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[Cancer Research 66, 915-920, January 15, 2006]
© 2006 American Association for Cancer Research

Cell, Tumor, and Stem Cell Biology

Effect of Rapamycin on Mouse Chronic Lymphocytic Leukemia and the Development of Nonhematopoietic Malignancies in Eµ-TCL1 Transgenic Mice

Nicola Zanesi1, Rami Aqeilan1, Alessandra Drusco1, Mohamed Kaou1, Cinzia Sevignani2, Stefan Costinean1, Laura Bortesi2,3, Gaspare La Rocca2, Pavel Koldovsky2, Stefano Volinia1, Rita Mancini1,4, George Calin1, Charles P. Scott2, Yuri Pekarsky1 and Carlo M. Croce1

1 Department of Molecular Virology, Immunology, and Medical Genetics, Comprehensive Cancer Center, Ohio State University, Columbus, Ohio; 2 Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania; 3 Department of Pathology, University of Verona, Verona; and 4 Faculty of Medicine and Surgery, University of Rome "La Sapienza," Rome, Italy

Requests for reprints: Carlo M. Croce, Department of Molecular Virology, Immunology, and Medical Genetics, Comprehensive Cancer Center, Ohio State University, 410 West 12th Avenue, Wiseman Hall, Room 385L, Columbus, OH 43210. Phone: 614-292-3063; Fax: 614-292-4080; E-mail: Carlo.Croce{at}osumc.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Chronic lymphocytic leukemia (CLL) is the most common leukemia in the world. The TCL1 gene, responsible for prolymphocytic T cell leukemia, is also overexpressed in human B cell malignancies and overexpression of the Tcl1 protein occurs frequently in CLL. Aging transgenic mice that overexpress TCL1 under control of the µ immunoglobulin gene enhancer, develop a CD5+ B cell lymphoproliferative disorder mimicking human CLL and implicating TCL1 in the pathogenesis of CLL. In the current study, we exploited this transgenic mouse to investigate two different CLL-related issues: potential treatment of CLL and characterization of neoplasms that accompany CLL. We successfully transplanted CLL cells into syngeneic mice that led to CLL development in the recipient mice. This approach allowed us to verify the involvement of the Tcl1/Akt/mTOR biochemical pathway in the disease by testing the ability of a specific pharmacologic agent, rapamycin, to slow CLL. We also showed that 36% of these transgenic mice were affected by solid malignancies, in which the expression of the Tcl1 protein was absent. These findings indicate that other oncogenic mechanism(s) may be involved in the development of solid tumors in Eµ-TCL1 transgenic mice. (Cancer Res 2006: 66(2): 915–20)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Chronic lymphocytic leukemia (CLL) is the most common human leukemia and most frequently affects individuals >50 years of age (1). It is a clinically heterogeneous disease characterized by accumulation of malignant long-living CD5+ B cells. Multiple molecular events likely contribute to malignant transformation (2). Because CLL occurs more commonly in people with at least one first-degree relative with CLL, familial aggregation of this disorder has been known for many years (3). Although several common genomic abnormalities in CLL have been identified (reviewed in ref. 4), no specific alterations in protein coding genes have been detected in CLL. By using engineered mouse models that phenocopy a human disease, strong evidence can be gathered relative to the involvement of a specific gene in the pathology. We have obtained such evidence for the TCL1 oncogene, located at 14q32.1, in CLL (5). Transgenic mice for the human TCL1 gene under the control of a tissue-specific µ immunoglobulin enhancer (Eµ-TCL1) developed clonal B cell expansions. These animals showed clones of CD5+ B cell populations initially in the peritoneum, then, at later stages, in the spleen and bone marrow. Elder mice eventually developed a CLL-like disease that shared many features with leukemic B cells of CLL patients (5).

Secondary malignancies are frequent complications in patients with CLL (6). Although other lymphoid malignancies are the most common, solid tumors have also been documented in CLL patients (7). Lung cancer, gastrointestinal carcinomas, melanomas, and other skin cancers are the most frequent solid tumors in this patient population (8–10). Moreover, Kyasa et al. (11) reported that secondary malignancies were the most common cause of death in patients initially diagnosed with CLL.

Tcl1 protein functions as a coactivator of Akt (12). One established Akt effector is mTOR, a serine/threonine kinase implicated in translation control that can be potently inhibited by the immunosuppressant rapamycin (13). mTOR mediates metabolic changes important for cell survival (14). Rapamycin has modest antitumor activity against PTEN-deficient tumors in mice (15, 16) and can potentiate drug-induced cell death in vitro (17, 18). To determine whether rapamycin inhibits mTOR, its activity can be indirectly assessed in extracts from untreated or treated tissues using antibodies that specifically recognize the phosphorylated form of a downstream mTOR target: the ribosomal S6 protein, which is phosphorylated by the S6 kinase (19).

In this study, we investigated whether the Eµ-TCL1 transgenic mouse model of CLL also resembled human disease in displaying secondary malignancies. Because CLL lymphocytes are mostly resting cells with a morphologically mature appearance that do not show any spontaneous proliferation in vitro (3), we did transplantation experiments of murine leukemic lymphocytes into syngeneic mice in order to expand CLLs in vivo. This system of in vivo CLL growth allowed us to study the involvement of the Tcl1/Akt pathway in the development of CLL by pharmacologic inhibition with the drug rapamycin.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Histopathology and immunohistochemistry. Eµ-TCL1 transgenic mice were studied and compared with nontransgenic siblings raised in identical conditions. Genotyping was done on tail DNAs by PCR as previously described (5). Animals were autopsied and tissues were fixed in 10% formalin and embedded in paraffin. Sections were stained with H&E according to standard protocols and analyzed by mouse pathologists (Frimorfo, Inc., Fribourg, Switzerland). Immunohistochemistry was done on representative sections. For the dewaxing step, the sections were heated for 1 hour at 55°C, followed by a rehydration step through a graded ethanol series and distilled water, immersed in PBS, and then treated with 0.1% trypsin solution in Tris buffer for 30 minutes at 37°C. Endogenous peroxidase was blocked with 10% normal serum. The 27D6/20 monoclonal antibody specific for recombinant human Tcl1 protein (20) was used as a primary antibody, and immunohistochemical staining was done by using streptavidin-biotin peroxidase labeling method according to the manufacturer's instructions (Histomouse-SP kit, Zymed, South San Francisco, CA).

Western blot analysis. Cell proteins were extracted with Nonidet P-40 lysis buffer, quantified by using the bicinchoninic acid kit (Pierce, Rockford, IL), size-fractionated on 12% Tris-glycine SDS-PAGE gels, and electrotransferred onto nitrocellulose (Bio-Rad, Hercules, CA). The membrane was blocked overnight in 10% nonfat dried milk in PBS/Tween 20. Expression was detected with the monoclonal antibody 27D6/20 for human Tcl1 protein (20) according to an enhanced chemiluminescence protocol (Amersham Pharmacia, Piscataway, NJ). Ponceau-S staining was used to verify equivalent protein loading. S6 and Phosho-S6 antibodies were purchased from Cell Signaling Technologies (Beverly, MA).

Cell preparations. Leukocytes were isolated from enlarged spleens and lymph nodes of ill Eµ-TCL1 transgenic mice. Tissues were dissociated in PBS medium between two frosted slides. Erythrocytes were lysed by brief treatment with 0.165 mol/L NH4Cl and then washed twice in PBS.

Fluorescence-activated cell sorting analysis. Cells (2 x 106), isolated from spleens and lymph nodes, were resuspended in fluorescence-activated cell sorting (FACS) medium (PBS/3% serum/0.1% NaN3), immunostained for surface expression of IgM. CD5 and B220 (BD Biosciences-PharMingen) for 1 hour at room temperature, washed with FACS medium thrice and then FACS analyzed using a BD Epics XL-MCL cytometer. Cells were gated as previously described (5).

Transplantation. B6/C3H F1 female mice (6-8 weeks old; The Jackson Laboratory, Bar Harbor, ME) were injected i.p. with 106 or 107 white cells from spleens and lymph nodes. For the control experiment, splenic white cells were isolated from a healthy transgenic donor. Mice were monitored for the development of leukemia and were killed or treated based on the appearance of clinical symptoms, in compliance with Federal and Institutional Animal Care and Use Committee requirements. One to four serial transplantations were done.

Drug treatment. The transplanted animals were treated with rapamycin (4 mg per kg i.p.) every other day in the prevention study or every day in the cure study. Rapamycin (LC Laboratories, Woburn, MA) was initially dissolved in 100% ethanol, stored at –20°C, and further diluted in an aqueous solution of 5.2% Tween and 5.2% polyethylene glycol 400 (final ethanol concentration, 2%) immediately before use. The mice were monitored at the time of each treatment. Morbidity was defined as the presence of detectable tumor or palpable lymph nodes.

Statistics. Differences in tumor incidence among the groups of mice were determined by Fisher's exact test (http://www.matforsk.no/ola/fisher.htm). Statistical tests were two-sided and were considered significant at P < 0.05. Survival of transplanted syngeneic mice was analyzed in the Kaplan-Meier format using log-rank (Mantel-Cox) test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Malignancies developed in Eµ-TCL1 mice. We studied the spontaneous tumor phenotype of 11 Eµ-TCL1 transgenic mice, and 12 wild-type littermates. Mice were kept under close supervision and were checked biweekly for signs of illness. All transgenic mice became visibly ill between 10 and 20 months of age, whereas only one of the control littermates became ill within the 20-month period. Eµ-TCL1 mice presented with enlarged spleen, lymph nodes, and livers, although not all the tissues were altered in the same way in all animals. In some mice, other tissues including salivary glands, kidneys, heart, esophagus, thymus, trachea and thoracic cavity were also altered at the macroscopic and/or microscopic level. Altered spleen and lymph nodes were analyzed by Western blot using antibodies specific for human Tcl1 protein (Fig. 1A, lanes 2, 3, 7-9). High expression of human Tcl1 protein was observed in altered lymphoid tissues of 10 out of 11 transgenic mice derived from the two founders, F3 and F10. Flow cytometric analysis of white cells from enlarged spleens and lymph nodes revealed the expansion of the CD5+/IgM+ population (Table 1) ranging between 18% and 91% in 10 of 11 mice (in the spleen of mouse no. 2, this population was 5%). Immunohistochemical analysis of enlarged lymphoid tissues with anti-Tcl1 (Fig. 1B) polyclonal antibodies confirmed the expansion of Tcl1+ populations in 10 of 11 transgenic mice. Histopathologic examination of all altered tissues, in addition to the other types of the above described analyses, classified the malignancies as a CLL-like disorder in 10 of 11 transgenic mice. The only control wild-type mouse that became ill developed a lymphoma unrelated to CLL.


Figure 1
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  		Figure 1. Expression of Tcl1 protein in mouse CLL tissues and solid neoplasms. A, Western blot analysis. Cell proteins were extracted from spleen, lymph nodes, and liver of ill Eµ-TCL1 transgenic mice and B6-C3H F1 syngeneic mice transplanted with leukemia cells. Top, lanes 2 and 3 are CLL spleen of transgenic mouse no. 1 and syngeneic mouse transplanted with the same leukemia, respectively. Lanes 7, 8, and 9 are CLL spleens of mice no. 3, 5, and 7, respectively. Lanes 1, 4, and 5 are liver, spleen, and lymph node, respectively, of transgenic mouse no. 2 affected by histiocytic sarcoma. Lane 10 is spindle cell sarcoma from mouse no. 6. Lane 6 is a positive control from the Supt 11 cell line. Bottom, Ponceau S solution staining of the membranes showing equal protein load on each lane. B and C, immunohistochemical analysis of lymphoid tissues in Eµ-TCL1 transgenic mice (magnification, x400). Anti-Tcl1 staining of CLL spleen (B), and histiocytic sarcoma (C) from mouse no. 5 (see Table 1).

 

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  		Table 1. Tumor phenotype in Eµ-TCL1 mice and B-CLL markers in spleen and lymph node white cells

 
The Eµ-TCL1 mouse no. 2 (Table 1) was diagnosed at 11 months of age with histiocytic sarcoma (Fig. 2A) affecting lymph nodes, pancreas, spleen, lungs, and liver. Analysis by Western blot (Fig. 1A, lanes 1, 4, 5, and 10) and immunohistochemistry in spleen, lymph nodes, and liver showed a very low level of Tcl1 protein expression (Fig. 1C). IgM+ and CD5+/IgM+ cell populations in the spleen were 8% and 5%, respectively. The very faint Tcl1 band could well be due to contamination of B cells. Consequently, this transgenic mouse developed a malignancy not only different from CLL but apparently unrelated to the Eµ-TCL1 transgene status.


Figure 2
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  		Figure 2. Histopathologic analysis of spontaneous solid tumors developed in Eµ-TCL1 transgenic mice (magnification, x400). A, spleen histiocytic sarcoma from mouse no. 2. B, small intestine histiocytic sarcoma from mouse no. 5. C, gallbladder spindle cell sarcoma from mouse no. 6. D, malignant pilomatricoma from mouse no. 10 (see Table 1).

 
Interestingly, an extensive pathologic analysis of nonlymphoid tissues in mice affected by CLL revealed that three other transgenic mice (nos. 5 and 6, each at 12 months of age, and no. 10 at 20 months, as shown in Table 1) developed malignancies such as histiocytic sarcoma in the intestine, spindle cell sarcoma of the gallbladder, and malignant pilomatricoma of the skin (Fig. 2B, C, and D, respectively), all unrelated to CLL and to Eµ-TCL1 gene expression. In total, 4 of 11 (36%) transgenic mice, derived from both founders F3 and F10, developed nonlymphoid malignancies compared with none in the wild-type group (P < 0.04).

CLL cell transplantation. White cells with expanded CD5+/IgM+ populations were isolated from enlarged spleens and lymph nodes of three Eµ-TCL1 mice (nos. 1, 3, and 5) diagnosed with CLL-like disease and transplanted in syngeneic mice. Median survival time for mice that received transplants of 107 and 106 white cells was 121 and 166 days, respectively (Fig. 3A). In all cases, autopsy confirmed that mice that received transplants died of complications including massive hepatomegaly and splenomegaly, peripheral leukemia, formation of ascitic fluid, enlarged neck, mediastinic and mesenteric lymph nodes, and leukemic infiltrates in multiple organs. Syngeneic control mice that received splenic white cells isolated from a healthy transgenic donor survived 250 days before being sacrificed. White cells from transgenic leukemic donors and transplants were indistinguishable by flow cytometry and resulted in an enriched CD5+/IgM+ population after one to four serial transplantations (data not shown). Multiple attempts using various culture conditions were not successful in establishing an in vitro CLL cell line, indicating that the leukemic cells had not undergone an immortalizing mutation. Cells from individual leukemias were maintained by serial transplantations into syngeneic recipient mice, thus revealing their ability to survive indefinitely in vivo, as shown in Fig. 3B and C.


Figure 3
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  		Figure 3. CLL cell transplantation. A, Kaplan-Meier plots showing survival of syngeneic mice that received Tcl1-positive B-CLL cell transplants. Syngeneic B6/C3H mice were injected i.p. with 107 (dashed line) and 106 (full line) white cells from spleens and lymph nodes of transgenic Eµ-TCL1 mice affected by B-CLL disease. Results are pooled from three independent leukemias with a total of 20 mice per inoculation group. In the control experiment (dotted line), 107 splenic white cells were isolated from a healthy transgenic donor and injected in five syngeneic mice. Animals were scored when moribund. B and C, gross pathology of syngeneic mice originally transplanted with B-CLL cells from mouse no. 3 (see Table 1). Recurrent in this leukemia, together with hepatomegaly and splenomegaly, are enlarged mesenteric lymph nodes that occur almost identically after one (B) or three (C) serial transplants.

 
Rapamycin treatment of a B-CLL-transplanted leukemia. We isolated human Tcl1-positive leukemia cells from mouse no. 4 (Table 1) and i.p. injected 1.5 x 107 cells per mouse in 15 syngeneic mice. Six weeks after injection, when all mice were still healthy, we started to administer rapamycin (4 mg/kg every other day) to five animals in the prevention study. As shown in Fig. 4A, 3 weeks later, 90% of the untreated controls showed clear signs of disease (Fig. 5A) and 80% (P = 0.02) of the mice treated with rapamycin were still healthy (Fig. 5B). Three of 10 control mice were given rapamycin on a daily basis after the first symptoms of disease appeared. In terms of mortality (Fig. 4B), after 9 weeks from the injection with leukemic cells, none of the rapamycin-treated mice (either in the prevention or in the cure group) had died, but only 29% of the untreated controls were alive (P = 0.007). Therefore, our treatment significantly prolonged the life of all treated animals. In this experiment, the delaying effect of rapamycin on mouse CLL was relatively short. After 11 weeks from the beginning of the experiment, seven of eight mice treated with rapamycin were dead compared with seven of seven control mice.


Figure 4
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  		Figure 4. A, morbidity in mice injected with B-CLL leukemia cells and treated with rapamycin. Control (C) and rapamycin-treated (R) mice after 6 and 9 weeks from injection with leukemic cells. Healthy mice (striped columns); ill mice (white columns). B, mortality in the same injected mice. Control (C) mice, rapamycin-treated after disease onset (T) mice, and rapamycin-treated before disease onset (R) mice, after 6 and 9 weeks (see text). Live mice (striped columns); dead mice (white columns).

 

Figure 5
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  		Figure 5. Effect of rapamycin treatment in mice injected with Tcl1-positive B-CLL cells. A, untreated control mouse injected i.p. with 1.5 x 106 leukemic cells. B, rapamycin-treated mouse injected i.p. with 1.5 x 106 leukemia cells and administered with rapamycin (4 mg/kg) every other day. C, Western blot analysis showing the expression of S6 kinase and phospho-S6 kinase in mouse CLL lymph nodes. Lanes 1, 2, and 4 are cell extracts from untreated mouse CLL lymph nodes. Lanes 3 and 5 are from rapamycin-treated CLL lymph nodes.

 
To investigate whether rapamycin indeed inhibits mTOR in mouse CLL, and whether our treatment targeted the correct biochemical pathway, we determined the phosphorylation status of the ribosomal protein S6 kinase in extracts from untreated or treated CLL mesenteric and mediastinic lymph nodes using an antibody that specifically recognizes the phosphorylated form of the ribosomal S6 protein. As shown in Fig. 5C, untreated CLL lymph nodes (lanes 1, 2, and 4) expressed phosphorylated S6, whereas rapamycin-treated lymph nodes (lanes 3 and 5) did not show any phosphorylation. On the other hand, all five specimens expressed equal amounts of unphosphorylated S6. Hence, rapamycin inhibits mTOR activity in our CLL model.


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this report, we studied spontaneous malignancies developed in Eµ-TCL1 transgenic mice over time. Although it was already known that these animals would develop a CLL-like leukemia (5), we found that 36% (4 of 11) of them also showed the onset of solid malignant tumors unrelated to the expression of the TCL1 transgene. Three of these four tumors appeared in relatively young mice (11-12 months), whereas no solid tumors were seen in wild-type littermates until the 20th month of age or later. Two of four malignancies were histiocytic sarcoma, a cancer of the macrophagic line known to be rare (2-5%) in 23-month-old mice (22), whereas in Eµ-TCL1 mice was present in 18% (2 of 11) of the animals at 11 to 12 months of age. Secondary malignancies are also frequent complications in patients with CLL (23). Although the most frequent events are secondary lymphoid malignancies, solid tumors were also documented in CLL patients. The National Cancer Institute's Surveillance Epidemiology and End Results program published a statistically significant 20% increase risk of secondary cancer in CLL (8). The highest risk among solid tumors was seen for skin cancer (6). Kyasa et al. (11) showed that a second malignancy was the most common cause of death in patients with CLL. The Eµ-TCL1 transgenic mouse no. 2 in Table 1 developed histiocytic sarcoma as the only malignancy without any sign of CLL. An interpretation of this result is that, in some cases, the development of the leukemia might be sufficient to impair the immune system, thus creating the conditions for other tumor development. A support to this interpretation comes from a case report in which CLL and Merkel cell carcinoma were diagnosed nearly simultaneously in a patient (24). The authors suggested that the outcome of Merkel cell carcinoma may be independent of any given feature of the preexisting CLL.

Possible explanations for the increased risk of secondary nonhematologic neoplasms in patients with CLL include genetic predisposition, immune deficiency, carcinogen exposure, and the effects of therapeutic treatment (9). In our CLL mouse model, 25% (1 of 4) of secondary malignancies were malignant pilomatrixoma, a type of skin cancer extremely rare in mice. The onset of this neoplasm, together with the other three sarcomas, might be the consequence of some immunodeficiency yet to be clarified. We believe that other factors like genetic predisposition, carcinogen exposure, and therapeutic treatment should not be an issue in our strictly controlled experimental conditions. In fact, transgenic and wild-type mice had the same genetic origin and none of these mice were exposed to carcinogens or therapies. Of course, we may not exclude that other, still unknown, mechanisms play a role in the increased frequency of these solid tumors. Thus far, two transgenic mouse models showing deregulated expression of Tcl1 in B cells (5, 25) were created. A recent report also described deregulated expression of APRIL, a tumor necrosis factor–like ligand, in another transgenic mouse model for CLL (26). Our model of CLL seems to be the only one that displays the onset of secondary malignancies in CLL-prone mice. In conclusion, we believe that this animal model of CLL may resemble the human condition more than previously described. In fact, not only the Eµ-TCL1 transgenic mouse develops a frank CLL-like leukemia, but it also resembles the human disease in its secondary and sometimes more lethal malignancies. Our findings suggest that immune defects associated with CLL might be associated with these nonhematologic neoplasms.

One of the applications of animal models for human cancers is to investigate the effectiveness of therapeutic approaches prior to human clinical trials. At the same time, the homogeneity of the genetic background of the model is an important factor to evaluate the effects of novel therapies. For this reason, we successfully established CLL transplants in syngeneic mice that allowed us at first to propagate, and later to therapeutically treat, the same leukemia in many different mice with the same genetic background. This system allows for the study of single leukemias and is a necessary step on the road to finding drugs that interfere specifically with the pathways involved in the disease.

Because Tcl1 protein is an Akt coactivator (12, 21), enhanced Akt kinase activity might be an important factor in CLL pathogenesis. In order to determine whether the development of mouse CLL is dependent on the Akt pathway, we investigated whether the disruption of Akt signaling through mTOR by the inhibitor rapamycin prevented or cured mouse CLL. Our treatment prolonged the life of all treated animals in either the prevention or in the therapeutic group. mTOR normally regulates translation by phosphorylating key components of the protein synthesis machinery, including the ribosomal protein S6 kinase (19). In our experiment, CLL lymph nodes of untreated mice expressed phosphorylated S6, whereas lymphoid tissues from rapamycin-treated mice did not show any phosphorylation. Consequently, rapamycin inhibits mTOR activity in our CLL mouse model. The delaying effect of rapamycin on mouse CLL in this experiment was relatively short. In the near future, we plan to use rapamycin in combination with other chemotherapeutic drugs to promote longer survival and to increase the disease-free period.

These results establish Tcl1 overexpression through Akt-mTOR signaling as a mechanism involved in CLL pathogenesis. Our findings also have important implications in choosing targeted therapeutics alone or in combination with conventional agents. In conclusion, our experiment provides new insights into a fundamental pathway of CLL biology and an in vivo validation for a strategy to prolong survival time in CLL-affected individuals.


   Acknowledgments
 
Grant support: NIH National Cancer Institute grant to C.M. Croce and The Sidney Kimmel Cancer Research Foundation grant to C.P. Scott.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We warmly thank Maricel Rocha, Holly Mc Breid, and Joshua Goldberg for technical assistance; and Dr. John P. Hagan for critical reading of the manuscript.

Received 9/23/05. Revised 10/31/05. Accepted 11/ 2/05.


   References
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 Introduction
 Materials and Methods
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 Discussion
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X.-j. Yan, E. Albesiano, N. Zanesi, S. Yancopoulos, A. Sawyer, E. Romano, A. Petlickovski, D. G. Efremov, C. M. Croce, and N. Chiorazzi
B cell receptors in TCL1 transgenic mice resemble those of aggressive, treatment-resistant human chronic lymphocytic leukemia
PNAS, August 1, 2006; 103(31): 11713 - 11718.
[Abstract] [Full Text] [PDF]

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