Inherited Variation and Polymorphism in DNA

August 3, 2017 Cytogenetics, Laboratory Medicine, Molecular Biology, Pharmacogenetics No comments

The original Human Genome Project and the subsequent study of now many thousands of individuals worldwide have provided a vast amount of DNA sequence information. With this information in hand, one can begin to characterize the types and frequencies of polymorphic variation found in the human genome and to generate catalogues of human DNA sequence diversity around the globe. DNA polymorphisms can be classified according to how the DNA sequence varies between the different alleles.

Single Nucleotide Polymorphisms

The simplest and most common of all polymorphisms are single nucleotide polymorphisms (SNPs). A locus characterized by a SNP usually has only two alleles, corresponding to the two different bases occupying that particular location in the genome. As mentioned previously, SNPs are common and are observed on average once every 1000 bp in the genome. However, the distribution of SNPs is uneven around the genome; many more SNPs are found in noncoding parts of the genome, in introns and in sequences that are some distance from known genes. Nonetheless, there is still a significant number of SNPs that do occur in genes and other known functional elements in the genome. For the set of protein-coding genes, over 100,000 exonic SNPs have been documented to date. Approximately half of these do not alter the predicted amino acid sequence of the encoded protein and are thus termed synonymous, whereas the other half do alter the amino acid sequence and are said to be nonsynonymous. Other SNPs introduce or change a stop codon, and yet others alter a known splice site; such SNPs are candidates to have significant functional consequences.

The significance for health of the vast majority of SNPs is unknown and is the subject of ongoing research. The fact that SNPs are common does not mean that they are without effect on health or longevity. What it does mean is that any effect of common SNPs is likely to involve a relatively subtle altering of disease susceptibility rather than a direct cause of serious illness.

Insertion-Deletion Polymorphisms

A second class of polymorphism is the result of variations caused by insertion or deletion (in/dels or simply indels) of anywhere from a single base pair up to approximately 1000 bp, although larger indels have been documented as well. Over a million indels have been described, numbering in the hundreds of thousands in any one individual’s genome. Approximately half of all indels are referred to as “simple” because they have only two alleles – that is, the presence or absence of the inserted or deleted segment.

Microsatellite Polymorphisms

Other indels, however, are multiallelic due to variable numbers of the segment of DNA that is inserted in tandem at a particular location, thereby constituting what is referred to as a microsatellite. They consist of stretches of DNA composed of units of two, three, or four nucleotides, such as TGTGTG, CAACAACAA, or AAATAAATAAAT, repeated between one and a few dozen times at a particular site in the genome. The different alleles in a microsatellite polymorphism are the result of differing numbers of repeated nucleotide units contained within any one microsatellite and are therefore sometimes also referred to as short tandem repeat (STR) polymorphisms. A microsatellite locus often has many alleles (repeat lengths) that can be rapidly evaluated by standard laboratory procedures to distinguish different individuals and to infer familial relationships. Many tens of thousands of microsatellite polymorphic loci are known throughout the human genome. Finally, microsatellites are a particularly useful group of indels. Determining the alleles at multiple microsatellite loci is currently the method of choice for DNA fingerprinting used for identity testing.

Mobile Element Insertion Polymorphisms

Nearly half of the human genome consists of families of repetitive elements that are dispersed around the genome. Although most of the copies of these repeats are stationary, some of them are mobile and contribute to human genetic diversity through the process of retrotransposition, a process that involves transcription into an RNA, reverse transcription into a DNA sequence, and insertion into another site in the genome. Mobile element polymorphisms are found in nongenic regions of the genome, a small proportion of them are found within genes. At least 5000 of these polymorphic loci have an insertion frequency of greater than 10% in various populations.

Coyp Number Variants

Another important type of human polymorphism includes copy number variants (CNVs). CNVs are conceptually related to indels and microsatellites but consist of variation in the number of copies of larger segments of the genome, ranging in size from 1000 bp to many hundreds of kilobase pairs. Variants larger than 500 kb are found in 5% to 10% of individuals in the general population, whereas variants encompassing more than 1 Mb are found in 1% to 2%. The largest CNVs are sometimes found in regions of the genome characterized by repeated blocks of homologous sequences called segmental duplications (or segdups).

Smaller CNVs in particular may have only two alleles (i.e., the presence or absence of a segment), similar to indels in that regard. Larger CNVs tend to have multiple alleles due to the presence of different numbers of copies of a segment of DNA in tandem. In terms of genome diversity between individuals, the amount of DNA involved in CNVs vastly exceeds the amount that differs because of SNPs. The content of any two human genomes can differ by as much as 50 to 100 Mb because of copy number differences at CNV loci.

Notably, the variable segment at many CNV loci can include one to as several dozen genes, that thusCNVs are frequently implicated in traits that involve altered gene dosage. When a CNV is frequent enough to be polymorphic, it represents a background of common variation that must be understood if alterations in copy number observed in patients are to be interpreted properly. As with all DNA polymorphism, the significance of different CNV alleles in health and disease susceptibility is the subject of intensive investigation.

Inversion Polymorphisms

A final group of polymorphisms to be discussed is inversions, which differ in size from a few base pairs to large regions of the genome (up to several megabase pairs) that can be present in either of two orientations in the genomes of different individuals. Most inversions are characterized by regions of sequence homology at the edges of the inverted segment, implicating a process of homologous recombination in the origin of the inversions. In their balanced form, inversions, regardless of orientation, do not involve a gain or loss of DNA, and the inversion polymorphisms (with two alleles corresponding to the two orientations) can achieve substantial frequencies in the general population.

[Clinical Art][Physiology] Iron Physiology

November 3, 2016 Cytogenetics, Hematology, Molecular Biology, Physiology and Pathophysiology No comments , , , , , , , , , , , , , , , , , , , , , ,

screen-shot-2016-11-02-at-10-03-03-pmGlobal Iron Homeostasis

Under normal conditions, dietary iron is usually 15-25 mg daily, of which 5%-10% (1-2 mg) is absorbed through the gastrointestinal (GI) tract and the same amount lost by desquanmation of GI epithelial cells, epidermal cells of the skin, and, in menstruating women, red bood cells. The average total body content of iron in men is 35-45 mg/kg; and lower in menstruating women. Most iron (about 1800 mg) is present in hemoglobin. Men and women, respectively, have approximately 2 or 1.5 g of erythrocyte iron. Iron is stored in cells, predominantly macrophages of the spleen, bone marrow, and liver, but also in hepatocytes, as ferritin or hemosiderin (partially denatured ferritin). At steady state, the serum ferritin level is a reasonably good reflection of total body iron stores. Total storage iron is approximately 1 g in men and 600 mg in women. Additional iron is found as myoglobin in muscle and in cytochromes and other enzymes.

PS: The loss of iron

Iron is eliminated only through the loss of epithelial cells from the gastrointestinal tract, epidermal cells of the skin, and, in menstruating women, red blood cells. On the basis of long-term studies of body iron turnover, the total average daily loss of iron has been estimated at ~1 to 2 mg in normal adult men and nonmenstruating women. Although iron is a physiologic component of sweat, only a tiny amount of iron (22.5 ug/L) is lost by this route. Urinary iron excretion amounts to <0.05 mg/day and is largely accounted for by sloughed cells. Menstruating women lose an additional, highly variable amount over each menstrual cycle, from 0.006 (average) to more than 0.025 mg/kg/day.

The release of iron into the circulation is regulated by ferroportin, expressed on the basolateral GI epithelial cell surface (and on cells of the reticuloendothelial system [RES] and hepatocytes). Ferroportin is downregulated by hepcidin, and when iron is low, hepcidin is low, allowing GI iron absorption to increase and stores to be mobilized from the RES. When iron is plentiful, hepcidin levels increase and result in decreased iron absorption and RES export. Hepcidin, in turn, is downregulated by the recently described hormone erythroferrone (ERFE), produced by erythroblasts during stress erythropoiesis. Absorbed iron is transported by transferrin and taken up into cells via the transferrin receptor. Each molecule of transferrin can bind two molecules of ferric (Fe3+) iron. Transferrin-bound iron turns over as iron is used, particularly by developing red blood cells in the bone marrow. The distribution of iron is influenced by multiple factors, and under normal conditions cells maintain a pool of labile iron by controlling uptake via expression of transferrin receptors and storage via ferritin. Most cells have no mechanism for iron efflux.

Iron balance is regulated such that the amount of iron absorbed equals the amount lost. There is, however, no physiologically regulated pathway for excretion of excess iron in iron overload.

Intestinal Iron Absorption

Iron is found in food as inorganic iron and heme (iron complexed to protoporphyrin IX). The typical diet consists of 90% inorganic and 10% heme iron, though diets in the industrial world can contain up to 50% heme iron from iron-rich meats. The bioavailability of inorganic but not heme iron is influenced by multiple factors such as other dietary constituents, for example, ascorbic acid (enhanced) and phytates and polyphenols in cereals and plants (inhibited). Iron absorption is strongly inhibited by tea, and less so by coffee.

PS: Intestinal Absorption of Iron

Iron is absorbed in the duodenum, and humans and other omnivorous mammals have at least two distinct pathways for iron absorption: one for uptake of heme iron and another for ferrous (Fe2+) iron. Heme iron is derived from hemoglobin, myoglobin, and other heme proteins in foods of animal origin, representing approximately 10% to 15% iron content in the typical Western diet, although heme-derived iron accounts for 2/3 of absorbed iron in meat-eating humans. Exposure to acid and proteases present in gastric juices frees the heme from its apoprotein. Heme is taken up by mucosal cells, but the specific receptor is still unkonwn. Once heme iron has entered the cell, the porphyrin ring is enzymatically cleaved by heme oxygenase. The liberated iron then probably follows the same pathways as those used by nonheme iron. A small proportion of the heme iron may pass into the plasma intact via heme exporter protein FLVCR (feline leukemia virus, subgroup C receptor), which transfers heme onto a heme-binding protein, hemopexin. Absorption of heme iron is relatively unafected by the overall composition of the diet.

Dietary nonheme iron is largely in the form of ferric hydroxide or loosely bound to organic molecules such as phytates, oxalate, sugars, citrate, lactate, and amino acids. Low gastric pH is thought be important for the solubility of inorganic iron. Dietary constituents may also have profound effects on the absorption of nonheme iron, making the bioavailability of food iron highly variable. Ascorbate, animal tissues, keto sugars, organic acids, and amino acids enhance inorganic iron absorption, whereas phytates, polyphenols, and calcium inhibit it. Depending on various combinations of enhancing and inhibitory factors, dietary iron assimilation can vary as much as tenfold.

The rate of iron absorption is influenced by several factors, including body iron stores, the degree of erythropoietic activity, blood hemoglobin and oxygen content, and the presence of inflammation. Iron absorption increases when stores are low or when increased erythropoietic activity is required, such as during anemia or hypoxemia. Conversely, the physiologically appropriate response to iron overload is downregulation of intestinal iron absorption; this downregulation fails in patients with hereditary hemochromatosis or chronic iron-loading anemias.

Iron is absorbed in the intestine via two pathways: one for inorganic iron and the other for heme-bound iron. Little is known about heme iron absorption. Nonheme iron in the diet is largely in the form of ferric-oxyhydroxides (Fe3+, ie, rust), but the intestinal epithelial cell apical iron importer, divalent metal transporter 1 (DMT1 or SLC11A2), transports only ferrous iron (Fe2+). DMT1 is a protein with 12 predicted transmembrane segments, which is expressed on the apical surface of absorptive enterocytes. Iron must therefore be reduced to be absorbed, and this is facilitated by duodenal cytochrome B (Dcytb), a heme-dependent ferrireductase. However, since knockout mice appear to have normal metabolism, Dcytb may not be the only ferrireductase enzyme involved in absorption of nonheme ironOnce transported across the apical border of the enterocyte, iron may be stored within the cell. For this purpose, iron is oxidized to Fe3+ by the H-subunit of ferritin and stored in this form. Eventually, the cell senesces and sloughs off into the feces, and stored iron is lost to the system. Alternatively, iron may be transported across the basolateral membrane into the portal circulation via ferroportin. Ferroportin 1 (FPN1) is the only known iron exporter in mammals and, like DMT1, transports only ferrous iron. FPN1 is a multi-transmembrane segment protein expressed on the basolateral surface of enterocytes. Aslo they are expressed in other tissues involved in handling large iron fluxes including macrophages, hepatocytes, and placental trophoblast. Similar to apical iron uptake, basolateral iron efflux is aided by an enzyme that changes the oxidation state of iron. Once reduced, ferrous iron is transported across the basolateral membrane by ferroportin, then oxidized to ferric iron by hephaestin. Intestinal iron absorption is regulated by hepcidin, which binds to ferroportin, inducing its internalization and degradation.

Cellular Iron Uptake, Storage, and Recycling

Each molecule of transferrin binds two ferric (Fe3+) iron atoms. Diferric transferrin (holotransferrin) binds to the transferrin receptor (TfR1) on target cells and enters by receptor-mediated endocytosis; it is then released from the TfR1 by acidification and transported into the cytoplasm by DMT1. The TfR is recycled to the cell surface.

Most iron in erythroid cells binds protoporphyrin to form heme, which complexes with globin proteins, forming hemoglobin. Erythrocytes survive in the circulation for approximately 120 days, after which aging red blood cells are phagocytized by macrophages of the RES. Hemoglobin is catabolized and iron released to transferrin via ferroportin, or stored within the RES as ferritin or hemosiderin.

PS: Reticuloendothelial System/RES



The main form of cellular iron storage is ferritin, a complex of subunits that binds iron and renders it insoluble and redox inactive. Circulating ferritin is present in a different subunit form than cellular storage ferritin. The function of circulating ferritin is incompletely understood.

Regulation of Iron Physiology

Because the total body iron content is largely determined by the efficiency of absorption of iron, the regulation of absorption has been of great interest for many years.

Hepcidin is a 25-amino-acid peptide produced in the liver and is the major regulator of iron absorption and storage. Hepcidin regulates cellular iron egress by binding to ferroportin, leading to its internalization and degradation. In this way, elevated levels of hepcidin inhibit iron absorption from the GI tract and promote storage (inhibit release) of iron within hepatocytes and macrophages. Hepcidin production is induced by interleukin (IL)-6 and IL-1 via the JAK/STAT pathway, and can be increased more than 100-fold in inflammatory states. The dysregulation of iron balance seen in the anemia of inflammation (anemia of chronic disease) can be attributed to an inappropriate increase in hepcidin levels, which leads to decreased circulating iron. Hepcidin levels are downregulated by anemia and iron deficiency. Hepcidin agonists and antagonists are under clinical development for the treatment of disorders of inappropriately low or high hepcidin levels, respectively.

Hepcidin, in turn, is negatively regulated by the recently described hormone ERFE. ERFE is produced by erythroblasts during stress erythropoiesis, feeding back on hepcidin and reducing its levels, thus allowing iron absorption and export via ferroportin to proceed.

The Molecular Mechanism for Hepcidin Regualtion

Systematic Mechanism

The two transferrin receptors TfR1 and TfR2, and HFE, an MHC class I-like membrane protein, may serve as holotransferrin sensors. HFE can interact with both transferrin receptors, but this interaction is modulated by holotransferrin concentrations. Because HFE and holotransferrin binding sites on TfR1 overlap, increasing concentrations of holotransferrin result in displacement of HFE from TfR1, and free HFE then interacts with TfR2. TfR2 protein is further stabilized by binding of holotransferrin. The holotransferrin/HFE/TfR2 complex then stimulates hepcidin expression through an incompletely understood pathway, possibly by potentiating BMP pathway signaling. HFE, however, may also regulate hepcidin expression without complexing with TfR2. The role of HFE or TfR2 in hepcidin regulation by iron is supported by genetic evidence: HFE and TfR2 mutations in humans or mice cause hepcidin deficiency and an adult form of hemochromatosis.

screen-shot-2016-11-03-at-3-19-33-pmThe BMP pathway with its canonical signaling via Smad proteins has a central role in the regulation of hepcidin transcription. BMP receptors are tetramers of serine/threonine kinase receptors, with two type I and two type II subunits. Recent data indicate that type I subunits Alk2 and Alk3 and type II subunit ActRIIA and BMPRII are specific BMP receptors involved in iron regulation. In the liver, BMP pathway signaling to hepcidin is modulated by a coreceptor hemojuvelin and, at least in mice, by the ligand BMP6. Loss of hemojuvelin or BMP6 in mice decrease hepcidin expression and impairs hepcidin response to acute or chronic iron loading. In humans, hemojuvelin mutations result in severe hepcidin deficiency and cause juvenile hemochromatosis. It remains to be clarified how BMP receptors and hemojuvelin interact with iron-sensing molecules that regulate hepcidin expression.

Local Mechanism (intestine)

In addtition to the regulaltion by systemic signals, iron absorption is subject to local regualtion by intracellular mechanisms in duodenal enterocytes. At least two mechanisms have been described: one related to the enterocyte iron levels and the other to the hypoxia pathway.

Regulation of the synthesis of multiple proteins involved in iron physiology, including TfR1, DMT1, FPN1, and ferritin, is controlled at a posttranscriptional level by influencing mRNA stability and translation. The mRNA of these proteins contain iron response elements (IREs), conserved nucleotide sequences with a stem-loop structure that binds iron regulatory proteins (IRPs)-1 and -2. The mRNAs for ferritin, DMT1, and FPN1 have IREs in the 5' untranslated region (UTR), and the mRNA for the TfR has multiple IREs in the 3' UTR. In low-iron states, IRP-1 is in a conformation that allows it to bind to IREs; for example, it binds the 3' IRE of the TfR mRNA, stabilizing it and allowing transcription of more TfR protein, and to the 5' UTR of ferritin mRNA, decreasing translation of ferritin for iron storage. Intracellular iron induces ubiquitination and degradation of IRP-2. Iron deficiency by this mechanism upregulates IRP activity via increased IRE binding, resulting in increased cellular iron uptake and decreased iron storage.

Dcytb mRNA does not contain an IRE but is strongly upregulated in iron-deficient duodenum, indicating additional regulation of transcription of iron-related genes. Hypoxia-inducible factor (HIF) transcriptin factors may serve as important local regulators of intestinal iron absorption. Iron deficiency induced HIF signaling in duodenum of mice, and caused increased Dcytb and DMT1 expression, and an increase in iron uptake. Accordingly, targeted deletion of Hif-2alpha in the intestine resulted in dramatic decrease in the expressions of DMT1-IRE and Dcytb mRNA. In vitro studies further demonstrated that HIF-2alpha directly binds to the promoters of DMT1 and Dcytb, activating their transcription.

Summary: Factors That Affecting Hepcidin Levels

Systemic mechanisms: 1) by transferrin levels, including holotransferrin/HFE/TfR2 complex + hemojuvelin (coreceptor) + BMP6 (ligand), which increases hepcidin gene expression; 2) by erythropoiesis, however, the mechanisms by which erythropoiesis regulates hepcidin production are not well understood, but it is thought that erythroid precursors in the bone marrow secrete a factor which exerts its effect on hepatocytes and causes hepcidin supression (so the ERFE previously mentioned?); and 3) by cytokines in inflammation state, via STAT-3 pathway (already described above).

Local mechanisms: 1) posttranscriptional level by influencing mRNA stability and tanslation, via IRPs; 2) HIF's impact on cis-acting regulatory elements of DMT1 and Dcytb genes.

Iron Cycle

screen-shot-2016-11-03-at-5-12-27-pmMost functional iron in the body is not derived from daily intestinal absorption (1 to 2 mg/day) but rather from recycling of iron (20 to 25 mg/day) from senescent erythrocytes and other cells. The most important source and destination of recycled iron is the erythron. At the end of a 4-month lifespan, effete erythrocytes are engulfed by reticuloendothelial macrophages, which lyse the cells and degrade hemoglobin to liberate their iron. This process is poorly understood, but it appears to involve the action of heme oxygenase for enzymatic degradation of heme. Some of this iron may remain stored in macrophages as ferritin or hemosiderin, but most is delivered to the plasma, and the rate of iron export is determined by the hepcidin-ferroportin interaction. In plasma, iron becomes bound to transferrin, completing the cycle. A small amount of iron, probably <2 mg, leaves the plasma each day to enter hepatic parenchymal cells and other tissue. Here, the iron is stored or used for synthesis of cellular heme proteins, such as myoglobin and the cytochromes.

Macrophage Iron Recycling

Although there are many types of tissue macrophages, those that participate in the catabolism of red blood cells can be subdivided into two categories. One type, exemplified by pulmonary alveolar macrophages, is able to phagocytose erythrocytes or other cells and convert the iron they contain into storage forms, but lacks the ability to return the iron to the circulation. This type of macrophage appears to retain the iron throughout its life span. The second type of macrophage, comprising the reticuloendothelial system, acquires iron in a similar fashion but is able to return it to the plasma. The latter macrophages, found espeically in the sinuses of the spleen and the liver, play a primary role in the normal reutilization of iron from destroyed red cells, allowing completion of the iron cycle shown in Figure 23.4.

Plasma Transport

The plasma iron-binding protein, transferrin, is a glycoprotein with a molecular weight of approximately 80 kDa. Transferrin is synthesized chiefly in the liver and actively secreted by hepatocytes, but lesser amounts are made in other tissues, including the central nervous system, the ovary, the testis, and helper T lymphocytes (CD4+ subset). The rate of synthesis shows an inverse relationship to iron in stores; when iron stores are depleted, more transferrin is synthesized, and when iron stores are overfilled, the level of transferrin decreases. Transferrin keeps iron nonreactive in the circulation and extravascular fluid, delivering it to cells bearing transferrin receptors.

Transferrin can be measured directly using immunologic techniques; and normal concentration in the plasma is approximately 2 to 3 g/L. Alternatively, transferrin is quantified in terms of the amount of iron it will bind, a measure called the total iron-binding capacity (TIBC: normal values for plasma iron and TIBC are given in Appendix A). In the average subject, the plasma iron concentration is 100 ug/dL, and the TIBC is 300 ug/dL. Thus, only about one third of the available transferrin binding sites are occupied, leaving a large capacity to deal with excess iron. Plasma iron concentration varies over the course of the day, with the highest values in the morning and the lowest in the evening. Levels of serum transferrin are more constant, and there is no apparent diurnal variation in TIBC. General practice has been to evaluate transferrin saturation using a first morning, fasting sample to standardize the results, but this may not be helpful.

Transferrin has two homologous iron-binding domains, each of which binds an atom of trivalent (ferric) iron. The iron atoms are incorporated one at a time and appear to bind randomly at either or both of the two sites. When binding is complete, the iron lies in a pocket formed by two polypeptide loops. One mole of anion, usually carbonate or bicarbonate, is taken up, and 3 moles of hydrogen ion are released from each mole of iron bouond. There are functional differences between teh two iron-binding sites, but it is not clear that these have physiologic importance.

Under physiologic circumstances, ferric iron binds to transferrin with very high affinity, with an affinity constant of ~1-6 x 1022 M-1. The affinity of iron-transferrin interaction is pH-dependent, decreasing as pH is lowered. Other transition metals, such as copper, chromium, manganese, gallium, aluminum, indium, and cobalt, can be bound by transferrin but with less affinity than iron.

Iron Delivery to Erythroid Precursors

The biologic importance of transferrin in erythropoiesis is illustrated by abnormalities observed in patients and mice with congenital atransferrinemia. When transferrin is severely deficient, red cells display the morphologic stigmata of iron deficiency. This occurs despite the fact that intestinal iron absorption is markedly increased in response to a perceived need for iron for erythropoiesis. Nonhematopoietic tissues avidly assimilate the non-transferrin-bound metal. Similarly, mutant mice lacking tissue receptors for transferrin die during embryonic development from severe anemia, apparently resulting from ineffective iron delivery to erythroid precursor cells.

Transferrin delivers its iron to developing normoblasts and other cells by binding to specific cell-surface receptors. The transferrin receptor (TfR1) is a disulfide-linked homodimer of a glycoprotein with a single membrane-spanning segment and a short cytoplasmic segment. It is a type II membrane protein, with its N terminus located within the cell. The native molecular weight of TfR1 is ~180 kDa. Each TfR1 homodimer can bind two tranferrin molecules. Diferric transferrin is bound with higher affinity than monoferric transferrin. As a result, diferric transferrin has a competitive advantage in delivering iron to the erythroid precursors. Apotransferrin has little affinity for the receptor at physiologic pH but considerable affinity at lower pH, an important factor in intracellular iron release.

TfR1 numbers are modulated during erythroid cell maturation, reaching their peak in intermediate normoblasts. Very few TfR1 molecules are found on burst-forming-unit erythroid cells, and only slightly greater numbers are found on colony-forming-unit erythroid cells. However, by the early normoblast stage, approximately 300,000 receptors are found on each cell, increasing to 800,000 at the intermediate stages. The rate of iron uptake is directly related to the number of receptors. The number decreases as reticulocytes mature, and late in maturation, erythroid cells shed all remaining receptors by exocytosis and by proteolytic cleavage. The shed receptors, referred to as soluble transferrin receptors (sTfR) is a sensitive indicator of erythroid mass and tissue iron deficiency.

After ligand and receptor interact, iron-loaded transferrin undergoes receptor-mediated endocytosis. Specialized endocytic vesicles form, which are acidified to a pH of 5 to 6 by the influx of protons. The low pH facilitates release of iron from transferrin and strengthens the apotransferrin-receptor interaction. Released iron is reduced by an endosomal ferrireductase, STEAP3, and transferred to the cytosol by DMT1. Because DMT1 must cotransport protons with iron atoms, vesicle acidification is also important for the function of this transporter. After the iron enters the cytosol, the protein components of the endosome return to the membrane surface, where neutral pH promotes the release of apotransferrin to the plasma.

After Entering into Erythroid Precursor Cells

In the normal subject, ~80% to 90% of the iron that enters erythoid precursor cells is ultimately taken up by mitochondria and incorporated into heme. Most of the remainder is stored in ferritin. Granules of ferritin may sometimes be detected using the Prussian blue reaction. Normoblasts with Prussian blue-positive (siderotic) granules are called sideroblasts, and, if the granules persist after enucleation, the mature cells are called siderocytes. In normal individuals, approximately half of the normoblasts are sideroblasts, each containing less than five small granules.

It is intriguing that even though erythroid precursors require large amounts of iron and heme for hemoglobin synthesis, they have been found to express the heme exporter FLVCR and the iron exporter ferroportin. Studies suggest that FLVCR is necessary for survival of erythroid precursors. Because heme is toxic to cells at high concentrations, it is thought that FLVCR functions as a safety valve to prevent accumulation of excess heme early during erythroid differentiation.

Erythorid precursors also express ferroportin but its role in erythroid maturation is yet unclear. It is interesting that erythoid cells preferentially express non-IRE ferroportin transcript during the stages of rapid iron uptake, thus avoiding any iron-induced increase in ferroportin translation dependent on the IRE/IRP system.

The Etiology of Primary Dyslipidemia

May 12, 2014 Cardiology, Cytogenetics, Physiology and Pathophysiology No comments , ,

Generally primary dyslipidemia are due to mutations of various genes. Thus these disorders of lipid are inheritable and have a obvious family history, which is important in dignosing the primary dyslipidemia. Primary disorders of lipid consist of disorders of elevated apoB-containing lipoproteins, disorders of low apoB-containing lipoproteins level, disorders of low HDL-C level, and disorders of high HDL-C level.

Primary Disorders of Elevated ApoB-Containing Lipoproteins

ApoB-containing lipoproteins include chylomicrons (apoB-48), VLDL (apoB-100), IDL (apoB-100), and LDL (apoB-100). To understand dyslipidemia, we here list the component of each lipoprotein.

Chylomicron consists of 3% of cholesterol, 90% of triglyceride, 6% of phospholipid, and 1% of protein.

VLDL consists of 22% of cholesterol, 55% of triglyceride, 15% of phospholipid, and 8% of protein.

IDL consists of roughly similar amounts of cholesterol and triglyceride.

LDL consists of 50% of cholesterol, 5% of triglyceride, 25% of phospholipid, and 20% of protein.

HDL consists of 20% of cholesterol, 5% of triglyceride, 25% of phospholipid, and 50% of protein.

Lipid disorders associated with elevated LDL-C and normal triglycerides

1. Familial Hypercholesterolemia (FH). FH is an autosomal codominant disorder characterized by elevated plasma levels of LDL-C and normal triglycerides, tendon xanthomas, and premature coronary atherosclerosis.

FH is caused by a large number (>1000) mutations in the LDL receptor gene, which can be divided into homozygous FH and heterozygous FH. The estimated incidence of homozygous FH is 1/1,000,000, and heterozygous FH of 1/500. The elevated levels of LDL-C in FH are due to an increase in the production of LDL from IDL (since a portion of IDL is normally cleared by LDLR endocytosis on the liver) and a delayed removal of LDL from the blood also by LDLR on the liver.

Fredrickson Classification of HyperlipoproteinemiasPS: 40%-60% of IDL is removed by the liver via LDLR with cofactor ApoE. Approximately 70% of circulating LDL is cleared by LDLR in the liver (also with cofactor ApoE). For more information about the metabolism of lipoproteins please refer

Individuals with two mutated LDL receptor alleles (homozygous) have much higher LDL-C levels than those with one mutant allele (heterozygous). Among homozygous FH individuals, patients can be classified into one of two groups based on the amount of LDL receptor activity measured in their skin fibroblasts: those patients with <2% of normal LDL receptor activity are called receptor negative and those patients with 2-25% normal LDL receptor activity are called receptor defective. In patients with homozygous FH total cholesterol levels are usually >500 mg/dL and can be higher than 1000 mg/dL.

Heterozygous FH is one of the most common single-gene disorders (occuring in approximately 1 in 500). The elevated level of LDL-C usually is 200-400 mg/dL and the triglyceride is normal.

2. Familial defective ApoB-100 (FDB)

FDB is a dominantly inherited disorder that clinically resembles heterozygous FH. This disease is rare in most populations except individuals of German descent, where the frequency can be as high as 1 in 1000. FDB is also characterized by elevated plasma LDL-C levels with normal triglycerides, tendon xanthomas, and an increased incidence of premature ASCVD (atherosclerotic cardiovascular disease).

FDB is caused by mutations in the LDL receptor-binding domain of apoB-100, most commonly due to a substitution of glutamine for arginine at position 3500 (Arg3500Glu). As a consequence of the mutation in apoB-100, LDL binds the LDL receptor with reduced affinity, and LDL is removed from the circulation at a reduced rate (both IDL and LDL, similar like FH).

Clinically, patients with FDB tend to have lower plasma levels of LDL-C than FH heterozygotes.

3. Autosomal dominant hypercholesterolemia due to mutations in PCSK9 (ADH-PCSK9 or ADH3)

ADH3 is a rare autosomal dominant disorder caused by gain-of-function mutations in proprotein converstase subtilisin/kexin type 9 (PCSK9). PCSK9 is a secreted protein that binds to the LDL receptor, resulting in its degradation. Normally, after LDL binds to the LDL receptor it is internalized along with the receptor. In the low pH of the endosome, LDL dissociates from the receptor and the receptor returns to the cell surface. The LDL is delivered to the lysosome. When PCSK9 binds the receptor, the complex is internalized and the receptor is redirected to the lysosome rather than to the cell surface.

The mutations causing enhanced activity of PCSK9 results in reduced number of hepatic LDL receptors and the hypercholesterolemia. Loss-of-function mutations in PCSK9 cause low LDL-C levels.

4. Autosomal recessive hypercholesterolemia (ARH)

ARH is a rare disorder due to mutations in a protein (LDLRAP) involved in LDL receptor-mediated endocytosis in the liver, which is characterized by hypercholesterolemia, tendon xanthomas, and premature coronary artery disease. In the absence of LDLRAP, LDL binds to the LDL receptor but the lipoprotein-receptor complex fails to be internalized.

The levels of plasma LDL-C tend to be intermediate between the levels present in FH homozygotes and FH heterozygotes. LDL receptor function in cultured fibroblasts is normal or only modestly reduced in ARH, whereas LDL receptor function in lymphocytes and the liver is negligible.

5. Sitosterolemia

Sitosterolemia is another rare autosomal recesive disease that can result in severe hypercholesterolemia, tendon xanthomas, and premature ASCVD. Misshapen red blood cells and megathrombocytes are visible on blood smear. Episodes of hemolysis are a distinctive clinical feature of this disease compared to other genetic forms of hypercholesterolemia.

Sitosterolemia is caused by mutations in either of two members of the ATP-binding cassette (ABC) half transporter family, ABCG5 and ABCG8. These genes are expressed in enterocytes and hepatocytes. The proteins heterodimerize to form a functional complex that pumps plant sterols such as sitosterol and campesterol, and animal sterols, predominantly cholesterol, into the gut lumen and into the bile. In normal individuals, <5% of dietary plant sterols are absorbed by the proximal small intestine and delivered to the liver. Absorbed plant sterols are preferentially secreted into the bile and are maintained at very low levels.

In sitosterolemia, the intestinal absorption of sterols is increased and biliary excretion of the sterols is reduced, resulting in increased plasma and tissue levels of both plant sterols and cholesterol. Incorporation of plant sterols into cell membranes results in misshapen red blood cells and megathrombocytes that are visible on blood semear.

6. Polygenic hypercholesterolemia

This condition is characterized by hypercholesterolemia due to elevated LDL-C with a normal plasma level of triglyceride in the absence of secondary causes of hypercholesterolemia.

Plasma LDL-C levels are generally not as elevated as they are in FH and FDB.

7. Elevated plasma levels of lipoprotein(a)

Unlike the other major classes of lipoproteins, that have a normal distribution in the population, plasma levels of Lp(a) have a highly skewed distribution with levels varying over 1000-fold range. Levels are strongly influenced by genetic factors, with individuals of African and South Asian descent having higher levels than those of European descent.

Lipid Disorders Associated with Elevated Triglycerides

1. Familial chylomicronemia syndrome

Genetic deficiency or inactivity of either LPL or ApoC-II (cofactor of LPL) results in impaired lipolysis and profound elevations in plasma chylomicrons. These patients can also have elevated plasma levels of VLDL, but chylomicronemia predominates. The fasting plasma is turbid, and if left at 4℃ for a few hours, the chylomicrons float to the top and form a creamy supernatant. In these disorders, fasting triglyceride levels are almost invariably>1000 mg/dL. Fasting cholesterol levels are also elevated but to a lesser degree.

LPL deficiency has autosomal recessive inheritance and has a frequency of approximately 1 in 1 million in the population. ApoC-II deficiency is also recessive in inheritance pattern and is even less common than LPL deficiency. Multiple different mutations in the LPL and apo-C-II genes cause these diseases. Obligate LPL heterozygotes have normal or mild-to-moderate elevations in plasma triglyceride levels, whereas individuals heterozygous for mutation in apoC-II do not have hypertriglyceridemia.

For unknown reasons, some patients with persistent and pronounced chylomicronemia never develop pancreatitis, eruptive xanthomas, or hepatosplenomegaly.

2. ApoA-V deficiency

ApoA-V circulates at much lower concentrations than the other major apolipoproteins. Individuals harboring mutations in both ApoA-V alleles can present as adults with chylomicronemia. The exact mechanism of action of ApoA-V is not known, but it appears to be required for the association of VLDL and chylomicrons with LPL.

3. GPIHBP1 deficiency

After LPL is synthesized in adipocytes, myocytes or other cells, it is transported across the vascular endothelium and is attached to a protein on the endothelial surface of capillaries called GPIHBP1. Homozygosity for mutations that interfere with GPIHBP1 synthesis or folding  cause severe hypertriglyceridemia

The frequency of chylomicronemia due to mutations in GPIHBP1 has not been established but appears to be very rare.

4. Hepatic lipase deficiency

HL is a member of the same gene family as LPL and hydrolyzes triglycerides and phospholipids in remnant lipoproteins and HDLs. HL deficiency is a very rare autosomal recessive disorder characterized by elevated plasma levels of cholesterol and triglycerides (mixed hyperlipidemia) due to the accumulation of circulating lipoprotein remnants (both IDL & chylomicrons remnants;supporting reference: Removal of chylomicron remnants in transgenic mice overexpressing normal and membrane-anchored hepatic lipase at and either a normal or elevated plasma level of HDL-C (HL hydrolyze TG and phopholipids of large HDL).

PS: HL hydrolyzes both TG and PL principally on remnant lipoproteins and HDL, and may facilitate the uptake of apoB-containing lipoproteins through interaction with HSPG (heparan sulfate proteoglycan).

5. Familial dysbetalipoproteinemia (FDBL)

Familial dysbetalipoproteinemia is characterized by a mixed hyperlipidemia due to the accumulation of remnant lipoprotein particles. ApoE is present in multiple copies on chylomicron and VLDL remnants and mediates their removal via hepatic lipoprotein receptors. The APOE gene ispolymorphic in sequence, resulting in the expression of the three common isoforms: apoE3, which is the most common; and apoE2 and apoE4, which both differ from apoE3 by a single amino acid.

ApoE4 is not associated with FDBL. ApoE2 has a lower affinity for the LDL receptor; therefore, chylomicron and VLDL remnants containing apoE2 are removed from plasma at a slower rate. Individuals who are homozygous for the E2 allele comprise the most common subset of patients with FDBL. Approximately 0.5% of the general population are apoE2/E2 homozygotes, but only a small minority of these individuals develop FDBL. In most cases, an additional, identifiable factor precipitates the development of hyperlipoproteinemia. The most common precipitating factors are high-fat diet, diabetes mellitus, obesity, hypothyroidism, renal disease, HIV infection, estrogen deficiency, alcohol use, or certain drugs.

Other mutations in apoE can cause a dominant form of FDBL where the hyperlipidemia is fully manifest in the heterozygous state, but these mutations are rare.

6. Familial hypertriglyceridemia (FHTG)

FHTG is a relatively common autosomal dominant diorder of unknown etiology (~1 in 500). FHTG is characterized by moderately elevated plasma triglycerides accompanied by more modest elevations in cholesterol. Some patients with FHTG have a more severe form of hyperlipidemia in which both VLDLs and chylomicrons are elevated (Type V hyperlipidemia), since VLDL and chylomicron compete for the same lipolytic pathway.

The major class of lipoproteins elevated in this disorders is VLDL, thus, patients with this disorder are often referred to as as having Type IV hyperlipoproteinemia. The elevated plasma levles of VLDL are due to increased production of VLDL, impaired catabolism of VLDL, or a combination of these mechanisms.

Increased intake of simple carbohydrates, obesity, insulin resistance, alcohol use, and estrogen treatment, all of which increase VLDL synthesis, can exacerbate this syndrome.

For unknown reasons, some patients with persistent and pronounced chylomicronemia never develop pancreatitis, eruptive xanthomas, or hepatosplenomegaly

FHTG appears not to be associated with increased risk of ASCVD in many families.

7. Familial combined hyperlipidemia (FCHL)

FCHL is generally characterized by moderate elevations in plasma levels of triglycerides (VLDL) and cholesterol (LDL) and reduced plasma levels of HDL-C. However, the disease typically has one of three possible phenotypes: (1). elevated plasma levels of LDL-C; (2). elevated plasma levels of triglycerides due to elevation in VLDL; or (3). elevated plasma levels of both LDL-C and triglyceride. And the lipoprotein profile can switch among these three phenotypes in the same individual over time and may depend on factors such as diet, exercise, and weight, etc.

Til today, the molecular etiology of FCHL remains poorly understood, and it is likely that defects in several different genes can cause the phenotype of FCHL.

Inherited Causes of Low Levels of HDL-C

1. Gene deletion in the ApoAV-AI-CIII-AIV locus and coding mutations in ApoA-I

Complete genetic deficiency of apoA-I due to deletion of the apoA-I gene result in the virtual absence of HDL from the plasma.

The genes encoding apoA-I, apoC-III, apoA-IV, and apoA-V are clustered together on chromsome 11, and some patients with no apoA-I have genomic deletions that include other genes in the cluster.

Missense and nonsense mutations in the apoA-I gene have been identified in some patients with low plasma level of HDL-C. ApoA-I is required for LCAT activity. With low levels and/or activity of mutant ApoA-I, LCAT activation is impaired, which precludes the normal esterifying of free cholesterol in the HDL and as a result the HDL is rapidly catabolismed from circulation.

2. Tangier disease (ABCA1 deficiency)

Tangier disease is a very rare autosomal codominant form of extremely low plasma HDL-C caused by mutations in the gene encoding ABCA1, a cellular transporter that facilitates efflux of unesterified cholesterol and phospholipids from cells to apoA-I, which form nascent HDL. Without ABCA1, the apoA-I secreted from the liver and intestine are poorly lipidated and as a result these apoA-I is immediately cleared from the circulation.

3. LCAT deficiency

LCAT deficiency is a very rare autosomal recessive disorder which is caused by mutations in LCAT, an enzyme synthesized in the liver and secreted into the plasma. In LCAT deficiency, the proportion of free cholesterol in circulating lipoproteins is greatly increased. Lack of normal cholesterol esterification impairs formation of mature HDL particles, resulting in the rapid catabolism of circulating apoA-I. Two genetic forms of LCAT deficiency have been described in humans: 1. complete deficiency or so-called classic LCAT deficiency, and 2. partial deficiency or fish-eye disease.

4. Primary hypoalphalipoproteinemia

Primary hypoalphalipoproteinemia is defined as a plasma HDL-C level below the tenth percentile in the setting of relatively normal cholesterol and triglyceride levels, with no apparent secondary causes of low plasma HDL-C and no clinical signs of LCAT deficiency or Tangier disease.

The metabolic etiology of this disease appears to be primarily accelerated catabolism of HDL and its apolipoproteins.

The Management of Multiple Myeloma in Younger Patients

September 19, 2013 Chemotherapy, Cytogenetics, Hematology, Therapeutics, Transplantation No comments , , , , , , , , , ,

Therapy for multiple myeloma (MM) has advanced with gratifying speed over the past 5 to 7 years and with this progress, a degree of uncertainty has arisen about optimal approaches to therapy, particularly in the newly diagnosed patients. Indeed, using mordern therapeutic strategies, living with MM for a decade or longer has now become a reality for a significant proportion of patients.


MM is characterized by neoplastic proliferation of plasma cells involving more than 10% of the bone marrow. Increasing evidence suggests that the bone marrow microenvironment of tumor cells plays a pivotal role in the pathogenesis of myelomas.

The malignant cells of MM, plasma cells, and plasmacytoid lymphocytes are the most mature cells of B-lymphocytes. B-cell maturation is associated with a programmed rearrangement of DNA sequences in the process of encoding the structure of mature immunoglobulins. It is characterized by overproduction of monoclonal immunoglobulin G (IgG), immunoglobulin A (IgA), and/or light chains, which may be identified with serum protein electrophoresis (SPEP) or urine protein electrophoresis (UPEP).

The role of cytokines in the pathogenesis of MM is an important area of research. Interleukin (IL)–6 is also an important factor promoting the in vitro growth of myeloma cells. Other cytokines are tumor necrosis factor and IL-1b.

The pathophysiologic basis for the clinical sequelae of MM involves the skeletal, hematologic, renal, and nervous systems, as well as general processes.

Development Progresses

Skeletal processes

Plasma-cell proliferation causes extensive skeletal destruction with osteolytic lesions, anemia, and hypercalcemia. Mechanisms for hypercalcemia include bony involvement and, possibly, humoral mechanisms. Isolated plasmacytomas (which affect 2-10% of patients) lead to hypercalcemia through production of the osteoclast-activating factor.

Destruction of bone and its replacement by tumor may lead to pain, spinal cord compression, and pathologic fracture. The mechanism of spinal cord compression symptoms may be the development of an epidural mass with compression, a compression fracture of a vertebral body destroyed by multiple myeloma, or, rarely, an extradural mass. With pathologic fracture, bony involvement is typically lytic in nature.

Hematologic processes

Bone marrow infiltration by plasma cells results in neutropeniaanemia, andthrombocytopenia. In terms of bleeding, M components may interact specifically with clotting factors, leading to defective aggregation.

Renal processes

The most common mechanisms of renal injury in MM are direct tubular injury, amyloidosis, or involvement by plasmacytoma.[14, 15] Renal conditions that may be observed include hypercalcemic nephropathy, hyperuricemia due to renal infiltration of plasma cells resulting in myeloma, light-chain nephropathy,amyloidosis, and glomerulosclerosis.

Neurologic processes

The nervous system may be involved as a result of radiculopathy and/or cord compression due to nerve compression and skeletal destruction (amyloid infiltration of nerves).

General processes

General pathophysiologic processes include hyperviscosity syndrome. This syndrome is infrequent in MM and occurs with IgG1, IgG3, or IgA. MM may involve sludging in the capillaries, which results in purpura, retinal hemorrhage, papilledema, coronary ischemia, or central nervous system (CNS) symptoms (eg, confusion, vertigo, seizure). Cryoglobulinemia causes Raynaud phenomenon, thrombosis, and gangrene in the extremities.


Some tests can afford important prognostic information and the subtypes of myeloma. These tests include classic CRAB measurements (calcium level, renal function, amemia, bone damage) , β2-microglobulin, albumin, lactate dehydrogenase (LDH), serum and urine monoclonal protein (24-hour) such as serum protein electrophoresis (SPEP), serum immunofixation electrophoresis (SIFE), 24 h urine protein electrophoresis (UPEP), urine immunofixation electrophoresis (UIFE), and so on, serum-free light chain assay.

Bone marrow examinations such as morphology, FISH (fluorescent in situ hybridization) analysis of key genetic events, metaphase cytogenetics are also mandatory at present.

Table 1. Genetic Tests to Be Performed in Myeloma Patients at Diagnosis.

With these tests, multiple myeloma can be divided into three subtypes, which are solitary plasmacytoma, smoldering myeloma (asymptomatic myeloma) and active myeloma (symptomatic myeloma).

Subtypes of Multiple Myeloma

According to the latest NCCN guideline MM can be categorized into three subgroups including solitary plasmacytoma, smoldering myeloma (asymptomatic), and active myeloma (symptomatic).

Solitary plasmacytoma

Solitary plasmacytoma is a large solitary focus of plasma cell proliferation. To simplify, solitary plasmacytomas can be divided into 2 groups according to location: Plasmacytoma of the skeletal system (SBP) or Extramedullary plasmacytoma (EMP). Similarly, the latest NCCN guideline for MM categorizes solitary plasmacytoma into solitary osseous or solitary extraosseous.

Criteria for identifying solitary bone plasmacytoma (SBP) vary among authors. Some include patients with more than one lesion and elevated levels of myeloma protein and exclude patients whose disease progressed within 2 years or whose abnormal protein persisted after radiotherapy. With the use of magnetic resonance imaging (MRI), flow cytometry, and polymerase chain reaction (PCR), the currently accepted criteria are as follows:

  • Single area of bone destruction due to clonal plasma cells
  • Bone marrow plasma cell infiltration not exceeding 5% of all nucleated cells
  • Absence of osteolytic bone lesions or other tissue involvement (no evidence of myeloma)
  • Absence of anemia, hypercalcemia, or renal impairment attributable to myeloma
  • Low, if present, concentrations of serum or urine monoclonal protein
  • Preserved levels of uninvolved immunoglobulins

Diagnostic criteria for extramedullary plasmacytoma (EMP) are as follows:

  • Tissue biopsy showing monoclonal plasma cell histology
  • Bone marrow plasma cell infiltration not exceeding 5% of all nucleated cells
  • Absence of osteolytic bone lesions or other tissue involvement (no evidence of myeloma)
  • Absence of hypercalcemia or renal failure
  • Low serum M protein concentration, if present

Smoldering myeloma

Smoldering myeloma describes a stage of disease of MM with no symptoms and no related organ or tissue impairment. According to the latest version of NCCN guideline for MM, criteria for the definition of smoldering myeloma are as follows:

  • M-protein in serum ≥30 g/L and/or
  • Bone marrow clonal plasma cells ≥10%
  • No related organ or tissue impairment (no end organ damage, including bone lesions) or symptoms.

Note that the M-protein refers to the monoclonal protein produced by MM cells.

Active/symptomatic myeloma

Criteria for the definition of active/symptomatic myeloma requires one or more of the following:

  • Calcium elevation (>11.5 mg/dL) [>2.65 mmol/L]
  • Renal insufficiency (creatinine >2 mg/dL) [177 µmol/L or more]
  • Anemia (hemoglobin <10 g/dL or 2 g/dL < normal)
  • Bone disease (lytic or osteopenic)

In the section of management of MM we will discuss the specific therapeutic approaches for these three subtypes of MM.

Prognosis and Genetics

Several factors can afford important prognostic information for multiple myeloma. They are β2-microglobulin, lactate dehydrogenase (LDH), cytogenetics, and plasma cell-specific FISH analysis (hyperdiploidy, t(4;14)(p16;q32), t(14;16)(q32;q23), 17p13, t (11;14)(q13;q32), 1q amplifications, 1p deletions, loss of 12p, gains of Cr5).

Table 2. Risk Classification Based on Baseline Testing

Of note that in the latest NCCN guideline about multiple myeloma several high-risk chromosomal aberrations in MM locates at 14q32, including three main ones that are t(11;14)(q13;q32), t(4;14)(p16;q32) and t(14;16)(q32;q23). Thus the risk incidence of t(11;14) is inconsistent with what was decribed in Table 2.

For this inconsistent two view I have sent an inquiry to NCCN and their answer was “We have reviewed your inquiry with the NCCN Guidelines Panel Chair, Dr. Kenneth Anderson. NCCN does not classify t(11;14) as high risk, it is only listed as a major group containing the 14q32 translocation. ”

And pay attention that patients with t(4;14), β2 microglobulin <4 mg/L and hemoglobin ≥10 g/dL may have intermediate risk disease.

Although the genetics can afford the prognosis of multiple myeloma, this approach still needs more evidence. At present the method is still the Durie-Salmon criteria or ISS criteria.

Table 3. Stage of Multiple Myeloma

As shown in the table 2 at left, the stage of multiple myeloma can be divided into three periods: stage I, stage II, and stage III.

The Management of Solitary Plasmacytoma

For those patients with osseous plasmacytoma, primary radiation therapy (45 Gy or more) to the involved field is the initial treatment and is potentially curative. Extraosseous plasmacytomas are treated initially with radiation therapy (45 Gy or more) to the involved field followed by surgery if necessary.

After radiation thearpy, patients with solitary plasmacytoma need follow-up. Blood and urine tests performed every 4 weeks initially to monitor response to the primary radiation therapy. If the patient achieves complete disappearance of the paraprotein then the frequency could be reduced to every 3-6 months or as indicated clinically. If the protein persists, then the monitoring should continue every 4 weeks. These tests include CBC, serum chemistry and those listed in the section of workup.

If progressive disease emerges, then the patient should be re-evaluated for recurrent plasmacytoma or myeloma, and systemic therapy administered as indicated.

The Management of Smoldering Myeloma

Although the activity of novel agents has advanced to the point that early interventions are now being explored in clinical trials for smoldering myeloma, there is still no evidence that early treatment will improve survival in asymptomatic and biochemically stable patients. A critical point is that up to 25% of smoldering myeloma patients will not require active treatment for 10 to 15 years. However, the majority will indeed progress during that time.

Once diagnosed, smoldering myeloma patients require frequent monitoring to allow treatment to begin before end-organ damage is evident. These tests are similar with solitary plasmacytoma, which are listed in the section of workup. If the disease progresses to symptomatic myeloma, these patients should be managed as active/symptomatic myeloma. We will discuss the management of active/symptomatic myeloma below.

The Management of Active/Symptomatic Myeloma

If the patients with MM progresse to active/symptomatic myeloma. Treatment should be initiated. Generally, we divide the treatment strategy into initial drug therapy, hematopoietic cell transplantation, and consolidation and maintenance thearpy after transplantation.

Therapeutic goal

There is a growing body of evidence showing an association between depth of response to therapy and improved long-term outcomes, including progressive-free survival (PFS) and overall survival (OS), in MM patients. Using conventional chemotherapy, it has been shown that there is a correlation between response before and after transplantation and that the quality of response after transplantation has a marked impact on outcome.

Importantly, studies suggest that if a patient achieves a complete response (CR), this must be durable and that the duration of CR is the best predictor of OS. However, some special cases makes the view that initially obtaining a CR in predicting long-term outcome questionable, for instance, group of rapidly responding but early relapsing patients, group of more indolent myelomas that revert to an “monoclonal gammopathy of uncertain significance like” profile after therapy, and group of myeloma patients with stable nonprogressive disease after induction therapy.

Initial drug therapy

Although success and long-term remission have been achieved in many transplantation-eligible patients using limited treatment regimens, such as thalidomide/dexamethasone, bortezomib/dexamethasone, and lenalidomide/dexamethasone, complete and very good partial response (VGPR) rates can be substantially increased by combining these various drugs in triplets or even using 4 drugs together.

On the right is the data of several clinical trials. I list all the detail of regimens below:

VAD: vincristine, adriamycin, and dexamethasone;

TD: thalidomide and dexamethasone;

RD: lenalidomide and dexamethasone;

PAD: bortezomib, doxorubicin, and dexamethasone;

VTD: bortezomib, thalidomide, and dexamethasone;

CVD: cyclophosphamide, bortezomib, and dexamethasone;

RVD: lenalidomide, bortezomib, and dexamethasone;

CVRD: cyclosphamide, bortezomib, lenalidomide, and dexamethasone.

A note of caution is that many of these studies are based on relatively small numbers of patients at single, or limited numbers, of centers, but cumulatively the message is consistent, with frequent, rapid, and deep responses seen.

Althought response rates are clearly improved with new drug cocktails, proving a consequent OS advantage is difficult and especially challenging given the large numbers of patients and the long duration of follow-up required. However, based upon response rates, depth of response achieved, and PFS as surrogates, 3-drug cocktails are currently the modality of choice in clinical practice, with use of RVD, CVD, or VTD as the most commonly chosen regimens outside of clinical trials.


Transplantation is a useful modality helping achieve or consolidating CR. But is it necessary to provide any consolidation chemotherapy before transplantation? If the patient is going to proceed to  transplantation, when do we implement the transplantation. However, because the goal of therapy is to maximize the depth and duration of remission, induction therapy can be continued in some patients for as long as the patient is responding and tolerating therapy, which might be instead of transplantation.

Generally ASCT is the primarily way of transplantation. Allo-SCT should infrequently be performed outside of clinical trials, as the risk of morbidity and early mortality of even nonmyeloablative transplantations is considerable.

Question one is whether to offer any consolidation chemotherapy before transplantation.

After initial induction thrapy, the subsequent approach is to provide further 4 to 6 cycles of induction threapy, then proceed eligible patients to ASCT. The reason to use stem cell transplantation is to provide a consolidation of remission after obtaining the best possible response to frontline treatment.

But a controversial area is what to do if the patient has already achieved a CR before transplantation. In this decision, the role of continued chemotherapy treatment versus proceeding to transplantation is less clear and an are of active research. Generally, in practice we prefer to proceed patients to transplantation without any further induction chemotherapy.

The reason to proceed to transplantation even achieving CR before transplantation is that current measures of CR are insufficiently sensitive and residual disease is in many, if not all, patients present but below the level of detection.

Question two is when do we offer stem cell transplantation to our patients who are eligible to this procedure. The timing of ASCT is also an area of active research. Patients are usually more fit for intensive therapy early in the course of the disease, but prior studies using conventional chemotherapy as induction demonstrated this a delayed ASCT had no adverse impact on OS and is feasible as part of salvage therapy in first relapse.

Maintenance therapy

Clinical studies found thalidomide maintenance to improve overall survival. Lenalidomide may offer the same advantages with less toxicity. Generally, it has become our practice to use maintenance routinely when patients have not achieved a CR after stem cell transplantation or when genetic risk markers suggest a very high risk of early relapse.

Figure 1. Respond Criteria for Multiple Myeloma

The Management of Therapy-related Acute Meyloid Leukemia

July 1, 2013 Chemotherapy, Cytogenetics, Hematology, Transplantation No comments

Until recently, the term “secondary leukemia” broadly included any AML with a history of prior malignancy as well as patients with any antecedent hematologic disorder and, in some series, any patient who presented with unfavorable cytogenetics.

Among therapy-related AML patients, 70% present with abnormalities of chromosome 5 or 7, which is the most typical presentation after the exposure to alkylating agents and/or ionizing radiation; 30% are those that arise after treatment with topoisomerase-2 inhibitors.

In general, the management of therapy-related AML is fraught with uncertainty because, among other reasons, most early studies included small numbers of patients and were retrospective. There have been no prospective randomized studies specifically directed at the treatment of therapy-related leukemias. Furthermore, the published data often included patients with myelodysplastic syndromes.

Induction Therapy

Hisytorically, it was presumed that every patient with therapy-related leukemia had an adverse prognosis and that standard induction therapy was inappropriated. However, there is no evidence that any induction therapy is superior to the standard 3+7 regimen. Among young adults, quite remarkably, prospective studies report an almost identical CR rate of 55% to 60% for patients treated with recognized unfavorable cytogenetics, and there are no reports that anything is better than this. Standard induction should remain the induction therapy regimen.

Postremission Therapy

Figure 1 AML in Patients Less Than 60 Years of Age.

It is still somewhat controversial whether thearpy-related AML has a prognosis that is intrinsically worse that de novo AML, independent of cytogenetics. In a very large database, the National Cancer Research Institute in Great Britain reported a significantly worse outcome for therapy-related AML than de novo AML, within each cytogenetic risk group (favourable, intermediate, and adverse). But, still, the management of patients with therapy-related AML should be guided by the cytogenetic and molecular features, which is the same as de novo AML.

Although there is a perception that any patient with therapy-related AML should be considered at high risk and referred to an allogeneic transplantation, there is no evidence that the long-term outcome for patients who present with a favorable karotype, with no adverse molecular features, is different from patients with the de novo AML. Thus, such patients with favorable category should not be referred to an allogeneic transplantation in CR1.