The number of people with sickle-cell disease in the United
States: national and state estimates
David C. Brousseau,1,2*Julie A Panepinto,3Mark Nimmer,1and Raymond G Hoffmann4
Sickle-cell disease is not a reportable condition, making it difficult to
ascertain the number of affected individuals. We estimated the number of
people with sickle-cell disease for the United States and each individual
state, adjusting for increased mortality. US Census population data for
eachof the 50 states plus the District of Columbia were obtained. The pub-
lished prevalence of sickle-cell disease for blacks and Hispanics of either
Mexican or non-Mexican ancestry was applied. Analysis revealed 89,079
(95% confidence interval: 88,494–89,664) people with sickle-cell disease in
the United States, 80,151 black and 8928 Hispanic. The state with the high-
est sickle-cell population was New York with 8308, followed by Florida
with 7539, and Texas with 6765 people with sickle-cell disease. This study
provides important information for researchers and policymakers attempt-
ing to better plan for the care of the sickle-cell population.
Sickle-cell disease is the most common inherited blood disorder in the
United States. It is responsible for approximately 113,000 hospitalizations and
$488 million dollars in hospitalization costs annually in the United States .
Although sickle-cell disease is a genetic disease diagnosed in this era by
newborn screening, it is not a reportable condition. Therefore, it is difficult to
ascertain the number of affected individuals in the United States. Newborn
screening is an effective tool for diagnosis, but its use was not widespread
until the late 1980s and 1990s , and it does not account for immigration.
Further complicating population estimates is the increased mortality associ-
ated with the disease, both compared to the mortality for the underlying
racial/ethnic group and when comparing more severe forms of the disease
(hemoglobin SS (HgbSS) and hemoglobin S beta thalessemia (HgbSB0)) to
less severe forms of the disease (hemoglobin SC (HgbSC) and HgbSB1) [3–
7]. Recent advances such as prophylactic penicillin and vaccines have
reduced mortality, but only for those young enough to have received the treat-
ments [8,9]. While no published studies have estimated the prevalence of
sickle-cell disease in the United States, one NIH estimate puts the number
‘‘between 50,000 and 75,000;’’ another at 80,000, and the Sickle Cell Disease
Association of America estimates the number to be ‘‘over 70,000’’ [10–12]
Census level-data and prevalence.Census data by age and race/ethnicity
were obtained for each individual state and the District of Columbia and
summed to equal the United States as a whole . Year 2005 was used as
the baseline. The applied prevalence rate of sickle-cell disease for blacks
was 289 per 100,000 live births . Prevalence rates for Hispanics were
89.8 Hispanic children of non-Mexican ancestry per 100,000 live births and
3.14 Hispanic children of Mexican ancestry per 100,000 live births .
These prevalence rates were applied to the Hispanic population numbers in
each state based on the state’s ratio of the number of Mexican births to
non-Mexican births, assuming birthrate was reflective of the current ethnic
population proportions. People identified as black-Hispanic (0.56% of the
population) were classified as black. We did not include sickle-cell disease
for whites (including Mediterraneans), Asian Indians, or other Asians in our
estimate. While the disease occurs in these populations, the prevalence is
very low, it is not possible to identify the country of origin from census data,
and the mortality data specific to these populations is not known.
Consistent with previous literature, 60% of children at birth were classified
as having HgbSS/HgbSB0and 40% were classified as having HgbSC/SB1
[8,15,16]. Other sickle-cell disease genotypes, because the prevalence of
these is very low, were excluded.
Mortality adjustment.We adjusted for mortality based on age and sickle-
Children (<18 years old): For children with HgbSC/HgbSB1, survival is simi-
lar to the underlying population . For children with HgbSS/HgbSB0, Quinn
et al.  reported a 6.4% sickle-cell disease-related mortality in children by
18 years of age. We used linear interpolation to obtain an adjustment by the
year. Although the majority of deaths occur early in childhood, this is coun-
tered by the use of the conjugate pneumococcal vaccine, which was started
in 2001 and would only affect those ?4 years old in our study.
Young adults (age 18–45 years): For young adults with HgbSC/HgbSB1,
there is no increase in sickle-cell-related mortality through 30 years of age
. From age 31 years through 45 years, there is a linear decrease in sur-
vival to 85% at 45 years of age; for similar ages, the black mortality rate is
5%, leaving an excess mortality of 10% between the ages of 31 and 45
years . For young adults with HgbSS/HgbSB0, published reports show
improved survival for those born during or after 1975 . For those born
during or after 1975 (18–30 years old), 89% survive to adulthood, and 71%
survive to age 30 years . With 75% of the deaths being sickle-cell related,
the survival in our population of 18–30 years old was 85.4% of predicted
based on population estimates. For those born before 1975 (31–45 years
old in our population), 79% survived to age 20 years . Ten-year survival
from age 20 years shows that 67% would live to 30 years old, 52% would
live to 40 years old, and 43% to 45 years old .
Middle-aged adults (age 46–65 years): For those with HgbSC/HgbSB1,
approximately 50% will be alive at age 65 years compared to 65% of blacks,
but this excess mortality occurred at younger ages . For those with
HgbSS/HgbSB0, 43% survived to age 45 years and only 10% will survive to
age 65 years .
Older adults (age > 65 years): For those with HgbSS/HgbSB0, about
1% fewer each year were alive from age 55 to 65 years and we contin-
ued with this rate. For those with HgbSC/HgbSB1, we used linear extrap-
olation to continue the adjustment to the mortality rate from 45 to 65
States were grouped by geographic census regions. A 95% confidence
interval (CI) was included for the national estimate based on a Poisson
assumption for the variability of the count data. In addition to the CI, two
sensitivity analyses were performed. The first analysis assumed that mortal-
ity was 10% lower for each age group than predicted from the literature; the
second, based on a small study showing a higher prevalence of severe dis-
ease in Hispanics, assumed that 80% of Hispanics had severe disease at
birth instead of 60% .
Population estimates, with mortality adjusted by age and sickle-cell type,
yielded a total year 2005 estimate of 89,079 (95% CI: 88,494–89,664) peo-
ple with sickle-cell disease in the United States, of which 80,151 were black
and 8,928 Hispanic (Fig. 1).
The distribution across age groups revealed 35,726 (40% of the popula-
tion) children with sickle-cell disease. Regional totals are shown in Fig. 1.
The South, with a sickle-cell population of 47,354 people, comprised 53% of
the sickle-cell population. The estimated sickle-cell population of New York
(8308 people), was more than three-fourths of that of the entire Western
The estimated number of people with sickle-cell disease in each state is
shown in Supporting Information Table I. The states with the highest esti-
mated number of people with sickle-cell disease were New York with 8308;
Florida with 7539; Texas with 6765; California with 6474; and Georgia with
5890. These five states comprised more than 43% of the total sickle-cell
Supporting Information Table II shows both the absolute and relative per-
centages of sickle-cell genotypes by age. The increased mortality for
HgbSS/HgbSB0leads to an alteration in the relative percentages of sickle-
C 2009 Wiley-Liss, Inc.
American Journal of Hematology
cell genotypes, with HgbSS/HgbSB0comprising 60% at birth, half at 30
years old, and approximately 25% of the sickle-cell population by 60 years
Our sensitivity analyses show small changes in the estimated sickle-cell
population. If our estimated mortality is 10% higher than actual mortality,
then the sickle-cell population is 3.1% higher and equals 91,840. If mortality
is correct, but Hispanics are more likely to have severe disease (80%
HgbSS/HgbSB0), there would be a decrease in Hispanics from 8928 to
8267, and the total sickle-cell population would be 88,418.
This study shows that the sickle-cell population is significantly higher than
previously estimated, with 89,079 people with sickle-cell disease in the Uni-
tes States. As of 2005, we estimate that approximately 10% of those with
sickle-cell disease are Hispanic. By using census level estimates, we
account for immigration in addition to births in the US. With the Hispanic
population being one of the largest growing populations in the United States,
the percentage of people with sickle-cell disease that are Hispanic will con-
tinue to increase.
In addition to the national estimates, we determined the number of people
with sickle-cell disease in each state. Previous studies attempting to esti-
mate the sickle-cell population have focused on a single state, the black
population only, and/or have not taken into account the increased mortality
of sickle-cell disease [3,17]. While these later two omissions would work in
opposite directions with regard to over or underestimation of a given state’s
sickle-cell population, they cast doubt on the accuracy of the estimate and
thus the overall study findings.
Another important finding is the relative proportions of sickle-cell geno-
types with increasing age. This is the first study to report the fact that above
age 40 years, more people with sickle-cell disease will have HgbSC/
HgbSB1. It is clear that early intervention into the lives of people with sickle-
cell disease is essential in order to maximize the benefit to those with the
most severe disease.
The study is limited by the fact that we did not include whites or Asians in
our analysis, potentially underestimating the sickle-cell disease population in
the Unites States. Despite this exclusion, our estimates are still higher than
previous national estimates. We also did not include information on Puerto
Rico; race/ethnicity data by age for Puerto Rico is not available on the US
In conclusion, the sickle-cell population is larger than previously reported,
with over 89,000 people with sickle-cell disease in the Unites States. The
national and statewide estimates will be useful to policy makers at both a
state and national level as they attempt to better understand the burden of
sickle-cell disease and plan for the management strategies in this population
with high rates of healthcare utilization.
1Department of Pediatrics, Children’s Research Institute
Medical College of Wisconsin, Milwaukee, Wisconsin
2Department of Population Health
Medical College of Wisconsin, Milwaukee, Wisconsin
3Department of Pediatrics, Section of Pediatric
Hematology/Oncology/Bone Marrow Transplant, Children’s Research Institute,
Medical College of Wisconsin, Milwaukee, Wisconsin
4Department of Pediatrics, Children’s Research Institute
Medical College of Wisconsin, Milwaukee, Wisconsin
*Correspondence to: David C. Brousseau, Department of Pediatrics, Medical
College of Wisconsin, CCC 550, 999 N 92nd St., Milwaukee, WI 53226
Additional Supporting Information may be found in the online version of this article.
Conflict of interest: Nothing to report
Published online 21 October 2009 in Wiley InterScience
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per100,000 livebirths, United
Figure 1.Sickle-cell disease population, by country, region, and state.
78American Journal of Hematology
Costs and length of stay for patients with and without sickle cell
disease after hysterectomy, appendectomy, or knee
Shital Kamble,1Marilyn J. Telen,2Michaela A. Dinan,1,3Chelsea A. Grussemeyer,1and Shelby D. Reed1,4*
Patients with sickle cell disease (SCD) who undergo surgical proce-
dures experience greater risk for preoperative and postoperative compli-
cations than patients without SCD; however, the impact of SCD on inpa-
tient resource use and costs has not been reported. [1–7] We recently
examined inpatient length of stay and total costs for patients with and
without SCD who underwent cholecystectomy or hip replacement and
found that patients with SCD had longer lengths of stay and incurred
higher costs.  In this study, we extend our previous work to surgical
procedures that are less commonly performed in patients with SCD:
hysterectomy, appendectomy, and knee replacement. Using a large
national database of inpatient stays, we found that patients with SCD
had significantly longer lengths of stay and incurred higher costs than
patients without SCD who underwent the same procedures, after adjust-
ment for patient, hospital, and procedural characteristics (P < 0.001).
Higher inpatient costs were largely attributable to longer hospital stays.
Future work should investigate the extent to which preoperative and
postoperative complications and other factors contribute to longer stays
among patients with SCD and how these factors might be addressed.
SCD is the most commonly inherited hemoglobinopathy, characterized by
anemia and unpredictable, acute complications that can rapidly become life
threatening [9–14]. SCD affects more than 70,000 people in the United
States and is predominant in African Americans, occurring in ?1 out of
every 500 African American births [9,15]. Compared to patients without
SCD, patients with SCD who undergo surgical procedures are at greater risk
for preoperative and postoperative complications, with SCD-related postop-
erative complications occurring in over 30% of patients, even after preopera-
tive blood transfusion [5–7,16]. In a health care environment increasingly
moving toward differentiated payments for lower- and higher-quality care, it
is important to understand patient and procedural factors that contribute to
higher complication rates, longer stays, and higher costs. We recently per-
formed an analysis comparing inpatient outcomes between patients with and
without SCD who underwent the two most commonly performed nonobste-
tric surgical procedures among patients with SCD in the United States: chol-
ecystectomy and hip replacement.  The study revealed that patients with
SCD who underwent either procedure incurred longer inpatient stays and
higher inpatient costs than patients without SCD.
In this study, we expanded our analysis to other less commonly performed
procedures (ie, hysterectomy, appendectomy, and knee replacement) in
patients with SCD and found similar results. Using the 2002–2006 Nation-
wide Inpatient Sample (NIS) from the Healthcare Cost and Utilization Proj-
ect, we identified 118 SCD and 85,073 non-SCD discharges for hysterec-
tomy, 69 SCD and 24,802 non-SCD discharges for appendectomy, and 62
SCD and 14,517 non-SCD discharges for knee replacement. For each pro-
cedure, patients with SCD were younger than patients without SCD (Table I;
P < 0.001 for each comparison). Hysterectomy and knee replacement were
less likely to be recorded as elective surgery for patients with SCD than for
patients without SCD [79.7% vs 86.0% (P 5 0.05) for hysterectomy; 77.4%
vs 93.1% (P < 0.001) for knee replacement]. Compared to patients without
SCD, patients with SCD were significantly more likely to receive blood trans-
fusions during the inpatient stay [28.0% vs 4.3% (P < 0.001) for hysterec-
tomy; 18.8% vs 2.5% (P < 0.001) for appendectomy; 30.6% vs 17.5% (P 5
0.007) for knee replacement]. Unadjusted comparisons of length of stay
revealed that patients with SCD had mean hospital stays that were approxi-
mately two times longer than for patients without SCD who underwent the
same procedure (P < 0.001 for each comparison). Mean inpatient costs fol-
lowed a similar pattern, ranging from 32% higher for knee replacement to
91% higher for appendectomy (P < 0.001 for each comparison). The aver-
age cost among patients with SCD who underwent appendectomy was
nearly $10,000 more than for patients without SCD. Percentages of patients
with SCD who died after appendectomy or knee replacement were also sig-
nificantly higher than for patients without SCD who underwent the same pro-
cedure [2.9% vs 0.3% (P 5 0.02) for appendectomy; 3.3% vs 0.1% (P 5
0.001) for knee replacement], but the absolute numbers of patients with
SCD who died were small (n 5 2 for each procedure).
After adjustment for patient, procedural, and hospital characteristics,
length of stay and inpatient cost estimates remained significantly higher for
patients with SCD than for patients without SCD (P < 0.001 for each com-
parison). Compared to patients without SCD, the adjusted length of stay for
patients with SCD was 35% longer for hysterectomy (length of stay ratio,
1.35; 95% confidence interval [CI], 1.23–1.49), 85% longer for appendec-
tomy (length of stay ratio, 1.85; 95% CI, 1.56–2.20), and 81% longer for
knee replacement (length of stay ratio, 1.81; 95% CI, 1.62–2.02). Compared
to patients without SCD, the adjusted cost estimates for patients with SCD
were 28% higher for hysterectomy (cost ratio, 1.28; 95% CI, 1.18–1.39),
61% higher for appendectomy (cost ratio, 1.61; 95% CI, 1.40–1.85), and
19% higher for knee replacement (cost ratio, 1.19; 95% CI, 1.08–1.32).
After including inpatient length of stay and discharge disposition in the
regression model for inpatient costs, we found that higher inpatient costs
were largely attributable to longer hospital stays for all three surgical proce-
dures. Inpatient costs for patients with SCD who underwent hysterectomy or
appendectomy remained 8% and 22% higher, respectively, than for patients
without SCD, after adjustment for inpatient length of stay and discharge dis-
position (cost ratio for hysterectomy, 1.08; 95% CI, 1.01–1.15; cost ratio for
appendectomy, 1.22; 95% CI, 1.11–1.33).
Among patients with SCD, sickle cell crisis occurred in 5.9% (7/118) of
patients who underwent hysterectomy, 31.9% (22/69) of patients who under-
went appendectomy, and 14.5% (9/62) of patients who underwent knee
replacement (unadjusted P < 0.001 across the three surgical procedures).
Due to the small number of patients with sickle cell crisis, we did not evalu-
ate the impact of this complication on length of stay, costs, or mortality.
In summary, after adjustment for patient, procedural, and hospital charac-
teristics, patients with SCD incurred significantly longer inpatient stays and
higher total costs for hysterectomy, appendectomy, and knee replacement
than patients without SCD. Patients with SCD who underwent hysterectomy,
appendectomy, or knee replacement were also significantly more likely to
have a procedure code for blood transfusion during the inpatient stay,
although the overall rates may be somewhat low due to undercoding of this
procedure . After accounting for inpatient length of stay and discharge
disposition, the impact of SCD on inpatient costs was attenuated for all three
surgeries, but costs remained significantly higher for appendectomy and hys-
terectomy. The higher costs for appendectomy and hysterectomy may be
associated with higher rates of sickle cell crisis and nonelective admissions
for these two procedures. The study results were consistent with previous
findings that focused on cholecystectomy and hip replacement , indicating
that the study results may be generalizable to other procedures.
This study used the 2002–2006 NIS, part of the Healthcare Cost and Uti-
lization Project sponsored by the Agency for Healthcare Research and Qual-
ity (AHRQ). The 2006 NIS consisted of complete discharge information for
1045 hospitals in 38 states in the United States . The NIS includes
patient demographic characteristics, primary and secondary diagnoses, pro-
cedures performed, discharge status, admission and discharge dates, and
inpatient charges. Diagnoses and procedures are coded using both Interna-
tional Classification of Diseases, Ninth Revision, Clinical Modification (ICD-
9-CM) and Clinical Classification Software (CCS) codes. Inpatient charges
were converted to costs using inpatient cost-to-charge ratios for individual
NIS hospitals provided by AHRQ [21,22].
American Journal of Hematology79
We selected discharges of adults aged 18 to 64 years who had undergone
hysterectomy (CCS code 124), appendectomy (CCS code 80), or knee
replacement (CCS code 152). To limit the possibility that differences in inpa-
tient outcomes between patients with and without SCD were attributable to
hospital-level factors, we limited the sample of patients without SCD for
each procedure to hospitals where at least one patient with SCD was dis-
charged for the same procedure during the study period. Discharges of
patients with SCD were identified using CCS code 61 as the primary or any
secondary diagnosis. Sickle cell crisis was identified using ICD-9-CM diag-
nosis code 282.42, 282.62, or 282.64. Laparoscopic hysterectomy was iden-
tified using ICD-9-CM procedure code 68.31, 68.41, 68.51, 68.61, or 68.71.
Laparoscopic appendectomy was identified using ICD-9-CM procedure code
47.01 or 47.11. We used the presence of at least one of ICD-9-CM proce-
dure codes 99.01 to 99.04 to identify patients who received at least one
blood transfusion during the hospitalization. In addition, we used the Elix-
hauser comorbidity algorithm, developed by AHRQ, to identify relevant
comorbid conditions [17,18].
Analyzing each procedure separately, we used descriptive statistics to
summarize patient, procedural, and hospital characteristics and patient
outcomes. Comparisons of categorical variables were based on chi-
square tests or Fisher exact tests; comparisons of continuous variables
were based on t tests. A generalized linear regression model was used
to evaluate the independent association of SCD with length of stay and
costs, after adjustment for potentially confounding variables. For length
of stay, we used a negative binomial distribution with a log link. For
costs, we specified the model using a gamma distribution with a log
link. There were too few inpatient deaths to model mortality. Covariates
included in each model for inpatient length of stay and costs were age
(as a continuous variable); sex (for appendectomy and knee replace-
ment); primary expected payer (categorized as private, Medicare, Med-
icaid, self-pay or no charge, or other); Elixhauser comorbid conditions
[17,18] (including congestive heart failure, valvular disease, pulmonary
circulation disease, peripheral vascular disease, paralysis, other neuro-
logical disorders, chronic pulmonary disease, diabetes with or without
chronic complications, hypothyroidism, renal failure, liver disease, peptic
ulcer disease, acquired immune deficiency syndrome, lymphoma, meta-
static cancer, solid tumor without metastasis, rheumatoid arthritis, coa-
gulopathy, obesity, weight loss, fluid and electrolyte disorders, chronic
blood loss anemia, deficiency anemias, alcohol abuse, drug abuse, psy-
choses, and depression); elective vs nonelective surgery; surgery type
(ie, laparoscopic vs open surgery for hysterectomy and appendectomy);
urban vs rural hospital location; hospital teaching status; hospital bed
size; hospital ownership (public, private, not specified); and US geo-
To evaluate the impact of SCD on inpatient costs after adjustment for dif-
ferences in length of stay and discharge disposition, we added length of
stay as a continuous variable and discharge disposition as a categorical var-
iable (i.e., routine vs non-routine status, with non-routine status including
transfer to short-term hospital, transfer to other, home health care, left
against medical advice, died, and alive but destination unknown). In addition,
we sought to determine the effect of sickle cell crisis on inpatient length of
stay and costs. However, we could not perform multivariable regression
analysis because of the small number of patients with SCD who had a diag-
nosis of sickle cell crisis.
We used SAS software 9.1.3 (SAS Institute Inc, Cary, North Carolina) for
all statistical analyses. The institutional review board of the Duke University
Health System approved the study.
The authors thank Damon Seils of Duke University for assistance with
manuscript preparation. Mr Seils did not receive compensation for his
assistance apart from his employment at the institution where the study
1Center for Clinical and Genetic Economics, Duke Clinical Research Institute,
Duke University School of Medicine Durham, North Carolina
2Duke Comprehensive Sickle Cell Center, Division of Hematology, Department of
Medicine, Duke University School of Medicine Durham, North Carolina
3Gillings School of Global Public Health, University of North Carolina Chapel Hil,
TABLE I. Characteristics and Outcomes of Patients With or Without Sickle Cell Disease Who Underwent Hysterectomy, Appendectomy, or Knee Replacement
(n 5 118)
(n 5 85,073)
(n 5 69)
(n 5 24,802)
(n 5 62)
(n 5 14,517)
Age, mean (SE), y
Male, n (%)
surgery, n (%)
Blood transfusion, n (%)
Costs, mean (SE), $
Length of stay,
mean (SE), d
Mortality, n (%)
SCD, length of
stay, ratio (95% CI)a
cost, ratio (95% CI)a
cost, ratio (95% CI)a,b
SCD, sickle cell disease; SE, standard error; CI, confidence interval.
aAdjusted for age, sex (for appendectomy and knee replacement), primary expected payer, 29 comorbidities Ref. [17,18], laparoscopic surgery (for hysterectomy and appendectomy), elective surgery, hospital location, hospital teaching sta-
tus, hospital bed size, hospital ownership, and US geographic region.
bAdjusted for inpatient length of stay and discharge disposition.
80 American Journal of Hematology
4Division of General Internal Medicine, Department of Medicine, Duke University
School of Medicine Durham, North Carolina
Contract grant sponsor: National Heart, Lung, and Blood Institute (Sickle Cell
Disease Clinical Research Network); Contract grant number: U10HL083698
Conflict of interest: Dr. Reed has made available a detailed listing of financial
disclosures at http://www.dcri.duke.edu/research/coi.jsp.
*Correspondence to: Shelby D. Reed, Center for Clinical and Genetic Economics,
Duke Clinical Research Institute, PO Box 17969, Durham, NC 27715.
Published online 27 October 2009 in Wiley InterScience
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Department of Health and Human Services. Diseases and Conditions
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nih.gov/health/dci/Diseases/Sca/SCA_WhoIsAtRisk.html. Accessed April 22,
16. Vichinsky EP, Haberkern CM, Neumary L, et al. A comparison of conserva-
tive and aggressive transfusion regimes in the perioperative management of
sickle cell disease. N Engl J Med 1995;333:206–213.
17. Elixhauser A, Steiner C, Harris DR, Coffey RM. Comorbidity measures for
use with administrative data. Med Care 1998;36:8–27.
18. Healthcare Cost and Utilization Project. Comorbidity Software, Version 3.3.
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19. Segal JB, Ness PM, Powe NR. Validating billing data for RBC transfusions: A
brief report. Transfusion 2001;41:530–533.
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Cytogenetic correlates of TET2 mutations in 199 patients with
Kebede Hussein,1Omar Abdel-Wahab,2,3Terra L. Lasho,1Daniel L. Van Dyke,1Ross L. Levine,2,3
Curtis A. Hanson,1Animesh Pardanani,1and Ayalew Tefferi1
TET2 is a putative tumor suppressor gene located at chromosome
4q24. TET2 mutations were recently described in several myeloid neo-
plasms but correlations with cytogenetic findings have not been
studied. Among a recently described cohort of patients with myelopro-
liferative neoplasms (MPN) who underwent TET2 mutation analysis,
199 had information on karyotype at diagnosis or time of TET2 testing:
71 polycythemia vera (PV), 55 primary myelofibrosis (PMF), 43 essen-
tial thrombocythemia (ET), 13 post-PV MF, 7 post-ET MF, and 10 blast
phase MPN. Forty eight patients (24%) exhibited abnormal karyotype:
15 favorable (sole 20q-, 13q-, or 19), 8 unfavorable (complex karyotype
or sole 18), and 25 ‘‘other’’ cytogenetic abnormalities. We found no
significant difference either in the incidence or type of cytogenetic
abnormalities between TET2 mutated (n 5 25) and unmutated (n 5
174) cases. Seventy nine patients, including 14 with TET2 mutations,
underwent follow-up cytogenetic testing and the findings were again
not affected by TET2 mutational status. We conclude that TET2
mutated MPN patients are not cytogenetically different than their TET2
The Ten-Eleven Translocation (TET) oncogen family members include
TET1, TET2, and TET3. The TET1 oncogene was first identified as a fusion
partner to mixed-lineage leukemia (MLL) gene during the chromosomal
translocation t(10;11)(q22;q23) in acute myelogenous leukemia [1,2]. The
TET1 gene product catalyzes conversion of 5-methylcytosine to 5-hydroxy-
methylcytosine, suggesting a role in epigenetic regulation . TET3 is
located at chromosome 2p13.1 and its function is unknown. TET2 is a puta-
tive tumor suppressor gene located at chromosome 4q24. Delhommeau
et al. first reported the occurrence of TET2 mutations in patients with
JAK2V617F-positive MPN . Subsequently, our group reported similar
mutations in MPN with and without JAK2V617F mutations, systemic masto-
cytosis, and other myeloid malignancies [5–8]. In this study, we looked for
possible correlations between TET2 mutations and cytogenetic abnormalities
This study was approved by the institutional review board. Inclusion crite-
ria included availability of bone marrow cytogenetic results at diagnosis or at
the time of blood sample collection for TET2 mutation analysis. Follow-up
cytogenetic data, where available, were also recorded. The specific MPN
diagnosis was established on the basis of the 2001 World Health Organiza-
tion (WHO) criteria . Both direct techniques and unstimulated culture
methods were used to harvest metaphases for analysis from bone marrow
aspirate . JAK2V617F mutation analysis was performed according to
previously published methods . Cytogenetic findings were assigned as
being favorable (sole 20q-, 13q-, or 19), normal, unfavorable (complex or
18), or ‘‘other cytogenetic abnormalities’’, based on recent prognostic stud-
ies in PMF [12,13]. High-throughput DNA sequence analysis was used to
screen for TET2 mutations in bone marrow-derived DNA as described previ-
A total of 199 MPN patients had information on both TET2 mutational sta-
tus and karyotype: 71 PV, 43 ET, 55 PMF, 13 post-PV MF, 7 post-ET MF,
and 10 with blast phase MPN. Overall, 48 (24%) patients exhibited an
abnormal karyotype: 15 favorable, 8 unfavorable, and 25 ‘‘others’’. TET2
mutations were identified in 25 (13%) patients. As reported before, patients
who harbored TET2 mutations were significantly older (P 5 0.001) than
those with wild-type TET2. In contrast, the distribution of gender, specific
MPN diagnosis, and JAK2V617F mutational status was similar between the
two groups (Table I). A similar number of patients, 7 (18%) and 41 (24%)
with mutated and wild-type TET2, respectively, harbored an abnormal karyo-
American Journal of Hematology81
type (P 5 0.6). The distribution of cytogenetic categories (favorable, normal,
unfavorable or ‘‘others’’) was also similar between the 2 groups (Tables I and
II). Similar observations were made when the results of follow-up cytogenetic
studies that were available in 79 patients were analyzed (Table I). We con-
clude that TET2 mutations do not affect initial or subsequent cytogenetic
findings in MPN.
1Division of Hematology, Hematopathology, and Cytogenetics, Departments of
Medicine and Laboratory Medicine, Mayo Clinic, Rochester, Minnesota
2Human Oncology and Pathogenesis Program, Memorial Sloan-Kettering Cancer
Center, New York, New York
3Leukemia Service, Department of Medicine, Memorial Sloan-Kettering Cancer
Center, New York, New York
Conflict of interest: Nothing to report.
Published online 9 October 2009 in Wiley InterScience
1. Lorsbach RB, Moore J, Mathew S, et al. TET1, a member of a novel protein
family, is fused to MLL in acute myeloid leukemia containing the t(10;11)(q22;
q23). Leukemia 2003;17:637–641.
TABLE I. Clinical and Cytogenetic Features at Diagnosis and During Follow-Up in Patients with Myeloproliferative Neoplasms Who Underwent TET2
Findings at diagnosis or at time of TET2 testing Findings at time of follow-up cytogenetic studies
VariableAll n (%)Mutated n (%) Wild-type n (%)P value All n (%)Mutated n (%) Wild n (%)P value
Cytogenetics n (%)
Cytogenetics n (%)
Gender n (%)
Age in years median (range)
Age n (%)
JAK2V617F mutation n (%)
Myeloproliferative neoplasm n (%)
199 (100)25 (13) 174 (87) 79 (100) 14 (18)65 (82)
0.6 12 (15)
0.001 43 (54)
TABLE II. Cytogenetic Findings in 25 TET2-Mutated Patients with Myeloproliferative Neoplasms
PV, polycythemia vera; PMF, primary myelofibrosis; ET, essential thrombocythemia; AML, acute myelogenous leukemia.
a(43–46, XX, 25, 26, add(7) (p13), del(11) (q13q23),213, add(13)(p13),216,220,221,222,13-7mar[cp20]).
82American Journal of Hematology
2. Ono R, Taki T, Taketani T, et al. LCX, leukemia-associated protein with a
CXXC domain, is fused to MLL in acute myeloid leukemia with trilineage dys-
plasia having t(10;11)(q22;q23). Cancer Res 2002;62:4075–4080.
3. Tahiliani M, Koh KP, Shen Y, et al. Conversion of 5-methylcytosine to
5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1. Science
4. Delhommeau F, Dupont S, Della Valle V, et al. Mutation in TET2 in myeloid
cancers. N Engl J Med 2009;360:2289–2301.
5. Tefferi A, Pardanani A, Lim KH, et al. TET2 mutations and their clinical corre-
lates in polycythemia vera, essential thrombocythemia and myelofibrosis.
7. Tefferi A, Lim KH, Abdel-Wahab O, et al. Detection of mutant TET2 in mye-
loid malignancies other than myeloproliferative neoplasms: CMML, MDS,
MDS/MPN and AML. Leukemia 2009;23:1343–1345.
8. Abdel-Wahab O, Mullally A, Hedvat C, et al. Genetic characterization of TET1,
TET2, and TET3 alterations in myeloid malignancies. Blood 2009;114:144–147.
9. Harris NL, Jaffe ES, Diebold J, et al. The World Health Organization classifi-
cation of neoplasms of the hematopoietic and lymphoid tissues: Report of the
Clinical Advisory Committee meeting–Airlie House, Virginia, November, 1997.
Hematol J 2000;1:53–66.
10. Dewald GW, Broderick DJ, Tom WW, et al. The efficacy of direct, 24-hour cul-
ture, and mitotic synchronization methods for cytogenetic analysis of bone
marrow in neoplastic hematologic disorders. Cancer Genet Cytogenet 1985;
11. Tefferi A, Lasho TL, Huang J, et al. Low JAK2V617F allele burden in primary
myelofibrosis, compared to either a higher allele burden or unmutated status,
is associated with inferior overall and leukemia-free survival. Leukemia 2008;
12. Hussein K, Huang J, Lasho T, et al. Karyotype complements the International
Prognostic Scoring System for primary myelofibrosis. Eur J Haematol 2009;
13. Hussein K, Van Dyke DL, Tefferi A. Conventional cytogenetics in myelo-
fibrosis: Literature review and discussion. Eur J Haematol 2009;82:329–
Endogenous thrombin potential in the assessment of
hypercoagulability in systemic lupus erythematosus
Bijal M. Mehta,1Adnan N. Kiani,2Catherine Chen,2Jayesh Jani,3Thomas S. Kickler,3and Michelle Petri2*
Endogenous thrombin potential (ETP) is a measurement of thrombin
formation capacity of plasma and may be increased in congenital and
acquired hypercoagulable states. We assessed whether ETP was asso-
ciated with antiphospholipid antibodies (aPL) and with thrombosis his-
tory in SLE. ETP was performed in 130 SLE patients using a Siemens
ETP Assay on BCS Coagulation System analyzer, that is equipped with
software to analyze different components of the coagulation wave
form, including lag-time (time to initiate thrombin generation), T-max
(estimate of enzymatic rate), C-max (measurement of peak height), and
area under the curve (total thrombin formation). Higher T-lag values
were found with deep venous thrombosis (DVT) (33.6 ± 15.9 vs. 22.9 ±
14.8, P 5 0.0018), myocardial infarction (MI) (43.6 ± 36.4 vs. 24.6 ± 15.1,
P 5 0.0855) and stroke (27.5 ± 13.5 vs. 24.5 ± 15.8, P 5 0.4883) than
without. T-max was also higher in patients with DVT (68.4 ± 21.9 vs.
56.5 ± 24.4, P 5 0.0300), and MI (123.8 ± 77.0 vs. 57.7 ± 22.1, P ?
0.0001) compared to those without. ETP T-lag and T-max were higher
for patients with aPL. ETP T-lag and T-max were associated with both
venous (DVT) and arterial (stroke, MI) thrombosis in SLE and with aPL.
This suggests that ETP measures should now be explored prospec-
tively to determine their predictive value for future thrombosis in SLE.
Systemic lupus erythematosus is a multisystem autoimmune disorder
mostly affecting young women . Thrombosis has been reported in 7.2–
12% of patients with SLE [2,3]. The proportionate mortality from thrombosis
in SLE is 26.7% . Multiple studies in SLE have shown a positive associa-
tion between antiphospholipid antibodies and thrombosis [3–6]. Because not
all patients with antiphospholipid antibodies suffer thrombosis, identification
of those with higher risk is important. The mechanism of anti phospholipid
antibody associated thrombosis appears to be multifactorial, including com-
plement activation , platelet activation [8,9], modulation of endothelial
function , and interference with the Protein C pathway through the devel-
opment of acquired protein C resistance and deficiencies of Protein C and
Protein S [11,12]. Increased expression of tissue factor in macrophages has
also been proposed .
The hemostatic system maintains a balance between procoagulant and anti-
coagulant pathways . The net effect of the hemostatic pathway is to gener-
ate thrombin to permit the conversion of fibrinogen to fibrin. A decrease in
thrombin generation, either acquired or inherited, can lead to impaired coagu-
lation and bleeding. In contrast, increased thrombin generation can lead to clot-
ting tendency and thrombosis. Hypocoagulation can readily be detected by
routine laboratory tests. In contrast, despite remarkable progress in defining
new risk factors for thrombosis, a laboratory test that globally measures overall
thrombin formation has not, until recently, been available .
Measurement of the thrombin generation curve is a well-established tool
in coagulation research, reflecting overall function of the blood coagulation
system . In platelet poor plasma, thrombin generation measurement
reflects all clotting deficiencies except for factor XIII, and is sensitive to oral
anticoagulants and heparin. Hyperprothrombinemia, absence of antithrom-
bin, protein C and S as well as activated protein C resistance increase
thrombin generation. Despite utilization of thrombin generation in the
research laboratory, the clinical application of measuring thrombin genera-
tion is only beginning. Recent improvement in reagents, coagulation equip-
ment, and software now permit the study of thrombin potential in many dif-
ferent clinical situations. Recently, this testing has been shown to be promis-
ing in assessing the risk of clotting recurrence [17,18]. However, this test
has not been approved for clinical use in the United States.
In our study, we hypothesized that the application of thrombin potential
might be useful in determining which SLE patients were at risk for thrombo-
sis. We now report the relationship of thrombin generation parameters to
clinical thrombosis in a cohort of patients with systemic lupus erythemato-
sus, including those with antiphospholipid antibodies.
Endogenous thrombin potential (ETP) is the amount of thrombin formed in
vitro under different conditions in a clotting reaction from the beginning to
the end of the reaction. It is thought that the ETP reflects the in vivo
capacity of an individual to generate thrombin.
Materials and Methods
Patients: One hundred and thirty patients with SLE were enrolled in this
study. These patients were enrolled from the Hopkins Lupus Cohort, a pro-
spective study of disease activity and predictors of morbidity and health sta-
tus. The study was approved by the Johns Hopkins University School of
Medicine Institutional Review Board. All patients gave written informed con-
sent. All patients had been seen quarterly (or more often if warranted) since
cohort entry, for assessment of disease activity (by the Physician’s Global
Assessment on a 0–3 visual analog, and the SELENA SLEDAI) [19,20], lab-
oratory tests (complete blood count, erythrocyte sedimentation rate, serum
creatinine, cholesterol, urinalysis, C3, C4, and anti-dsDNA), anticardiolipin
(ELISA, INOVA) and dilute Russell viper venom time . Cardiovascular
risk factors and thrombotic history were also assessed. Of these 130
patients, 24 had a history of deep venous thrombosis (DVT), 15 had a cere-
brovascular accident (CVA) in the past, and two had a myocardial infarction
(MI) in the past. The specimens were collected at the time of their routine
visit, and there was no relation to occurrence of thrombotic events. 24
patients were on warfarin and one was on heparin at the time of their visit
when blood work was done for measurement of ETP. 98 and 60 patients
had elevated anticardiolipin antibody and elevated diluted russel viper venom
time (dRVVT) respectively, at some point during their work up. The dRVVT
test was done according to manufacturer’s procedure and employed a confir-
matory step with addition of phospholipid for those patients with a prolonged
dRVVT (Siemens, Marburg Germany).
American Journal of Hematology 83
ETP was performed using the ETP kit by Siemens (Marburg Germany)
and a Siemens BCS analyzer. To determine ETP, the conversion kinetics of
a synthetic thrombin substrate were measured by the release of a chromo-
phore in the plasma sample at a wavelength of 405 nm. The thrombin for-
mation was started by the addition of Dade Innovin Reagent and calcium
chloride. The thrombin formation curve was obtained by mathematical deri-
vation of the measured formation kinetics, whose integral (described as the
‘‘surface area under the curve’’) corresponds to ETP. In addition to being
converted by thrombin, the substrate was also converted by the resulting
thrombin-a2-macroglobulin complex. To determine the ETP, the reaction rate
of the thrombin-a2-macroglobulin complex was calculated based on the total
conversion and then subtracted. The necessary calculations were carried out
automatically by the BCS System with the available ETP analysis procedure.
The development of new software by Siemens Diagnostics, Marburg, Ger-
many permits the plotting and analysis of the entire curve of thrombin forma-
tion, i.e., the thrombogram. (See Fig. 1) . The parameters that can be
derived from the thrombin generation curve are the following: the lag time,
endogenous thrombin potential (area under the curve), peak height of
thrombin which reflects the maximum amount of thrombin generated from
the plasma, and time to peak thrombin formation .
Samples were collected in 3.2% citrate. After collection, the samples were
spun at 2500g on an Ependorf centrifuge to remove any platelets or micro-
particles. The samples were then frozen at 2708C until testing. At the time
of testing the samples were thawed in a 378C waterbath, and then again
spun at 2500g before testing.
All results for continuous variables are expressed as means ± SD, unless
specified otherwise. Continuous variables were analyzed with a two-sided t-
test and categorical variables were compared by the Pearson chi-square or
Fishers exact test. Oneway ANOVA was performed for normally distributed
variables. Statistical analysis was performed using JMP (v5.0.1, SAS Insti-
tute, Cary, NC). A P-value of 0.05 was taken as statistically significant.
Results and Discussion
Data were obtained from 130 patients with SLE. The patients were 94%
female, 6% male, 5% Asian, 2% Hispanic, 38% African-American, 50%
Caucasian, and 4% other ethnicities. The mean age was 45 years. The ETP
reference ranges in normal patient plasma (20 males and 20 females age
25–60 years), were T-lag (12.5 ± 21.3), T-max (36.4 ± 60.0), C-max
(124.5 ± 188.5), and AUC (265.4 ± 637.8). The reference ranges were col-
lected from normal laboratory employees and done on platelet poor plasma.
Tables I and II summarize the results of ETP T-lag and T-max of our
patients. SLE patients with deep venous thrombosis (DVT) and myocardial
infarction (MI) had greater T-lag (the time to initiation of activation of throm-
bin) and T-max (the estimate of enzymatic rate) values than patients without.
ETP T-lag and T-max were also higher in SLE patients with the lupus antico-
agulant and anticardiolipin. The presence of lupus anticoagulant and anticar-
diolipin had no significant association with the C-max levels (the highest
point, or peak, of the thrombin generation curve). The AUC for SLE patients
with DVT, MI and stroke was lower than in patients without DVT, MI, or
stroke. The AUC was also lower in patients with the lupus anticoagulant and
anticardiolipin. Similarly, it was lower in those patients with venous and arte-
rial thrombosis, than in those without thrombosis.
Anticoagulants like warfarin as well as heparin would produce decreased
area under the curve, C-max, and prolong the lag time. As the amount of
thrombin formed decreases in patients who are adequately anticoagulated,
the T-max should be lower and not higher.
Past studies have shown increased thrombin generation in women on oral
contraceptive pills and with a history of venous thromboembolism [22,23] or
with a history of venous thromboembolism only . In these studies
[23,24], ETP was measured in the presence of thrombomodulin (TM). How-
ever, one of the above studies excluded patients with antiphospholipid syn-
drome  and another study  excluded patients with antiphospholipid
antibodies/lupus anticoagulants. Our study is the first large study of ETP in
SLE, and the first study to report on the different parameters of the thrombin
generation curve in SLE.
A recent study  has shown that measurement of whole blood thrombin
generation assay, peak height, and ETP were higher in patients with a his-
tory of venous thromboembolism compared to controls. However, in our
study, we used platelet poor plasma. Use of platelet rich plasma in the test-
ing of thrombin generation also reflects the contribution of platelets in the
generation thrombin. A major disadvantage of using platelet rich plasma is
that only fresh samples can be used to produce reliable results.
In contrast to a previous study, we did not find higher ETP AUC levels in
SLE, nor in patients with known hypercoagulable states, such as antiphos-
pholipid antibodies . Instead, we found that the T-lag and T-max were
TABLE I. T-lag: Time to Initial Inflection of the Curve—Reference
Range in Normal Patient Plasma: 12.5 ± 21.3
Historical variables Factor negativeFactor positiveP-value
All thrombosis ever
Myocardial infarction ever
Digital Gangrene ever
21.7 ± 12.9 (95)
22.9 ± 14.8 (106)
33.4 ± 18.6 (35)
33.6 ± 15.9 (24)
24.6 ± 15.1 (128)
24.5 ± 15.8 (115)
24.8 ± 15.6 (128)
19.8 ± 7.7 (70)
43.6 ± 36.4 (2)
27.5 ± 13.5 (15)
25.0 ± 17.4 (2)
30.8 ± 19.7 (60)
21.8 ± 11.7 (33)
23.3 ± 14.2 (71)
25.8 ± 16.5 (97)
26.8 ± 18.0 (47)
Same day visit variables Rsquare adjP-value
TABLE II. T-max: Time to Maximum Thrombin Generation—Refer-
ence Range in Normal Patient Plasma: 36.4 ± 60.0
Historical variablesFactor negativeFactor positiveP-value
All thrombosis ever
Digital Gangrene ever
57.8 ± 24.0 (95)
56.5 ± 24.4 (106)
70.9 ± 26.4 (35)
68.4 ± 21.9 (24)
57.7 ± 22.1 (128)123.8 ± 77.0 (2)
57.7 ± 24.3 (115)
58.7 ± 24.4 (128)
51.1 ± 19.2 (70)
66.3 ± 24.2 (15)
54.3 ± 27.0 (2)
67.5 ± 26.7 (60)
53.4 ± 25.8 (33)
56.6 ± 22.7 (71)
60.5 ± 23.7 (97)
62.4 ± 27.4 (47)
Same day visit variablesRsquare adjP-value
time to initial inflection of the curve, T-max, time to maximum thrombin generation;
C-max, maximum amount of thrombin generated; AUC, total thrombin generated,
ETP. [Color figure can be viewed in the online issue, which is available at www.
A schematic representation of a thrombin generation curve. Lag-time,
84American Journal of Hematology
higher in SLE patients with antiphospholipid antibodies. We anticipated that
the T-max would simply reflect the AUC and we were surprised to observe
discordant results between the AUC and T-max. We do not have an explana-
tion for this. Because a relatively low amount of phospholipid is used to ini-
tiate thrombin formation, it is conceivable that the prolonged lag time reflects
the effect of antiphospholipid antibody in blocking the effect of phospholipid.
While the increased T-max may reflect accelerated formation of thrombin,
the prolonged T-lag may actually contribute toward the finding. In the calcu-
lation of T-max both the lag time and slope of the thrombin generation curve
determine T-max. If indeed there is a higher rate of thrombin formation, the
significance of this is unclear. It is conceivable that this may reflect an initial
burst of thrombin formation that is dampened by anti-thrombin or other natu-
ral thrombin inhibitors. Regardless of this, overall our findings do not indicate
that under the conditions we employed could we demonstrate increased
thrombin potential. The limitation of our in vitro studies to reflect in vivo phe-
nomenon, is that we are not using whole blood which includes the contribu-
tion of platelets and platelet microparticles.
Clearly the pathogenesis of thrombosis associated with antiphospholipid
antibodies in SLE is multifactorial, involving not just the procoagulant pro-
teins, the cellular constituents involving hemostasis, the anticoagulant mech-
anisms and fibrinolytic pathway. We know that the thrombotic complications
are ameliorated by anti-thrombin agents including heparin and warfarin. This
clearly argues for a key role of thrombin in the pathogenesis of the compli-
cations associated with antiphospholipid antibodies.
As we begin to correlate the results of thrombin generation tests with a
variety of clotting and bleeding disorders, we are likely to gain a better
understanding of the meaning of different parts of the thrombogram. The
analysis of wave forms, of which the thrombogram is an example, is a new
way to dissect out the complex interactions of coagulation proteins. The
challenge is to correlate these parameters with clinical events or other bio-
chemical or functional measurements of coagulation.
Our studies show that there are changes in the thrombogram associated
with antiphospholipid antibodies and history of thrombosis in SLE. Future
prospective study is now warranted to determine the predictive value of ETP
measures for future thrombosis.
1Cleveland Clinic, Department of Rheumatic and Immunologic Diseases,
2Division of Rheumatology, Johns Hopkins University School of Medicine,
3Pathology Department, Special Coagulation Laboratory,
Johns Hopkins University School of Medicine, Baltimore, Maryland
*Correspondence to: Michelle Petri; 1830 East Monument Street, Suite 7500,
Baltimore MD 21205, USA., Telephone: 410-955-3823, Fax no: 410-614-0498.
Contract grant sponsor: National Institute of Health; Contract grant number:
NIH AR 43727; Contract grant sponsor: General Clinical Research Center;
Contract grant number: MO1-RR00052; Contract grant sponsor: Siemens Medical
Diagnostics, Marburg, Germany.
Conflict of interest: Nothing to report.
Published online 14 October 2009 in Wiley InterScience (www.interscience.wiley.com).
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HIV-negative, HHV-8-unrelated primary effusion lymphoma-like
lymphoma: report of two cases
Tsuyoshi Takahashi, Akira Hangaishi, Go Yamamoto, Motoshi Ichikawa,
Yoichi Imai, and Mineo Kurokawa*
Primary effusion lymphoma (PEL) is a rare type of lymphoma confined to
the body cavities, such as pleural, pericardial, and peritoneal cavities.
PEL is usually associated with human herpes virus 8 (HHV-8) and human
immunodeficiency virus (HIV) infection, however, there are some reports
of HIV-negative and HHV-8-unrelated cases. Recently, these cases are
described as HHV-8-unrelated PEL-like lymphoma. Here, we report two
American Journal of Hematology 85
such cases. In both cases, no lymphadenopathy or organ involvement
with lymphoma was found. Surface marker revealed that they were both
CD20 positive lymphoma. Systemic chemotherapy with CHOP regimen
with rituximab was effective and gradually led to disappearance of the
lymphoma. HHV-8-unrelated PEL-like lymphoma is truly a distinct clinical
entity and the prognosis of it seems to be better than PEL.
PEL is a very rare type of non-Hodgkin lymphoma that involves only body
cavities . According to the World Health Organization (WHO) classification
of hematological malignancies, PEL is classified as a subtype of diffuse
large B-cell lymphoma that is closely associated with human herpes virus-8
(HHV-8) and HIV . On the other hand, it has been reported that there are
some patients with HHV8-negative and HIV-negative PEL that highly
expresses B-cell markers, which are described as HHV-8-unrelated PEL-like
lymphoma . The reports of HHV-8-unrelated PEL-like lymphoma are anec-
dotal and the character of the lymphoma is not well known yet. Here, we
report two cases of HHV-8-unrelated PEL-like lymphoma who were success-
fully treated with R-CHOP and review of the literature.
A 82-year-old man went to an outpatient clinic because ofedema of his lower
extremities in January, 2008. He was found to have massive pericardial effu-
sion, left pleural effusion, and sign of cardiac decompensation. Soon after
admission, the patient was treated with drainage of the pericardial and pleural
effusion. On cytological examination of the pleural and pericardial effusion,
middle to large-sized atypical lymphoid cells were observed. The cells were
positive for CD20 and CD79a, but negative for CD3. The immunoglobulin light
chain restriction was also observed. He was suspected to have PEL and intro-
duced to our hospital. When he was admitted, he had massive left pleural effu-
sion and moderate pericardial effusion. The serum lactose dehydrogenase
(LDH) level was 214 IU/L. Tests for hepatitis C virus (HCV) and HIV antibody
were negative. Cytological evaluation of the pleural effusion demonstrated mid-
dle to large-sized atypical lymphoid cells with prominent nucleoli. The cell block
preparation of pleural effusion revealed that atypical lymphoid cells were nega-
tive for HHV-8, but positive for EBER-ISH and EBNA2. The pleural effusion
test for HHV-8 using polymerase chain reaction (PCR) method was also nega-
tive. No mass or lymphoma cells were detected on whole body CT scan, FDG-
PET, and bone marrow biopsy. He was diagnosed as HIV-negative HHV-8-
unrelated PEL-like lymphoma. The patient was treated with six courses of che-
motherapy consisting of rituximab, cyclophosphamide, doxorubicin, vincristine,
and prednisolone (R-CHOP). After six courses of R-CHOP, the pleural effusion
and pericardial effusion became little left. Now, 12 months passed since the
last chemotherapy, and although the slight effusion is still left, disease status
has continued to be stable without further treatment. Chest X-rays when the
patient was diagnosedandnow are shown inFig.1A.
A 73-year-old man had edema of his lower thighs and he was diagnosed as
having pericardial effusion, pleural effusion, and ascites on whole body CT
scan. In January 2009, he had shortness of breath and came to the out-
patient clinic of our hospital. He was diagnosed as cardiac decompensa-
tion with massive pericardial effusion and treated with drainage of it. On
the cytological examination of the pericardial effusion, large atypical lym-
phoid cells with prominent nucleoli were observed. The cell block prepa-
ration of the pericardial effusion revealed that the cells were positive for
CD20, but negative for CD3, HHV-8, and EBER-ISH. No masses or lym-
phoma cells were detected on whole body CT scan, FDG-PET, and bone
marrow biopsy. Tests for HCV and HIV antibody were negative. He was
diagnosed as HIV-negative HHV-8-unrelated PEL-like lymphoma. The
patient was treated with six courses of R-CHOP therapy. After repeated
courses of R-CHOP, the pericardial effusion and pleural effusion gradu-
ally decreased. However, after five courses of R-CHOP, liver dysfunction
appeared. The ultrasonographic examination revealed that he had con-
gestion of the liver due to recurred pericardial effusion. Aspiration of fluid
from pericardium was performed twice. However, invasion of lymphoma
cells were not detected in evaluation of cytology and flow cytometric
analysis at this time. After that, liver dysfunction resolved and pericardial
effusion was stable with slight pleural effusion. He was performed six
round of R-CHOP treatment then discharged. Chest X-rays when the
patient was diagnosed and now are shown in Fig. 1B.
PEL was originally described in 1989 as B-cell lymphomatous effusion
in a body cavity without detectable tumor masses and associated with
HHV-8 and HIV infection, mostly occurs in immunodeficiency status [1–3].
However, this entity has been reported in a small number of cases associ-
ated with HIV-negative HHV-8-unrelated PEL-like lymphoma [4–6]. The
PEL lymphoma cells are usually negative for pan-B-cell markers, such as
CD19, CD20, and CD79a. On the other hand, HIV-negative HHV-8-unre-
lated PEL-like lymphoma cells highly express B-cell markers. In our cases,
the lymphoma cells also expressed CD20 and CD79a. As for the patho-
genesis of PEL-like lymphoma, Tanaka et al. reported that some of these
were EBV positive . HCV had also been suggested to be an etiological
agent . Both of the present cases were HCV negative, although case 1
was EBV positive and case 2 was negative.
As to treatment, there is no standard chemotherapeutic regimen recom-
mended for HIV-negative HHV-8-unrelated PEL-like lymphoma because of small
numbers of reports. CHOP-like regimen had been frequently given in these
cases. Recently, rituximab, an anti-CD20 monoclonal antibody, has been incor-
porated into the standard chemotherapy for many B-cell NHLs showing CD20
positivity. In both of our cases, we used rituximab containing regimen because
the lymphoma cellswere CD20 positive and it was effective in both cases.
The prognosis of PEL is poor and the median survival of PEL is less than
6 months, whereas the prognosis of HIV-negative HHV-8-unrelated PEL-like
lymphoma may be better than that [9,10]. In our cases, one is alive for 21
months and another is alive for 9 months after their diagnoses. Thus prog-
nosis of PEL-like lymphoma seems to be better than that of PEL as reported
previously. In light of the cases from literature and our present ones, PEL
and HIV-negative HHV-8-unrelated PEL-like lymphoma may have different
pathogenesis, immunophenotypic features, and prognosis.
Department of Hematology and Oncology, Graduate School of Medicine,
University of Tokyo, Tokyo, Japan
*Correspondence to: Mineo Kurokawa, Department of Hematology and Oncology,
Graduate School of Medicine,
University of Tokyo, 7-3-1 Hongo,
Bunkyo-ku, Tokyo 113-8655, Japan.
Conflict of interest: Nothing to report.
Published online 16 October 2009 in Wiley InterScience (www.interscience.wiley.com).
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panel) and now (right panel).
Chest X-rays of the cases. A: Chest X-rays when case 1 was diagnosed (left panel) and now (right panel). B: Chest X-rays when case 2 was diagnosed (left
86 American Journal of Hematology
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Tumor flare reactions and response to lenalidomide in patients
with refractory classic Hodgkin lymphoma
Gaetano Corazzelli,1Rosaria De Filippi,1,2Gaetana Capobianco,1Ferdinando Frigeri,1
Vincenzo De Rosa,3Giancarla Iaccarino,1Filippo Russo,1Manuela Arcamone,1Cristina Becchimanzi,1
Stefania Crisci,1Gianpaolo Marcacci,1Barbara Amoroso,4Secondo Lastoria,5and Antonio Pinto1*
Patients with Hodgkin lymphoma (HL) failing salvage stem cell trans-
plantation are candidates to investigational strategies. Lenalidomide
represents an attractive option as it targets several signaling path-
ways, which regulate survival of HL cells and their microenvironmental
interactions. We report the occurrence of Grades 2 and 3 tumor flare
reactions in the first three patients entered a lenalidomide-based com-
passionate program for treatment-refractory HL. Flares occurred in
concomitance of the scheduled week-off lenalidomide and upon with-
drawal of symptomatic steroid treatment, and were associated with
changes in B-cell regulatory cytokines and the concurrent expansion
of polyclonal B-cells. Flares mimicked tumor progression but were
effectively managed with anti-inflammatory treatment and followed by
a clinical response, suggesting that they may mirror the pleiotropic
actions of lenalidomide on HL microenvironment.
Patients with Hodgkin’s Lymphoma (HL) recurring after stem cell trans-
plantation are incurable and candidates to investigational agents . Lenali-
domide, a thalidomide-derivative highly active in myeloma and other B-cell
malignancies, represents an attractive option due to its pleiotropic effects on
tumor cells and their microenvironment . In this sense, signaling pathways
regulating survival of HL cells and their interactions with bystander immune
and stromal cells in tumor tissues overlap with those modulating proliferation
and cross-talk of malignant plasma cells in the bone marrow (BM) microenvir-
onment. These involve receptors/ligands of the tumor necrosis factor (TNF)
superfamily, cytokines, chemokines and adhesion/co-stimulatory and proangio-
genic molecules . Lenalidomide targets several of these pathways and
might exert a direct effect on tumor cells by inhibiting Akt phosphorylation and/
or cyclin-dependent kinases [2,4]. Based on these evidences a lenalidomide-
based compassionate program for biopsy-proven treatment refractory HL was
launched at our Institution. We herein describe the occurrence of a tumor flare
reaction (TFR) in the first three patients entered the program.
The Institutional Ethic Committee approved (April 2008), a program of oral
lenalidomide at the dose of 25 mg from day 1 to day 21 of a 28-day cycle
until progression. Lenalidomide (Revlimid1) was purchased from Celgene
(Summit, NJ), and the patients were treated after written informed consent
and the approval of a pregnancy-prevention and risk-management plan.
Response evaluation was planned after three courses and scored according
to the revised International Criteria integrated by fluorine-18-fluorodeoxyglu-
cose (18F-FDG) positron emission tomography . Changes in superficial
nodal sites were monitored by bi-weekly ultrasonography, expressed as the
change in sum of the products of the greatest diameter (SDP) from baseline
and plotted as bar-graph histogram against time. Serum levels of interleukin
(IL)-6 and -7, a proliferation-inducing ligand (APRIL) and the B-lymphocyte
stimulator (BAFF/BlyS) were concurrently evaluated as these cytokines are
highly expressed in HL microenvironment, by regulating proliferation/activa-
tion of normal B-lymphocytes and tumor cells, and correlate with clinical–
pathological features and outcomes [3,6]. Serial serum samples were stored
frozen at –808C and assayed in parallel by ELISA kits for IL-6 and IL-7
(DRG, Mountainside, NJ), APRIL (Bender Medsystems, Burlingame, CA),
BAFF/BlyS (R&D Systems, Minneapolis, MN) according to manufacturer
instructions. Serum free light chains (sFLC) were also assayed to qualify
systemic B-cell expansions . Immunonephelometric sFLC testing was per-
formed according to manufacturer instructions (Freelite, The Binding Site,
TABLE I. Patient Characteristics, Toxicity, And Response to Lenalidomide
1 38/MSN6 Autologous
IVB 941 SD 138Grade 2 anemia
Grade 2 fatigue
2 34/MSN7 IVB1102 SD124Grade 2 anemia
Grade 2 thrombopenia
Grade 3 anemia
Grade 2 thrombopenia
Grade 2 tumor flare
Grade 2 fatigue
Grade 3 tumor flare
Grade 2 fatigue
Grade 2 diarrhea
NS, nodular sclerosis; SCT, stem cell transplantation; PS, performance status; SD, stable disease; PR, partial remission.
aAccording to CTCAE v3.0.
American Journal of Hematology 87
sites during lenalidomide therapy. Data is presented as percentage variation in the sum of products of the greatest diameter (SDP) of measurable lesions from baseline
and plotted as bar-graph histogram against time. Target lesions were evaluated by biweekly ultrasonography as follows: patient 1, bilateral supraclaevear lymph nodes
(left panel); patient 2, left laterocervical, lumboaortic and bilateral iliac and inguinal lymph nodes and spleen (middle panel); patient 3, bilateral retroclavicular, right axillary,
lumboaortic, bilateral iliac and inguinal lymph nodes and spleen (right panel). (B) Changes in circulating B-cell stimulatory cytokines during therapy with lenalidomide.
Serum samples were collected at baseline (before the first course), before starting the second and third courses of lenalidomide and at one month after the third course.
Data is presented as serum concentrations in picogram per milliliter, for each patient: patient 1, left panel; patient 2, middle panel; patient 3, right panel. (C) Changes in
serum free light chains (FLC) levels (normal ranges, k: 3.3–19.4 mg/L; l: 5.71–26.3 mg/L) during therapy with lenalidomide. Data is presented as serum concentrations
in milligram per liter, for each patient: patient 1, left panel; patient 2, middle panel; patient 3, right panel. In all instances, the serum FLC ratio, calculated as k/l (free k
concentration divided by free l) remained within the normal reference range (0.26–1.65), except for the second time point of patient 1, where a free k/l ratio of 1.82 was
calculated. (D) Patient 1, computed tomography scan of the thorax at the start of lenalidomide therapy (left panel). Computed tomography (middle panel) and18F-FDG-
PET (right panel) scans of the same thorax area during tumor flare reaction (TFR) occurrence (i.e., day 130 from starting of treatment). The infiltrative tissue at the right
sternocostal–osteochondral junction, with lytic microlacunar erosion of the bone margin associated with small retrosternal confluent nodulations (left panel), evolved into a
markedly edematous inflammatory area of hypodensity, which deformed the anterior chest wall (middle panel) and strongly retained18F-FDG (right panel), so mimicking
tumor progression. The TFR extensively involved the muscoloaponeurotic planes of the pectoral region with inflammatory edema spreading to pleurocostal planes and
mediastinum. (E) Patient 3,18F-FDG-PET-documented partial response to lenalidomide after three complete courses. Note that the marked reduction in number, size,
and intensity of18F-FDG uptake of disease sites, including spleen and extranodal lesions.
Lenalidomide-related development of tumor flares and responses in patients with Hodgkin lymphoma. (A) Dimensional changes in disease-involved target
88American Journal of Hematology
Patients’ characteristics, response outcomes and toxicity to lenalidomide
(NCI Common Toxicity Criteria v3.0), are detailed in Table I.
Patient 1 had baseline bilateral supraclavicular nodal involvement and a
retrosternal confluent adenopathy infiltrating the right sternocostal–osteo-
chondral junction. He was under continuous methylprednisone (16 mg/day)
for persistent fever and sweats. Two weeks after starting lenalidomide, a
30% decrease in SDP of superficial lymph nodes was observed (Fig. 1)
along with B symptoms resolution, leading to complete steroid taper at day
119. Shortly before the second course (day 126) the patient presented with
a re-enlargement of the superficial nodes and a massive painful swelling
which deformed the right anterior chest wall. Imaging documented a mark-
edly edematous hypodensity area extensively involving pectoral muscoloapo-
neurotic planes and spreading to pleurocostal planes and mediastinum,
which avidly retained18F-FDG (Fig. 1). A Grade 2 TFR was diagnosed, and
tramadol, naproxen and methylprednisone (8 mg/day) were administered
while continuing on full-dose lenalidomide. Ten days later, pain and swelling
resolved and two further courses were given leading to a stepwise decrease
of nodal sizes down to 58% of pretreatment values (Fig. 1), consistent with
In patient 2, disease involved multiple nodal (laterocervical, internal
mammary, bilateral lung hilar, lumboaortic, iliac, and bilateral inguinal) and
extranodal (dorsal and lumbar spine, femurs) sites. Despite steroid-related
diabetes, the patient was under methylprednisone (24 mg/day), due to profuse
sweating, fever and analgesic-requiring generalized bone pain. He had throm-
bocytopenia (68.000 mL21), a hypocellular BM and a massively enlarged
spleen (158 3 153 3 58 mm) with miliary/micronodular lesions. After 2 weeks
of lenalidomide, B symptoms subsided, nodal lesions shrank by 30% (Fig. 1)
and steroids were withdrawn by day 119, also to reduce insulin. At day 127,
the patient was admitted with a painful nodal re-enlargement (SDP 120%), a
gross right groin lesion (maximum diameter, 69 mm) and severe back and pel-
vic pain. A Grade 2 TFR was diagnosed and treatment with oxycodone and
ketoprofen led to complete symptoms resolution, including bone and groin
pain disappearance at analgesics weaning. Following courses were delivered
at full doses, yielding to disease stabilization (39% reduction in overall SDP)
and normalization of platelets to 194.000 mL21(Fig. 1).
In patient 3, disease involved nodal (bilateral retroclavicular, axillary, lum-
boaortic-iliac, and inguinal) and extranodal (lungs, sternal ribs) sites (Fig. 1).
Spleen was enlarged (169 3 149 3 63 mm) with multiple nodulations. The
patient displayed thrombocytopenia (77.000 mL21), anemia (Hgb 7.7 g/dL) and
suffered from cough, exertion dyspnea and persistent fever requiring methyl-
prednisone (16 mg/day). Lenalidomide was withheld on day 118 due to afe-
brile microbiological-negative Grade 2 diarrhea, controlled by octreotide ace-
tate. Meanwhile, a 26% reduction of nodal sites was recorded along with cough
resolution and improvement of dyspnea, allowing steroid withdrawal by day
124. The second course was started at 25 mg/day, but 5 days later the patient
developed a disabling chest bone pain exacerbated by finger pressure and
breathing, and crippling gait impairment due to a wide and painful inflammatory
swelling of left inguinal lymph nodes involving the whole groin and the upper
thigh. A Grade 3 TFR was diagnosed and lenalidomide lowered to 15 mg/day.
Tramadol, naproxen and methylprednisone (8 mg/day) were administered,
leading to complete clinical resolution after 7 days. The third course was deliv-
ered at 15 mg/day, leading to a partial remission with reduction in number, size
and18F-FDG uptake of all disease sites including spleen (Fig. 1), and a stable
recovery of platelets (>130.000 mL21) and hemoglobin (>8.5 g/dL).
In none of the patients lenalidomide was discontinued. Best responses were
recorded after three courses, and the progressions were documented on days
1138, 1124, and 1164 (Table I). As show in Fig. 1, development of TFRs dis-
played a close temporal association with rise in serum levels of cytokines stim-
ulating B-cell proliferation and activation, such as IL-6, IL-7, BAFF/BLyS, and
APRIL. Concurrently, sFLC testing disclosed the elevation of both k and l
sFLC levels with normal k/l ratios, consistent with an inflammatory-like gener-
alized B-cell activation and polyclonal expansion . Upon lenalidomide contin-
uation, serum levels of cytokines and of polyclonal sFCL stepwise decreased
to normal limits. The compassionate program was stopped after enrolment of
these first three patients to be replaced by an ongoing phase I/II study.
We have described three cases of very likely TFR in HL patients given
lenalidomide. Following early shrinkage of nodal lesions, patients dis-
played distinctive signs of TFR syndrome with a sudden-onset painful re-
enlargement of tumor-involved sites (up to 50%), accompanied by inflam-
matory edema of overlaying skin, and disabling bone pain at disease-
bearing skeletal sites. This occurred at days 126 (patient 1), 127 (patient
2), and 133 (patient 3), in concomitance of the scheduled week-off lenali-
domide and after steroids had been withdrawn to verify for response on
systemic symptoms or reduce insulin. Symptoms and signs resolved
upon anti-inflammatory/analgesic treatment and never recurred. Although
disease progression could have been deemed, lenalidomide was continued
and all patients attained an objective response. It would have been other-
wise surprising for a tumor progression of such magnitude to display a so
prominent and painful inflammatory picture and promptly reverse with anti-
inflammatory agents only.
The occurrence of a lenalidomide-related TFR syndrome has been
reported in about 50–60% of patients with chronic lymphocytic leukemia
(CLL), reaching a Grades 3 and 4 severity in 8–9% of the cases [8–10]. In
CLL patients, development of lenalidomide-induced TFRs was frequently
associated with steroid taper/suspension [9,10] and the presence of large
tumor volumes , while low-dose prednisone prophylaxis was shown to
reduce severity of flares [2,9,11]. In our cases, a straightforward association
with steroid taper/withdrawal and the presence of large tumor masses was also
evident. Given the nature of our study, we are unable to provide conclusive
figures on the incidence rate of TFRs in HL or document whether TFRs may
also occur in HL patients receiving lenalidomide without steroids. As patients
with progressive HL may typically receive steroids as part of their treatment,
final results of ongoing trials will be needed to unravel these issues [12–14].
Development of TFRs has been related to lenalidomide-induced upregula-
tion of immune-stimulatory molecules on tumor cells, triggering a prominent
inflammatory cytokine release syndrome, and activation of bystander
immune cells [2,8,9]. We documented that serum levels of B-cell-activating
cytokines were abnormally elevated concurrently with TFR development and
displayed a stepwise decrease at resolution. Flares were also associated to
the transient elevation of sFLC levels with a normal free k/l ratio, consistent
with a generalized expansion of polyclonal B-cells such as occurring in sys-
temic inflammatory disorders [7,15]. This may reflect the activity of elevated
B-cell-activating cytokines and/or a direct effect on bystander B-cells. As
lenalidomide promotes survival of normal, but not neoplastic, B-cells , it
may have contributed to the expansion of reactive B-lymphocytes primed by
the proinflammatory ‘milieu’ of HL . While the contribution of other
immune effectors was not explored in our study, the association of flares
with steroid suspension further supports an underlying immune-modulatory
Lenalidomide-induced TFRs may also occur in HL patients. They mimic
early tumor progression but are manageable and followed by clinical
responses upon drug continuation. While conclusive data in HL are expected
from ongoing studies, our observations suggest that TFRs may reflect the
pleiotropic actions of lenalidomide on HL microenvironment.
This work was supported in part by Ministero della Salute, Ricerca Finaliz-
zata FSN, IRCCS, Rome, Italy. We thank Dr. Alessandro Marchei for help in
Hematology, NationalCancerInstitute,Fondazione‘G.Pascale’,IRCCS,Naples, Italy
2Department of Cellular and Molecular Biology and Pathology, Faculty of
Biotechnological Sciences, Federico II University, Naples, Italy
3Radiology Unit, National Cancer Institute, Fondazione ‘G. Pascale’, IRCCS,
4The Binding Site, Rome, Italy
5Nuclear Medicine Unit, National Cancer Institute, Fondazione ‘G. Pascale’,
IRCCS, Naples, Italy
Gaetano Corazzeli and Rosaria De Fillippi contributed equally to the study
*Correspondence to: Antonio Pinto, MD, Hematology-Oncology and Stem Cell
Transplantation Unit, National Cancer Institute, Fondazione G. Pascale, IRCCS,
Via Mariano Semmola, I-80131, Naples, Italy.
Conflict of interest: B.A. acts as a consultant to the Binding Site. All other authors
declare no competing financial interests.
Published online 21 October 2009 in Wiley InterScience
American Journal of Hematology 89
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