the M protein) and • ≥ 10% bone marrow lymphoplasmacytic infiltration (usually intertrabecular) by small lymphocytes that exhibit plasmacytoid or plasma cell differentiation and a typical immunophenotype (e.g., surface IgM+, CD5±, CD10−, CD19+, CD20+, CD23−) that satisfactorily excludes other lymphoproliferative disorders, including chronic lymphocytic leukemia and mantle cell lymphoma • Evidence of anemia, constitutional symptoms, hyperviscosity, lymphadenopathy, or hepatosplenomegaly that can be attributed to the underlying lymphoproliferative disorder	Owen et al²³⁷; Baldini et al²³⁸; Gobbi et al²³⁹; Kyle et al^240,³¹⁸
Light-chain MGUS	All criteria must be met: • Abnormal free light chain (FLC) ratio (<0.26 or > 1.65) • Increased level of the appropriate involved light chain (increased κ FLC in patients with ratio > 1.65 and increased λ FLC in patients with ratio < 0.26) • No immunoglobulin heavy-chain expression on immunofixation • Absence of end organ damage such as hypercalcemia, renal insufficiency, anemia, and bone lesions (CRAB) that can be attributed to the plasma cell proliferative disorder	Dispenzieri et al²²²
Solitary plasmacytoma	All four criteria must be met: • Biopsy-proven solitary lesion of bone or soft tissue with evidence of clonal plasma cells • Normal bone marrow with no evidence of clonal plasma cells • Normal skeletal survey and MRI of spine and pelvis (except for the primary solitary lesion) • Absence of end organ damage such as hypercalcemia, renal insufficiency, anemia, or bone lesions (CRAB) that can be attributed to a lymphoplasma cell proliferative disorder	Dimopoulos et al^226,300
Systemic AL amyloidosis	All four criteria must be met: • Presence of an amyloid-related systemic syndrome (e.g., renal, liver, heart, gastrointestinal tract, or peripheral nerve involvement) • Positive amyloid staining by Congo red in any tissue (e.g., fat aspirate, bone marrow, or organ biopsy) • Evidence that amyloid is light-chain related established by direct examination of the amyloid (possibly using mass spectrometry (MS)–based proteomic analysis, or immunoelectron microscopy; note that immunohistochemistry results to type amyloid may be unreliable), and • Evidence of a monoclonal plasma cell proliferative disorder (serum or urine M protein, abnormal free light chain ratio, or clonal plasma cells in the bone marrow) • Note: Two to 3% of patients with AL amyloidosis will not meet the requirement for evidence of a monoclonal plasma cell disorder listed above; the diagnosis of AL amyloidosis must be made with caution in these patients.	Rajkumar et al³¹⁹
POEMS Syndrome	All four criteria must be met • Polyneuropathy • Monoclonal plasma cell proliferative disorder (almost always λ) • Any one of the following three other major criteria: 1. Sclerotic bone lesions 2. Castleman disease 3. Elevated levels of vascular endothelial growth factor (VEGF)* • Any one of the following six minor criteria: 1. Organomegaly (splenomegaly, hepatomegaly, or lymphadenopathy) 2. Extravascular volume overload (edema, pleural effusion, or ascites) 3. Endocrinopathy (adrenal, thyroid, pituitary, gonadal, parathyroid, pancreatic)† 4. Skin changes (hyperpigmentation, hypertrichosis, glomeruloid hemangiomata, plethora, acrocyanosis, flushing, white nails) 5. Papilledema 6. Thrombocytosis/polycythemia	Dispenzieri et al³⁰⁸; Dispenzieri³²⁰

Note: Not every patient meeting the above criteria will have POEMS syndrome; the features should have a temporal relationship to each other and no other attributable cause. Anemia and/or thrombocytopenia are distinctively unusual in this syndrome unless Castleman disease is present.

POEMS, Polyneuropathy, organomegaly, endocrinopathy, monoclonal protein, skin changes.

^*The source data do not define an optimal cutoff value for considering elevated VEGF level as a major criterion. We suggest that VEGF measured in the serum or plasma should be at least threefold to fourfold higher than the normal reference range for the laboratory that is doing the testing to be considered a major criterion.

^†To consider endocrinopathy as a minor criterion, an endocrine disorder other than diabetes or hypothyroidism is required, because these two disorders are common in the general population.

Modified from Kyle RA, Rajkumar SV. Criteria for diagnosis, staging, risk stratification and response assessment of multiple myeloma. Leukemia 2009;23:3–9.

Multiple myeloma accounts for approximately 10% of hematologic malignancies.5,6 The annual incidence, age-adjusted to the 2000 U.S. population, is 4.3 per 100,000.⁷ An estimated 22,000 new cases and 11,000 new deaths were expected to occur in the United States in 2012.⁸ Multiple myeloma is twice as common in blacks compared with whites and slightly more common in males than females.⁹ The median age at diagnosis is 66 years,¹⁰ and only 2% of patients are younger than 40 years of age.

Almost all patients with myeloma are believed to evolve from an asymptomatic premalignant stage termed monoclonal gammopathy of undetermined significance (MGUS).11,12 MGUS is present in more than 3% of the population older than the age of 50 and progresses to myeloma or related malignancy at a rate of 1% per year.^13,14 In some patients, an intermediate asymptomatic but more advanced premalignant stage referred to as smoldering multiple myeloma (SMM) can be recognized clinically.¹⁵

Pathogenesis

Transition from Normal Plasma Cell to MGUS

Antigenic Stimulation and Immunosuppression

MGUS is characterized by evidence of genomic instability on molecular genetic testing. The trigger for this genomic instability is not well understood, but current evidence suggests that antigenic stimulation may be a key factor (Fig. 104-1). Unlike normal plasma cells, human myeloma cell lines and primary myeloma cells express a broad range of Toll-like receptors (TLRs). TLRs are normally expressed by B lymphocytes and are essential for these cells to recognize infectious agents and pathogen-associated molecular patterns (PAMP), which then initiates the host-defense response.^18–18 The aberrant expression of TLRs by plasma cells may enable them to respond to TLR-specific ligands, resulting in an abnormal and perhaps sustained response to infection. It has been shown that TLR-specific ligands cause increased myeloma cell proliferation, survival, and resistance to dexamethasone-induced apoptosis. These effects are mediated in part by autocrine interleukin (IL)-6 production.^16,17

Figure 104-1 Pathogenesis of myeloma. IL-6, interleukin-6; MGUS, monoclonal gammopathy of undetermined significance; MIP-1α, macrophage inflammatory protein-1α; OPG, osteoprotegerin; RANKL, receptor activator of nuclear factor-κB ligand; VEGF, vascular endothelial growth factor.

IL-6 is a major growth factor for plasma cells,¹⁹ and there is overexpression of CD126 (IL-6 receptor α-chain) in MGUS compared with normal plasma cells.^20,21 Thus, abnormal TLR expression and/or overexpression of IL-6 receptors in plasma cells may be early, initiating events that lead to an abnormal response to infection and act as sustained, autocrine IL-6–dependent, proliferative triggers for plasma cells. During this process, plasma cells may acquire one of various cytogenetic alterations that result in a limited clonal plasma cell proliferative process, namely MGUS.

Immunosuppression either by promoting evasion of tumor surveillance or by promoting antigenic stimulation may also contribute to the initiation of monoclonal gammopathies. Monoclonal proteins have been reported in the context of immunosuppressive states such as bone marrow/stem cell transplantation, organ transplantation, and human immunodeficiency virus (HIV) infection.22–25 Patients undergoing renal transplantation develop M proteins depending on the level of immunosuppression to which they are subjected.²⁵

Cytogenetic Changes

Over 90% of MGUS is associated with cytogenetic changes possibly precipitated by infection or immune dysregulation as discussed earlier, likely during IgH switch recombination or somatic hypermutation.²⁶ Approximately 50% of patients with MGUS have primary translocations in the clonal plasma cells involving the immunoglobulin heavy chain (IgH) locus on chromosome 14q32 (IgH-translocated MGUS/SMM) (see Fig. 104-1).^5,26 The most common partner chromosome loci and genes dysregulated in these translocations are 11q13 (CCND1 [cyclin D1 gene]), 4p16.3 (FGFR3 and WHSC1), 6p21 (CCND3 [cyclin D3 gene]), 16q23 (MAF), and 20q11 (MAFB).^29–29 It is likely that these translocations play an important pathogenetic role in the resultant limited clonal proliferation that is clinically manifested as MGUS.

Approximately 45% of cases of MGUS are associated with trisomies, usually of the odd numbered chromosomes with the exception of 13; and the origin of the remaining 5% or fewer of MGUS is not clear.26,30 These categories of MGUS that lack evidence of IgH translocations are referred to as non–IgH-translocated MGUS. In one recent study it was shown that there is a small overlap wherein a subset of patients has both types of translocations as well as trisomies; this condition is likely present at the MGUS stage, but the sequence and prevalence are not well known (Table 104-2).³¹

Table 104-2

Revised Primary Molecular Cytogenetic Classification of Myeloma

FISH Abnormality	Gene/Chromosome(s) Affected	Percentage of Myeloma Patients
Trisomy without IgH abnormality	One or more trisomies of odd numbered chromosomes	42
IgH abnormality without trisomy		30
t(11;14)	CCND1	15
t(4;14)	FGFR3 and WHSC1	6
t(14;16)	MAF	4
t(14;20)	MAFB	<1
Unknown partner/deletion of IgH region	(CCND1 [cyclin D1]), 4p16.3 (FGFR3 and WHSC1), 6p21 (CCND3 [cyclin D3]), 16q23 (MAF), and 20q11 (MAFB)	5
IgH abnormality with trisomy*		15
t(11;14)	CCND1 (cyclin D1)	3
t(4;14)	FGFR3 and WHSC1	4
t(14;16)	MAF	1
t(6;14)	CCND3 (cyclin D3)	<1
Unknown partner/deletion of IgH region		7
Monosomy 14 in absence of IgH translocations or trisomy		4.5
Other cytogenetic abnormalities in absence of IgH translocations or trisomy or monosomy 14		5.5
Normal		3

IgH, Immunoglobulin H.

^*In addition to the effect of the translocated gene, there is the added effect of trisomies of odd-numbered chromosomes in this group.

Modified from Kumar S, Fonseca R, Ketterling RP, et al. Trisomies in multiple myeloma: impact on survival in patients with high-risk cytogenetics. Blood 2012;119:2100 American Society of Hematology.

Deletion of chromosome 13, a major prognostic factor in multiple myeloma, is seen in up to 50% of patients with MGUS by interphase fluorescent in-situ hybridization (FISH); hence, the presence of this abnormality cannot be used to differentiate MGUS from multiple myeloma.²⁹

Progression of MGUS to Malignancy

The constant rate of progression of MGUS to myeloma, macroglobulinemia, or related malignancy in a recent epidemiological study over a period spanning 30 to 35 years strongly suggests a simple, random, two-hit genetic model of malignancy.¹³ The risk of progression is similar regardless of the known duration of antecedent MGUS, suggesting that the second-hit responsible for progression is a random event, not cumulative damage.

The specific second-hit that initiates the cascade of events associated with progression is unknown. Several abnormalities have been detected with progression in both the plasma cell and its microenvironment that likely play a role in the progression of MGUS, but little is known about the sequence of events (see Fig. 104-1). RAS mutations, CDKN2A methylation, abnormalities involving the MYC family of oncogenes, secondary translocations, and TP53 mutations have all been identified in clonal plasma cells in association with progression to the symptomatic stage.²⁷ It is possible that the second-hit responsible for disease progression may be different in IgH-translocated versus non–IgH-translocated MGUS, and even within the various subtypes of IgH-translocated MGUS, depending on the partner chromosome involved. For example, amplification of chromosome 1q21 has been noted in over 40% of patients with SMM and myeloma compared with 0% in MGUS,³² suggesting that such amplification (e.g., by trisomy 1) may play a role in progression, perhaps in non–IgH-translocated MGUS.

The bone marrow microenvironment undergoes marked changes with progression, including induction of angiogenesis,³³ suppression of cell-mediated immunity,³⁴ and paracrine loops involving cytokines such as interleukin-6 and vascular endothelial growth factor (VEGF).³⁵ As in solid tumors, the transition from MGUS to multiple myeloma may involve an angiogenic switch. In solitary plasmacytoma, which can be considered to be analogous to localized stage I solid tumor, induction of angiogenesis at the time of diagnosis has been shown to be a predictor of progression to myeloma, suggesting a pathogenetic role for the process in disease progression.³⁶ Furthermore, there is a gradual increase in degree of bone marrow angiogenesis along the disease spectrum from MGUS to SMM to symptomatic myeloma.³³ In one study, approximately 60% of myeloma bone marrow plasma samples stimulated angiogenesis in an in vitro angiogenesis assay, compared with 0% of SMM and 7% of MGUS (P < .001).³⁷

Pathogenesis of Bone Lesions

The progression of MGUS to myeloma is characterized by the development of bone lesions. The pathogenetic mechanisms involved are complex and involve a combination of osteoclast activation coupled with osteoblast inhibition. Understanding these mechanisms has resulted in clinical trials with specific agents to prevent or delay the formation of bone lesions in myeloma.

There are several important mechanisms that mediate increased osteoclast activation.³⁸ There is an increase in the receptor activator of nuclear factor κB (NF-κB) ligand (RANKL) expression by osteoblasts and possibly plasma cells. This is accompanied by decreased stromal cell secretion of the RANKL decoy receptor osteoprotegerin (OPG).^39,40 In addition, the binding of OPG to RANKL is also inhibited by syndecan-1 (CD138) secreted and or shed by myeloma cells. The net result of these facts is an increase in the RANKL/OPG ratio. This causes increased osteoclast activation mediated through the NF-κB pathway. A second factor governing the activation of osteoclasts is the release of macrophage inflammatory protein (MIP)-1α and MIP-1β by myeloma cells. Both these cytokines cause osteoclast activation primarily by increasing RANKL expression in stromal cells. Finally, there is increased expression of stromal derived factor-1α (SDF-1α) by stromal cells and myeloma cells. SDF-1α causes osteoclast activation by binding to chemokine (C-X-C motif) receptor 4 (CXCR4) on osteoclast precursors. In addition to these three factors, several other cytokines such as IL-1β and IL-6 are also thought to play a role in osteoclast activation and bone resorption.

Osteoblast inhibition in myeloma is believed to be primarily related to increased dickkopf 1 (DKK1) expression by myeloma cells.⁴¹ DKK1 binds to Wnt receptors and inhibits the Wnt signaling pathway by preventing the normal binding of Wnt glycoproteins to Wnt receptors.³⁸ The inhibition of Wnt signaling prevents the intracellular stabilization of β-catenin, leading to the phosphorylation and degradation of β-catenin through the proteasome pathway. Normally, β-catenin plays an important role in osteoblast activation and its absence reduces the activity of osteoblasts. In addition to its key role in causing osteoblast inhibition, increased levels of DKK1 may also contribute in some measure to osteoclast activation. Other factors such as IL-3 and IL-7 may contribute to osteoblast inhibition. The combination of osteoclast activation and inhibition of osteoblast differentiation is believed to be the mechanism behind the development of osteolytic lesions in myeloma.

Clinical Presentation

The most common presenting symptoms of myeloma are fatigue and bone pain.¹⁰ Osteolytic bone lesions and/or compression fractures are the hallmark of the disease and can be detected on routine radiography, magnetic resonance imaging (MRI), computed tomography (CT), or combined fluorodeoxyglucose-labeled positron emission tomography (FDG-PET)/CT (Figs. 104-2 through 104-4). Bone pain may present as an area of persistent pain or be migratory, often in the lower back and pelvis. Pain may be sudden in onset when associated with a pathological fracture and is often precipitated by movement. Extramedullary expansion of bone lesions may cause nerve root or spinal cord compression. Anemia occurs in 70% of patients at diagnosis and is the primary cause of fatigue. Hypercalcemia is found in one fourth of patients, whereas the serum creatinine value is elevated in almost one half of patients.

Figure 104-2 Osteolytic lesions in the skull on plain radiographs in a patient with myeloma.

Investigation

A complete blood cell count, urinalysis, and determination of serum creatinine, calcium, β₂-microglobulin, albumin, C-reactive protein, and lactate dehydrogenase levels are needed for diagnosis, prognosis, and staging. If available, peripheral blood flow cytometry to detect and quantify circulating plasma cells should be done. In addition, patients require tests to identify and quantitate M proteins, bone disease, and bone marrow involvement. Specialized tests are also performed on the bone marrow for risk stratification.

Identification of Monoclonal Proteins

Myeloma is characterized by the presence of monoclonal immunoglobulins in the serum and/or urine. Monoclonal immunoglobulins are commonly referred to as monoclonal proteins, M proteins, or paraproteins. The presence of M proteins is indicative of a clonal plasma cell proliferative disorder such as myeloma, MGUS, Waldenström macroglobulinemia, and so on; additional tests are required to distinguish between the various plasma cell disorders.

M proteins can be detected by serum protein electrophoresis (SPEP) in 82% of patients with myeloma and by serum immunofixation (IFE) in 93% of patients.¹⁰ Up to 20% of patients with myeloma lack heavy-chain expression in the M protein and are considered to have light-chain myeloma. The M protein in these patients is always detected in the urine but can be absent in the serum even by immunofixation, making it imperative that protein electrophoresis and immunofixation are always done on both the serum and urine in all patients in whom myeloma is suspected. Addition of urine protein electrophoresis (UPEP) and urine IFE will increase the sensitivity of detecting M proteins in patients with myeloma to 97%. Most (60%) of the remaining patients who are negative for M proteins on serum and urine electrophoresis and immunofixation studies will have evidence of clonal paraproteins on the serum free light chain (FLC) assay. Currently, only 1% to 2% of patients with myeloma will have no detectable M proteins on any of these tests. These patients have true nonsecretory myeloma.

Serum Protein Electrophoresis and Immunofixation

Agarose gel SPEP and immunofixation are the preferred methods of detection of serum M proteins (Fig. 104-5). M proteins appear as a localized band on SPEP.⁴² After recognition of a localized band suggestive of an M protein on SPEP, immunofixation is necessary for confirmation and to determine the heavy- and light-chain class of the M protein. In addition, immunofixation is more sensitive than SPEP and allows detection of smaller amounts of M protein and should therefore be performed whenever myeloma, amyloidosis, macroglobulinemia, or a related disorder is suggested. The size of the M protein is measured using SPEP. For purposes of clinical trials and monitoring, the M protein is considered to be “measurable” if it is 1 g/dL or more in the serum and or greater than or equal to 200 mg/d in the urine.

Figure 104-5 A, Serum protein electrophoresis (PEL) and immunofixation (IFE) showing a normal pattern with no evidence of M protein. B, Serum protein electrophoresis (PEL) showing M protein in the γ region, which is IgG λ on immunofixation (IFE).

Quantitative Immunoglobulin Studies

Quantitation of serum immunoglobulins is performed with a rate nephelometer in patients in whom M proteins are detected. It is an added parameter that can be followed particularly in patients with myeloma and macroglobulinemia, in whom at times the M protein size estimated on SPEP may be unreliable (e.g., small β-migrating proteins and IgA/IgM proteins that tend to polymerize).

Urine Protein Electrophoresis and Immunofixation

Typically, screening for suspected monoclonal gammopathies has included UPEP and immunofixation in addition to the serum studies discussed earlier because a subset of patients with myeloma and amyloidosis may have an M protein restricted to the urine and absent on serum studies. In such patients, the diagnosis of plasma cell dyscrasia would be missed if urine studies are not performed. A 24-hr urine specimen is required for UPEP and immunofixation. Urine M protein levels are measured on UPEP and used in monitoring disease progression and response to therapy.

Serum Free Light-Chain Assay

The serum FLC assay (Freelite, The Binding Site Limited, Birmingham, UK) provides an important tool to quantify monoclonal light chains secreted by myeloma cells, especially in patients who secrete small amounts of intact monoclonal immunoglobulin.⁴³ This automated nephelometric assay allows quantitation of free kappa (κ) and lambda (λ) chains (i.e., light chains that are not bound to intact immunoglobulin) secreted by plasma cells. An abnormal κ/λ FLC ratio indicates an excess of one light chain type versus the other and is interpreted as a surrogate for clonal expansion based on extensive testing in normal volunteers and patients with myeloma, amyloidosis, and renal dysfunction.^43,44

The normal serum free κ level is 3.3 to 19.4 mg/L, and the normal free λ level is 5.7 to 26.3 mg/L.⁴⁵ The normal ratio for FLC κ/λ is 0.26 to 1.65. The normal reference range in the FLC assay reflects a higher serum level of free λ light chains than would be expected given the usual κ/λ ratio of 2 for intact immunoglobulins. This occurs because the renal excretion of free κ (which exists usually in a monomeric state) is much faster than that of free λ (which is usually in a dimeric state). ^43,44 Patients with a κ/λ FLC ratio less than 0.26 are typically defined as having monoclonal λ free light chain and those with ratios greater than 1.65 are defined as having a monoclonal κ free light chain. If the FLC ratio is greater than 1.65, κ is considered to be the “involved” FLC and λ the “uninvolved” FLC, and vice versa if the ratio is less than 0.26.

A study of 428 patients demonstrated that the urine studies can be eliminated when screening for the presence of monoclonal plasma cell disorders by using the serum FLC assay in combination with SPEP and immunofixation.⁴⁶ If a monoclonal plasma cell disorder is identified on screening, urine studies should be done to aid monitoring disease progression and response to therapy over time. Besides its role as a substitute for urine studies in the screening of plasma cell disorders, the FLC assay is used to predict prognosis in MGUS, SMM, and solitary plasmacytoma.^49–49 It is also used to monitor patients with oligosecretory or nonsecretory myeloma, primary amyloidosis, and patients with the light-chain only form of myeloma.^44,50–52 To use the FLC assay to monitor disease progression, the baseline FLC ratio must be abnormal and the involved FLC level 100 mg/L or higher.⁵²