A. De Crescenzo

A. Gueli

C. Tarella

D. Gottardi

H. Hu

I. Ricca

AIR Universita degli studi di Milano

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CASE REPORT
Blood Transfus 2013; 11: 311-4  DOI 10.2450/2012.0166-12
© SIMTI Servizi Srl
Efficacy of rituximab-bendamustine in cold agglutinin haemolytic anaemia 
refractory to previous chemo-immunotherapy: a case report
Angela Gueli1,2, Daniela Gottardi1, Huijing Hu1, Irene Ricca3, Alberto De Crescenzo1,  Corrado Tarella1,2
1Haematology and Cell Therapy Unit, Umberto I Hospital, Turin; 2Department of Medical Oncology, University 
of Turin, Turin; 3Division of General Medicine, Cottolengo Hospital, Turin, Italy
Introduction
Primary cold agglutinin disease (CAD) is a 
haemolytic anaemia caused by high titres of cold 
antibodies, usually IgM, directed against red blood 
cells1,2. Cold agglutinins are present at low titres in 
all normal individuals, but at high concentrations, the 
agglutinins can have pathological consequences. In 
the cool peripheral circulation, cold agglutinin-coated 
red blood cells activate the complement cascade to 
the C3b/C3d stage and in the hepatic circulation C3b+ 
erythrocytes trigger phagocytosis by macrophages. 
An autoimmune disorder  is responsible for the high 
titres of circulating cold antibodies and CAD accounts 
for approximately 13-15% of cases of autoimmune 
haemolytic anaemia3. Moreover, CAD has been found 
to be associated with a clonal lymphoproliferative 
disorder in many cases. Indeed, the cold agglutinins 
are monoclonal IgM in more than 90% of CAD 
patients and the presence of a lymphoid neoplasm, in 
particular B-cell non-Hodgkin's lymphoma (NHL), has 
been observed in approximately 75% of patients with 
primary CAD4,5. 
Treatments, including corticosteroids or alkylating 
agents, which are effective in other forms of autoimmune 
haemolytic anaemia are usually ineffective in CAD. On 
the other hand, half of the patients with CAD respond 
to rituximab alone, a drug that has markedly improved 
the prognosis of patients with B-cell lymphomas. 
Moreover, combining rituximab with fludarabine has 
further improved the outcome of CAD patients, with a 
75% overall response rate being achieved in a recent, 
prospective trial6.
Most CAD patients are elderly and their advanced 
age makes the use of potentially harmful therapies 
questionable. It is, therefore, reasonable to search 
for less toxic regimens for CAD patients. Recently, 
bendamustine, a molecule analogous to fludarabine, 
has been successfully employed in the treatment of 
low-grade B-cell NHL7,8. Compared to fludarabine, 
bendamustine has fewer side effects and an excellent 
tolerability. Thus, bendamustine with rituximab might 
be an effective chemo-immunotherapy option for elderly 
patients with CAD. The case report here presented, gives 
support to the use of bendamustine in CAD. 
Case report 
A 74-year old Caucasian male with worsening severe 
anaemia was referred to our Centre in January 2009. The 
patient had a past medical history of poliomyelitis at the 
age of 4 years. He also reported a recent diagnosis of 
benign prostatic hypertrophy and hypertension, treated 
with ACE-inhibitors. On physical examination, the 
patient was remarkably pale with conjunctival icterus; he 
had bilateral lower limb oedema and mild tachycardia. 
There was no significant peripheral lymphadenopathy 
or evidence of hepatosplenomegaly.
The complete blood count revealed anaemia with 
a haemoglobin concentration of 7 g/dL, an increased 
reticulocyte count (218109/L) and normal white blood 
cell and platelet counts. The suspected haemolysis was 
confirmed by the marked increase of serum lactate 
dehydrogenase (LDH) at 1,667 U/L along with an 
undetectable serum haptoglobin and a raised level of 
indirect bilirubin (2.9 mg/dL). Analysis of the urine 
showed an increased level of urobilinogen along with 
moderate haemoglobinuria. The direct Coombs' test was 
strongly positive for complement C3d and a high titre 
(1/2,048) of anti-I cold antibody was detected. The direct 
Coombs' test was negative for IgG and no monoclonal 
IgM was detected by serum electrophoresis. 
A bone marrow biopsy showed a hypercellular 
marrow with erythroid hyperplasia and a slight 
interstitial excess of small, CD20-positive lymphocytes. 
Immunophenotyping of the bone marrow aspirate 
revealed the presence of a B-cell clonal population, 
which was negative for CD5 and CD10. These cells 
accounted for approximately 50% of bone marrow 
lymphocytes. Computed tomography of the chest 
and abdomen did not reveal any lymphadenopathies. 
Virological markers of hepatitis C virus and human 
immunodeficiency virus were negative, while antibodies 
to the core antigen of hepatitis B virus were detected. 
Based on these findings, a diagnosis of CAD associated 
with a CD5-negative B-cell lymphoproliferative disorder 
was made. 
The patient was referred to our Centre under steroid 
therapy (prednisone 1 mg/kg), started in the preceding 
2 weeks. This steroid therapy had been of little if any 
benefit, with the patient's parameters of haemolytic 
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Gueli A et al
Blood Transfus 2013; 11: 311-4  DOI 10.2450/2012.0166-12
anaemia remaining unchanged. Thus, following 
admission, the dose of prednisone was progressively 
tapered down and the treatment was stopped within 
2 months. Taking into consideration the co-existing 
B-cell NHL, treatment was started with rituximab, an 
anti-CD20 monoclonal antibody. Rituximab was 
prescribed as weekly infusions of 375 mg/m2. However, 
after two infusions, a further drop of haemoglobin 
to 6.8 g/dL was recorded. Additional therapy was, 
therefore, required, and a combination of rituximab 
with cyclophosphamide, vincristine and prednisolone 
(R-CVP) was chosen, based on a report demonstrating 
the efficacy of R-CVP in indolent B-cell NHL9. 
Following R-CVP, due to the persistence of both 
severe anaemia and a high titre of cold agglutinins, the 
patient underwent multiple plasmapheresis procedures 
and two red cell transfusions. A transient reduction of 
haemolysis was observed while the patient was under 
daily plasmapheresis and his haemoglobin level reached 
the value of 10.7 g/dL along with a progressive decrease 
of serum LDH and bilirubin level. Three plasmapheresis 
procedures were performed. However, haemolysis 
rapidly recurred a few days after discontinuation of the 
plasmapheresis. The persistent haemolysis necessitated 
a modification of the chemo-immunotherapy approach 
and a regimen of rituximab, oxaliplatin, dexamethasone 
and cytarabine (R-OxDHA) was started. Recent studies 
showed the excellent tolerability of the OxDHA scheme, 
in which oxaliplatin replaces the highly nephrotoxic 
cisplatin that was included in the original DHAP 
scheme10-12. In a preliminary experience by our group, the 
R-OxDHA combination resulted unexpectedly effective 
in indolent B-cell NHL and chronic lymphocytic 
leukaemia13. Indeed, both the original DHAP and the 
modified Ox-DHA regimens are known to have potent 
anti-tumour activity and are particularly suitable for 
patients with refractory or recurrent NHL. Unfortunately 
our patient with CAD had a poor response to the 
R-OxDHA combination, with further progression of 
haemolysis, a serum LDH value spiking at >1,500 U/L 
and increasingly severe anaemia (haemoglobin value 
down to 6 g/dL).
Two additional plasmapheresis procedures were 
performed and three transfusions of incompletely 
matched packed red blood cells were given with 
minimal benefit. The patient needed further therapy. 
Fludarabine was a possible option for this patient, 
since it has been proposed as the most suitable drug 
in the case of autoimmune haemolytic anaemia 
associated with lymphoproliferative disease refractory 
to conventional chemo-immunotherapy5,6,14. However, 
the side effects of fludarabine, alone or in combination, 
cannot be minimised, particularly in elderly patients 
previously exposed to immunosuppressive treatments. 
Thus, bendamustine was considered as an alternative. 
In the first course the dose of bendamustine was 70 
mg/m2/die for 2 consecutive days every 4 weeks, 
whereas the dose from the second course was 90 mg/
m2/die. Rituximab was given at the standard dose 
of 375 mg/m2 with each course of bedamustine. 
The patient had an unexpectedly rapid and marked 
response. Indeed, already after the first course of 
rituximab-bendamustine, the patient's haemoglobin 
concentration increased progressively and no more red 
blood cell transfusions were necessary. The patient was 
discharged and his treatment was continued in a Day 
Hospital regimen. The changes in haemoglobin and 
serum LDH values in the various phases of treatment 
are summarised in Figure 1.
The patient was re-assessed in July 2009, after 
three courses of rituximab-bendamustine. The bone 
marrow biopsy showed complete remission of 
the lymphoma. Minimal residual disease analysis 
showed a residual, very weak band in the polymerase 
chain reaction for Ig clonal rearrangement. No 
evidence of hepatosplenomegaly was found in the 
ultrasound examination. Cold agglutinin antibody 
resulted positive with a titre of 1/1,024 and the 
patient's haemoglobin was 13 g/dL. Serum LDH, 
bilirubin and haptoglobin levels were normalised 
(Figure 1). In consideration of: (i) the excellent 
response; (ii) the advanced age of the patient; (iii) 
the multiple immunosuppressive treatments received; 
and (iv) the infantile poliomyelitis infection, the 
rituximab-bendamustine treatment was discontinued 
and the patient was placed under clinical observation 
without any additional therapy for the CAD.
Figure 1 - Changes of haemoglobin (Hb) and serum lactate 
dehydrogenase (LDH) values during the different 
phases of treatment. 
R-CVP: rituximab + cyclophosphamide, vincristine, 
prednisolone; R-OxDHA: rituximab + oxaliplatin, 
dexamethasone, cytarabine; see text for details of drug doses 
and schedules.
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Blood Transfus 2013; 11: 311-4  DOI 10.2450/2012.0166-12
Rituximab-bendamustine in cold-agglutinin haemolytic anaemia
In June 2012, 3 years after the   rituximab-bendamustine 
treatment, the patient is still transfusion-independent and 
in continuous clinical remission of the previous CAD. The 
last complete blood count revealed a haemoglobin level 
of 14.4 g/dL, with a reticulocyte count of 34109/L and 
persistently normal white blood cell and platelet counts. 
Evaluation of serum cold agglutinins showed residual 
positivity with a titre of 1/128. Normal values of LDH, 
haptoglobin, reticulocytes and bilirubin confirmed the 
complete remission of the haemolytic anaemia. 
Discussion 
We report here the favourable outcome of an 
elderly patient with CAD treated with rituximab and 
bendamustine. CAD is an autoimmune haemolytic 
anaemia which is frequently associated with B-cell 
NHL. In fact, the patient described in this report was 
diagnosed with a low-grade lymphoma. The peculiarity 
of this case was the refractoriness of the disease to most 
treatments known to be quite effective in autoimmune 
anaemia associated with B-cell NHL, such as steroids, 
rituximab monotherapy and rituximab combined with 
chemotherapy. It is known that rituximab is a key drug 
in the treatment of B-cell NHL and it has been shown 
to be frequently effective in immune cytopenias15-18. 
The most striking feature of this case was the good 
response to bendamustine given together with rituximab. 
The patient's haemolysis resolved rapidly following 
treatment with bendamustine and three courses of the 
rituximab-bendamustine combination were sufficient 
to induce a complete clinical response, which is still 
maintained more than 3 years after treatment completion. 
Finally, the tolerability of the rituximab-bendamustine 
salvage treatment was surprisingly good, considering 
the advanced age of the patient and the multiple 
previous immunosuppressive treatments. This case 
report confirms that bendamustine is an effective anti-
lymphoma drug, which is exceptionally suitable for 
elderly patients with low-grade NHL.
Bendamustine is an alkylating agent with cytotoxic 
activity and was Þrst synthesised in the early 1960s in 
Jena, in the former East German Democratic Republic, 
by Ozegowski and Krebs19. The intention was to 
synthesise a nitrogen mustard compound that was less 
toxic than, but at least as effective as, other alkylating 
agents. Bendamustine has structural similarities to both 
alkylating agents and purine analogues and in general 
it is very well tolerated. In fact, the compound proved 
to be quite effective and it was extensively used over 
a 20-year period in East Germany in cancer patients. 
Since its licensing in Europe, bendamustine has been 
increasingly employed in the management of NHL, 
either as a single agent or in different combinations, 
primarily in association with rituximab. Several studies 
have shown that bendamustine is clinically active 
in patients with disease refractory to conventional 
chemotherapy20,21. It has distinct mechanistic features, 
which probably contribute to its high clinical efficacy. 
In fact, due to its partial cross-reactivity with other 
chemotherapeutic agents, bendamustine is among the 
drugs most extensively employed in the treatment 
of relapsed/refractory NHL. Moreover, there is now 
great interest in the potential role of bendamustine as 
initial therapy, given its promising efficacy and good 
safety profile. At present, bendamustine has been 
approved as first-line therapy for chronic lymphocytic 
leukaemia and studies are ongoing regarding its 
front-line use for low-grade follicular lymphoma21.
The case reported here suggests a possible role for 
bendamustine in CAD and, in general, in autoimmune 
haemolytic anaemias associated with NHL. Significant 
advances have been made in the treatment of CAD22. 
However, there are still several unresolved issues, 
including the problem of treatment-related toxicity, 
the chances of achieving long-lasting responses and 
the need to find efficient second-line therapy. The 
excellent outcome of the CAD patient described here 
suggests that prospective trials should be considered to 
investigate the safety and efficacy of the combination of 
bendamustine-rituximab as a novel therapeutic option, 
suitable as both first- and second-line treatment for 
elderly patients with autoimmune haemolytic anaemia 
associated with a B-cell lymphoproliferative disease. 
Keywords: haemolytic anaemia, cold agglutinin 
disease, bendamustine, non-Hodgkin's lymphoma.
The Authors declare no conflict of interest.
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Arrived: 20 July 2012 - Revision accepted: 8 November 2012
Correspondence: Corrado Tarella
S.C.D.U. Ematologia e Terapie Cellulari
A.O. Ordine Mauriziano-Umberto I
Corso Turati 62
10128 Torino, Italy
e-mail: corrado.tarella@unito.it


Efficacy of rituximab-bendamustine in cold agglutinin haemolytic anaemia refractory to previous chemo-immunotherapy : a case report

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Efficacy of rituximab-bendamustine in cold agglutinin haemolytic anaemia refractory to previous chemo-immunotherapy : a case report

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