Clinical features, Incidence and Risk factors
The clinical diagnosis is based on a triad of hepatomegaly, sudden weight gain, and jaundice. The cytoreductive conditioning is given 6-10 days before the day of hemopoietic cell infusion which is called day 0. Increases in liver size and tenderness, renal sodium retention and weight gain are usually the first signs at 10-20 days after the start of cytoreductive therapy. Following regimens containing cyclophosphamide (CY), one of the most commonly used conditioning agents, weight gain starts at day 1, and hyperbilirubinemia on day 6.The incidence and severity of SOS varies significantly depending on the different conditioning regimens and patient variables such as underlying liver inflammation and fibrosis , age and individual pharmacogenetic variability in drug metabolism of busulfan (BU)and CY. SOS develops in 5-40% of patients undergoing HCT during the first few weeks after being conditioned with myeloablative regimens. The cause is the toxic injury from the cytoreductive chemotherapy, especially after regimens containing CY, total body irradiation (TBI) and gemtuzumab ozogamicin (Mylotarg). The frequency and severity of SOS have declined in the last few years because: 1)clinicians have retreated from the strategy of dose escalation conditioning regimens such as CY and 13.2 GY TBI to the use of non-myeloablative regimens or those that do not contain CY; 2) pharmacokinetic monitoring of plasma BU levels adjusted to stay below a toxic threshold; 3)a steep decline of chronic hepatitis C, a major risk factor for severe SOS in HCT populations , 4) HCT done earlier in healthier patients; 5) and removal of drugs e.g. norethisterone and acetominophen that increase the risk of SOS. Fatality rates for SOS after HCT are dependent on both the clinical definition of SOS and what constitutes severe SOS. Depending on the conditioning regimen used, most patients recover from SOS (70% from Cy containing regimens, 84% when caused by other alkylating agents). Over the three decades that HCT has been employed worldwide, thousands of HCT recipients have died of SOS, yet, among hepatologists it rarely has recognized as one of the list of leading causes of liver failure!

Experimental studies
In a rat monocrotaline model with histologic characteristics similar to human SOS, the first morphologic changes noted by electron microscopy is the loss of SEC fenestrae and the appearance of gaps in the SEC barrier. In vitro microscopy has shown that the SEC round up and red cells begin to penetrate into the space of Disse beneath the rounded up endothelial cells and dissect off the sinusoidal lining. The sinusoidal lining cells, including Kupffer cells and stellate cells, as well as SEC, embolize downstream and obstruct sinusoidal flow. By the time hepatocyte necrosis is observed (the first histologically identifiable finding of VOD in man), there is extensive denudation of the sinusoidal lining.

In vitro studies have shown that SEC are far more susceptible than hepatocytes to the alkylating drugs that produce SOS., The common drugs that produce SOS ( BU, CY, and TBI) profoundly deplete sinusoidal endothelial cell glutathione prior to death as well as damaging DNA. Following monocrotoaline, support of SEC glutathione prevents cell death, but not if it is discontinued. One possible explanation for the rounding up of the SEC may be increased activity of matrix metalloproteinases (MMP) which degrade the extracellular matrix on the abluminal side of the sinusoidal endothelial cell, allowing the cells to become detached from the space of Disse. In the experimental model, increased MMP activity correlates with the rounding up of SEC, while inhibition of MMP activity by administering glutathione suppresses this. In the in vivo model, hepatic vein nitric oxide decreases in parallel with the changes in the sinusoidal flow, whereas nitric oxide precursors prevent the morphological and clinical features. The role of coagulation in the pathophysiology is controversial. DeLeve & McCluskey's elegant cinemicroscopy and scanning em.studies did not demonstrate fibrin deposition. However, a more recent study with the same model by Copple, using a highly specific antibody for cross-linked fibrin and SEC, found that Z3 SEC loss and fibrin deposition preceeded hepatic parenchymal cell injury. One obvious question, why the lesion is confined to Z3, is not fully understood. In the case of CY, the activation by cytochromes occurs principally in Z3 exposing the SEC, the cells most susceptible to toxic agents, to the highest concentration of toxic metabolite. The combination of chemoirradiation induced "endotheliopathy" leads to marked increase in intrahepatic resistance and decreased blood flow. The injury to SEC leads to decreased NO accompanied by increased stellate cell production of endothelin -1 with activation/proliferation, expression of smooth muscle proteins, sinusoidal constriction and fibrogenesis (see Rockey 2003). Also, the mechanical obstruction at the small post-sinusoidal pores may play a critical role in the localization.

In sum, the general concept is of glutathione depletion by toxic metabolites, compounded by DNA damage leading to SEC death with disruption of sinusoidal cells and obstruction to sinusoidal blood flow.

Histological features of human SOS
The sequence of histological changes with SOS that occur after HCT were gleaned from chronological analysis of many biopsies and necropsies, The trichrome stain is essential for outlining sinusoids, venules and hepatic cords, while other stains (VVG and reticulum) are complementary. All sections should be viewed completely to identify characteristic histologic changes of SOS that may not be present at all levels. The histological data do not elucidate the primary site of injury or molecular events that occur in the sinusoids before clinical signs of SOS appear. Whether SOS occurs after conditioning with cytoreductive agents or Mylotarg (a humanized anti/CD33 conjugated to the cytotoxic agent calicheamicin), the first recognizable changes by light microscopy do not appear until 7-10 days after the agents are given. These first changes consist of zone 3 congestion and hemorrhage in the space of Disse and concentric widening of the subendothelial space of small venules by edema and entrapped red cells and occasionally hepatocytes. Presumably, the elevated sinusoidal pressure, as reflected by elevated wedged venous pressure gradients results in Z3 hepatocyte ischemic necrosis along with detachment and embolized sinusoidal cells. The necrosis of perivenular hepatocytes is reflected by immunohistochemistry (IHC) with markedly diminished Z3 hepatocyte immunostaining with anti-cytokeratin 35βH11. Additional ICC studies demonstrate deposition of FVIII vWF, but not platelet antigens, in the periadventitial zone, within the concentrically widened subendothelial zone of venules, but not within the venular lumen, corresponding to clogging of pores that drain sinusoids to the venules. Obstruction of the pores draining the sinusoids into the venules can be appreciated on thin plastic embedded sections. Electron microscopic studies show closure of fenestre in sinusoidal endothelial cells and accumulation of extracellular material, collagen.

Within two weeks of the onset of signs of SOS, ICC with α-smooth muscle actin antibodies for activated (and presumably proliferated)stellate cells demonstrates a marked increase in the number of stellate cells lining the zone 3 sinusoids. At this time, extracellular deposits of matrix can be seen in the subendothelial spaces and the sinusoids. The areas of hepatocyte drop-out or necrosis contain numerous monocytic-marked cells presumably reflecting monocytic infiltration which have become Kupffer cells. ICC stains for SEC show a marked loss in the damaged Z3 sinusoids. The severity of symptoms,i.e. bilirubin elevation before day 20 and ascites was significantly correlated with the number of acini involved. In a subset of patients, a bimodal course with initial improvement in symptoms, presumably from less congestion and hepatocyte necrosis, is followed by a steady and inexorable downward course with, likely reflecting proliferation of stellate cells and progressive collagenization of the sinusoids. In those patients with severe SOS who survive beyond day 40-50, there is coalescence of extinguished perivenular zones with fibrous bridging between central veins, simulating cardiac cirrhosis.

Differential Diagnosis
Several other conditions may elevate the bilirubin after transplant, including the sepsis syndrome with fluid overload, congestive heart failure, overwhelming viral hepatitis from herpes simplex,herpes zoster, or adenovirus, hemolysis and hyperacute graft-versus-host disease (GVHD). Marked early elevations of serum AST and ALT, a reflection of centrilobular hepatocyte necrosis, is a poor prognostic indicator of survival from SOS, but, like bilirubin, their specificity may limit their clinical use. Imaging studies are useful for demonstrating hepatomegaly and attenuated hepatic venous flow. A model developed for the treatment of severe SOS predicts the outcome of SOS following CY-based regimens using as variables the rate of increase of both bilirubin and weight gain in the first two weeks following HCT. Nontheless, when there is uncertainty in the clinical diagnosis of SOS, transvenous liver biopsy (TVLB) allows both histological evaluation of SOS and hepatic venous wedge pressure gradient. A wedged venous pressure gradient >10 mmHg is highly specific for VOD. TVLB can be done safely if the platelet count is over 30,000/μl. We have utilized a transfemoral approach with forceps style biopsies because of a lower rate of bleeding complications and possibility of avoiding pre-existant thrombosus in the superior vena cava that would prevent a transjugular approach. At least six, and preferably more, small forceps biopsy pieces should be obtained to ensure adequate sampling of venular and perivenular regions.

Clinicopathological correlates
In retrospective autopsy studies, 20-30% of cases with occluded venules were without clinical symptoms. Furthermore, 45% of patients with clinically mild to moderate SOS and 25% of those with severe SOS did not have occluded venules . These data suggest that widespread occlusion of central veins is associated with more severe disease with ascites whichexacerbates the circulatory impairment at the level of the sinusoids. A coded review of histologic features in a cohort of 76 necropsy patients found several additional perivenular lesions to correlate with signs of SOS in the absence of venular occlusion. The strongest statistical correlations in clinically severe SOS were Z3 changes of hepatocyte necrosis, sinusoidal fibrosis, eccentric thickening of the subendothelial zone of the venule, phlebosclerosis, and an overall estimate of venular involvement/narrowing. Moreover, the number of such histological changes present was proportional to the clinical severity of SOS. High doses of the anti-myelogenous leukemia drug, Mylotarg, is particularly prone to cause SOS after HCT. Sinusoidal fibrosis may be the predominant lesion.

More than one etiology for liver disease may be present. When both GVHD and SOS may occur in the same liver, clinicopathologic correlation is needed to assess the relative magnitude of either process. GVHD rather than VOD is the most likely contributing factor to the liver dysfunction which first develops after day 30.

Treatment and Prevention of SOS
Non-relapse mortality following myeloablative conditioning therapy in allogeneic HCT is highly correlated with the total serum bilirubin. A total serum bilirubin of >10 mg/dl was associated with 83% mortality at day 30, while a total serum bilirubin >19 mg/dl was associated with 87% mortality at day 30 and 100% at day 60. Patients with severe SOS typically die with renal cardiopulmonary failure and syndrome rather than of liver failure. There is extensive literature on treatment, briefly reviewed in the reference by DeLeve in Seminars in Liver Disease, 2002. A large and conflicting literature exists on the use of anticoagulation for severe SOS. To date, the most promising agent on the horizon is defibrotide, a single-stranded polydeoxyribonucleotide derived from animal tissue which has binding sites in vascular endothelium, up-regulates the release of prostacyclin, prostaglandin E2, thrombomodulin, and TPA in vivo and in vitro, as well as profibrinolytic effects by decreasing thrombin generation tissue factor expression, PAI-1 release and endothelin activity.. In an uncontrolled study of 88 patients with severe SOS, treatment with defibrotide resulted in complete resolution in 36% of such patients. Among the 29 patients who underwent autopsy, findings consistent with VOD were confirmed in 85% of the biopsies taken and in 76% of the autopsies, with some cases considered to have an inadequate sample or showing hepatic necrosis or centrilobular congestion, suggestive of SOS. A randomized prospective study using defibrotide started prior to transplantation, now under way, should establish its efficacy in preventing or reducing VOD after HCT.

Prevention of SOS
The most certain way to prevent VOD is by reducing or eliminating the high doses of cytoreductive therapy. The increasing uses of non-myeloablative conditioning for a number of marrow disorders has in fact eliminated SOS in this cohort. However, for some diseases, myeloablative conditioning is still a necessity. Pharmacokinetic monitoring of plasma BU indicates that levels are influenced by the regimen, age and underlying disease. A strong relationship exists between elevated plasma Bu concentrations and decreased patient survival. Doses of busulfan are now monitored during conditioning and adjusted from rising above the threshold where SOS is much more likely to occur.

CY is currently the most commonly used agent in preparation for HCT, generally in combination with either BU or TBI. In vivo following activation by liver cytochromes the toxic metabolites formed are the active antitumor DNA cross-linking agent phosphormide mustard and the potent liver toxin acrolein. Pharmacokinetic measurements CY is done using a chemically stable acid reporter metabolite chloroethylphosphoramide musturd (CEPM) which reflects the toxic metabolites. A strong association exists between the degree of CEPM exposure, the peak total bilirubin at day 20 and a six-fold increase of non-relapsed mortality at one year in those with CEPM levels in the highest quartile. The reporter metabolite demonstrated a 16-fold variation among different patients. Related studies in rats, suggest this variability may be related to the transport of the gluthathione conjugated metabolite, glutathionyl cyclophosphamide out of the hepatocyte by the bile canalicular multiorganic ion transporter ABCC2. A trial to reduce the mortality caused by the CY/TBI regimen is underway using pharmocokinetic adjustment of the second CY dose based on the first day's exposure to the reporter molecule CPEM, targeting the total exposure to a value consistent with low toxicity and reliable engraftment.

Many thanks to my colleagues George McDonald, John Slattery and Laurie DeLeve

References

1. DeLeve LD, Shulman HM, McDonald GB. Toxic injury to hepatic sinusoids: Sinusoidal Obstruction Syndrome(Veno-Occlusive Disease). Seminars in Liver Disease; 2002; 22: 27-41.
2. Richardson PG, Murakami C, Wei LJ, et al. Multi-institutional use of defibrotide in 88 patients post stem cell transplant with severe veno-occlusive disease and multi-system organ failure; response without significant toxicity in a high risk population and factors predictive of outcome. Blood;2002; 100: 4337-4343
3. McCune JS, Gibbs JP, Slattery JT. Plasma concentration monitoring of busulfan-Does it improve outcome? Clin Pharm; 2000; 39: 155-165
4. McDonald GB, Slattery JT, Bouvier ME, et al. Cyclophosphamide metabolism, liver toxicity, and mortality following HCT. Blood; 2003 in press (available online)
5. Hemopoietic Cell Transplantation 3rd edition in press editors Blume KG, Forman SJ, Appelman FR Chapters 23 and 58
6. Copple BL, Barner A, Ganey PE and Roth RA. Endothelial injury and fibrin deposition in rat liver after monocrotaline exposure. Toxicological Sciences 2002; 65: 309-318
7. Rockey DC, Vascular mediators in the injured liver. Hepatology 2003;37:4-12.