- Email: [email protected]
E2F4 as susceptibility factor in the early onset colorectal cancer
Med Clin (Barc). 2016;146(5):230–232
www.elsevier.es/medicinaclinica
Scientific letters E2F4 as susceptibility factor in the early onset colorectal cancer夽 E2F4 como factor de susceptibilidad en el cáncer colorrectal de inicio temprano Dear Editor: E2F is a family of 6 transcription factors (E2F6 E2F1) that regulates the expression of genes involved in the development of the cell cycle, and whose dysregulated synthesis induces the neoplastic transformation of some cell lines.1 E2F4 is the one expressed most abundantly, accounting for 50% of these proteins. In resting cells, E2F4 is mainly located in the nucleus, where it forms a complex with p130.2 The cell entry in the division process coincides with the active transport of the protein from the nucleus to the cytoplasm.3 The E2F4 protein contains a sequence of 13 consecutive serine residues in its transactivation domain.4 These are encoded by a series of triplets (AGC) whose number is frequently altered in colorectal cancers (CRC) with microsatellite instability phenotype.5,6 The E2F4 proteins encoded by genes with an altered number of AGC triplets show increased transactivator capacity and are capable of producing a cell proliferation stimulation.7 Furthermore, E2F4 overexpression has proliferative and tumorigenic effects.8 It has been reported that E2F4 alleles with a number of triplets other than 13 in the germline favour the development of CRC.9,10 The aim is to analyze the relationship between the presence of E2F4 gen alleles with a number of ACG triplets other than 13 and greater susceptibility to develop CRC. 44 patients with CRC diagnosed before the age of 51 years were selected. None of the 44 patients had germline mutations (Lynch syndrome). DNA extraction was performed from peripheral blood (germline) and tumour tissue (tumour line). The Nushift (EMSA) kit by Geneka Biotechnology, Inc. was used to test the transcription factors of the E2F family. (Cat. N◦ 2006690). The DNA of the tumour line is used as a control to assess that there is no difference with the germline due to microsatellite instability. Immunohistochemistry was performed using a standard technique. The DNA extracted from the peripheral blood of 344 healthy unrelated individuals was used as control group. Given the low allele frequencies, they were grouped according to the size of variation, regardless of whether they were insertions or deletions: V (1) variations of a triplet (both, insertions and deletions); V (2) of 2 triplets, and so on. Statistics: 2 test where possible and Fisher exact test in the other cases. The tumour line DNA showed no differences with germline DNA regarding the microsatellite instability under study. In terms of the total E2F4 altered alleles in the germline, 9% is observed in the CRC
夽 Please cite this article as: Ríos A, Rodríguez JM, Carbonel P, Parrilla P. E2F4 como factor de susceptibilidad en el cáncer colorrectal de inicio temprano. Med Clin (Barc). 2016;146:230–231.
˜ S.L.U. All rights reserved. 2387-0206/© 2015 Elsevier Espana,
group (8 out of 88 alleles) compared to 7% of the population group (48 out of 688) (p > 0.05) (Table 1). Regarding the distribution of the alleles in the population group, there were more insertions than deletions, while in the CRC group their numbers were similar. As shown in Table 1, there is an imbalance in the distribution of allele frequencies, being more frequent those representing larger variations in the CRC population. Thus, in the population group, 75% (36/48) of the variations correspond to insertions and/or deletions of one single triplet, while this variation only represents 12% in the CRC group (1/8) (p = 0.003). The assay of mRNA synthesized by different E2F4 alleles showed no differences in the expression or the half-life of the same. The in situ E2F4 protein assay by immunohistochemistry showed greater E2F4 protein concentration in the preparations derived from tumour tissue from individuals with altered E2F4 alleles. There is a gradation from high to low, which correlates with the greatest difference regarding 13. In humans, the AGC repetitive sequence of E2F4 is polymorphic in 4–6% of the population.9,10 Thus, in our study, no differences were observed in the frequency with which the E2F4 alleles were identified other than 13 repeats in the CRC group when compared to the population group (9 versus 7%). However, differences are observed in the distribution of the different alleles. Thus, in the population group, 75% of the variations (36/48) corresponded to expansions and contractions of a single triplet, whereas in the CRC group, these were only 12% (1/8). Furthermore, the distribution of alleles between the two populations is different for all major variations of a triplet, allowing speculation on the hypothesis that the alleles which move away from 13 in more than one triplet provide increased susceptibility to CRC development. The MRNA assay in individuals carrying different-sized alleles showed no difference in the expression of each one of them, nor in their half-life. Immunohistochemistry was used to assess the influence of this polymorphism in the distribution and concentration of E2F4 protein at cellular level, observing an increase in staining intensity when the E2F4 alleles were smaller in the germline. The staining was exclusively cytoplasmic, primarily affecting glandular cells, especially neoplastic cells. Likewise, the staining was strong in macrophages, in all cases, probably due to their phagocytic activity on tumour cells. The absence of nuclear staining is, possibly, a result of the inability of the antibody used in the immunohistochemical technique to recognize its epitope when E2F4 is dimerized with any of the DP proteins and bound to DNA. The regulatory activity of E2F4 is partly modulated by its cellular location (mainly nuclear in resting cells and cytoplasmic in dividing cells). Therefore, the presence of E2F4 in the cytoplasm of glandular cells could be related to the high proliferative activity of these cells, especially the neoplastic ones. The diversity of mechanisms by which the transcription factors of the E2F family are able to regulate gene expression makes interpreting the consequences of E2F4 accumulation difficult. In this case the activity of the protein seems to be closely linked to the active transport through the nuclear membrane, mediated by
Scientific letters / Med Clin (Barc). 2016;146(5):230–232 Table 1 Allelic frequencies for each of the variations in the general population and in colorectal carcinoma cases.
V (1) V (2) V (3) V (4) V (5) V (6) a
Control (n = 688)
Colorectal cancer (n = 88)a
36 (30i + 6d) 5 (41 + 1d) 3 (2i + 1d) 1 (1d) 3 (2i + 1d) 0
1 (1i) 2 (1i + 1d) 1 (1d) 0 3 (2i + 1d) 1 (1d)
This is twice the number of patients, as the analysis involves alleles.
members of the pRB family as a result of their accumulation, which may alter the cell cycle regulatory activity of other members of the E2F family. Using paraffin-embedded specimens has disadvantages, like the poor quality of the DNA extracted or the inability to analyze mRNA or proteins, except when immunohistochemistry techniques are applied. However, it allows to select the different tumour tissues areas in a more precise way, and in some cases, address the sequencing of not very extensive DNA fragments. In conclusion, there is no evidence supporting that the expansion or contraction in the number of triplets of the E2F4 gene have a trigger effect on the tumour development process of CRC, although there is insufficient data to suspect that the alleles that differ from the common of 13 repeats (AGC) in more than one triplet can provide some degree of susceptibility to the development of CRC, possibly as a result of the alteration in protein concentration. Conflict of interests The authors declare no conflict of interest. References 1. Xu G, Livingston DM, Krek W. Multiple members of the E2F transcription factor family are the products of oncogenes. Proc Natl Acad Sci U S A. 1995;92:1357–61.
Cyclosporin-induced thrombotic thrombocytopenic purpura. Report of three cases夽 Púrpura trombótica trombocitopénica inducida por ciclosporina. Estudio de 3 casos Dear Editor, Thrombotic thrombocytopenic purpura (TTP) was first described by Moschcowitz in 1925.1 TTP should be suspected when a patient presents with: microangiopathic haemolytic anaemia with peripheral blood schistocytes,2 haptoglobin reduction and increased LDH,3 thrombocytopenia,4 acute renal failure, neurological disorders and, in some cases, fever.5 Cyclosporine can cause renal thrombotic microangiopathy with clinical manifestations of TTP (reaction described in the summary of product characteristics as rare: ≥1/10,000, <1/1000).6 This form of TTP is often, but not always, reversible with treatment discontinuation.6,7 3 patients undergoing hematopoietic stem cell transplant who received cyclosporine as immunosuppressive therapy are reported.
夽 Please cite this article as: Selvi Sabater P, Espuny Miró A, Rizo Cerdá AM, de la Rubia Orti JE. Púrpura trombótica trombocitopénica inducida por ciclosporina. Estudio de 3 casos. Med Clin (Barc). 2016;146:231–232.
231
2. Lindeman GJ, Gaubatz S, Livingston DM, Ginsberg D. The subcellular localización of E2F4 is cell cycle dependent. Proc Natl Acad Sci U S A. 1997;94:5095–100. 3. Gaubatz S, Lees JA, Lindeman GJ, Livingston DM. E2F4 is exported from the nucleus in a CRM1-dependent manner. Mol Cell Biol. 2001;21:1384–92. 4. Paquin MC, Cagnol S, Carrier JC, Leblanc C, Rivard N. ERK-associated changes in E2F4 phosphorylation, localization and transcriptional activity during mitogenic stimulation in human intestinal epithelial crypt cells. BMC Cell Biol. 2013;14:33. 5. Yoshitaka T, Matsubara N, Ikeda M, Tanino M, Hanafusa H, Tanaka N, et al. Mutations of E2F4 trinucleotide repeats in colorectal cancer with microsatellite instability. Biochem Biophys Res Commun. 1996;227:553–7. 6. Paquin MC, Leblanc C, Lemieux E, Bian B, Rivard N. Functional impact of colorectal cancer-associated mutations in the transcription factor E2F4. Int J Oncol. 2013;43:2015–22. 7. Ikeda M, Orimo H, Moriyama H, Nakajima E, Matsubara N, Mibu R, et al. Close correlation between mutations of E2F4 and hMSH3 genes in colorectal cancers with microsatellite instability. Cancer Res. 1998;58:594–8. 8. Garneau H, Paquin MC, Carrier JC, Rivard N. E2F4 expression is required for cell cycle progression of normal intestinal crypt cells and colorectal cancer cells. J Cell Physiol. 2009;221:350–8. 9. Souza RF, Yin J, Smolinski KN, Zou TT, Wang S, Shi YQ, et al. Frequent mutation of the E2F-4 cell cycle gene in primary human gastrointestinal tumors. Cancer Res. 1997;57:2350–3. 10. Matsubara A, Yoshitaka T, Matsuno T, Ikeda M, Isozaki H, Tanaka N, et al. Multiple tumors and a novel E2F-4 mutation. A case report. Digestion. 2000;62:213–6.
Antonio Ríos a,b,c,∗ , José Manuel Rodríguez a,b,c , Pablo Carbonel d , Pascual Parrilla a,b,c a
Departamento de Cirugía, Universidad de Murcia, Murcia, Spain Servicio de Cirugía General y de Aparato Digestivo, Hospital Clínico Universitario Virgen de la Arrixaca, Servicio Murciano de Salud, El Palmar, Murcia, Spain c Instituto Murciano de Investigación Biosanitaria Virgen de la Arrixaca (IMIB), Murcia, Spain d Centro de Bioquímica y Genética Clínica, Instituto Murciano de Investigación Biosanitaria Virgen de la Arrixaca (IMIB), Hospital Clínico Universitario Virgen de la Arrixaca, Servicio Murciano de Salud, El Palmar, Murcia, Spain b
∗ Corresponding
author. E-mail address: [email protected] (A. Ríos).
Patient 1. Male, 61 years of age at the time of transplantation, diagnosed with stage IVB diffuse large B-cell non-Hodgkin lymphoma, who underwent allogeneic peripheral blood stem cell transplant from an unrelated donor, HLA-compatible and ABO. The patient started on day +8 with increased bilirubin, and after ruling out the hepatic veno-occlusive disease, intravascular haemolysis data were observed with increased LDH levels reaching 972 IU/l, decreased platelet count (9 × 109 /L), haemoglobin decrease (90 g/l), haptoglobine decrease (0.05 g/l) and presence of schistocytes in peripheral blood. In addition, there is evidence of renal involvement with increased creatinine levels in blood. Patient 2. Female, 14 years of age at the time of transplantation, diagnosed of acute lymphoblastic leukaemia with 11q abnormalities and skin infiltration. Compatible with the diagnosis of acute lymphoblastic leukaemia ALL-L2 (FAB), Pre-B acute leukaemia (EGIL B-III), Precursor B-cell ALL (WHO). She underwent allogeneic peripheral blood stem cell transplant from an unrelated donor, HLA-compatible and ABO-compatible. The patient began on day +30 with data which demonstrated haemolysis with increased LDH levels reaching 1368 IU/l, decreased platelet count (30 × 109 /L), haemoglobin decrease (75 g/l), haptoglobine decrease (0.01 g/l) and presence of schistocytes in blood smear. Patient 3. Male, 46 years of age at the time of the second transplant, diagnosed with acute lymphoblastic leukaemia. Compatible with the diagnosis of acute lymphoblastic leukaemia ALL-L2 (FAB),
www.elsevier.es/medicinaclinica
Scientific letters E2F4 as susceptibility factor in the early onset colorectal cancer夽 E2F4 como factor de susceptibilidad en el cáncer colorrectal de inicio temprano Dear Editor: E2F is a family of 6 transcription factors (E2F6 E2F1) that regulates the expression of genes involved in the development of the cell cycle, and whose dysregulated synthesis induces the neoplastic transformation of some cell lines.1 E2F4 is the one expressed most abundantly, accounting for 50% of these proteins. In resting cells, E2F4 is mainly located in the nucleus, where it forms a complex with p130.2 The cell entry in the division process coincides with the active transport of the protein from the nucleus to the cytoplasm.3 The E2F4 protein contains a sequence of 13 consecutive serine residues in its transactivation domain.4 These are encoded by a series of triplets (AGC) whose number is frequently altered in colorectal cancers (CRC) with microsatellite instability phenotype.5,6 The E2F4 proteins encoded by genes with an altered number of AGC triplets show increased transactivator capacity and are capable of producing a cell proliferation stimulation.7 Furthermore, E2F4 overexpression has proliferative and tumorigenic effects.8 It has been reported that E2F4 alleles with a number of triplets other than 13 in the germline favour the development of CRC.9,10 The aim is to analyze the relationship between the presence of E2F4 gen alleles with a number of ACG triplets other than 13 and greater susceptibility to develop CRC. 44 patients with CRC diagnosed before the age of 51 years were selected. None of the 44 patients had germline mutations (Lynch syndrome). DNA extraction was performed from peripheral blood (germline) and tumour tissue (tumour line). The Nushift (EMSA) kit by Geneka Biotechnology, Inc. was used to test the transcription factors of the E2F family. (Cat. N◦ 2006690). The DNA of the tumour line is used as a control to assess that there is no difference with the germline due to microsatellite instability. Immunohistochemistry was performed using a standard technique. The DNA extracted from the peripheral blood of 344 healthy unrelated individuals was used as control group. Given the low allele frequencies, they were grouped according to the size of variation, regardless of whether they were insertions or deletions: V (1) variations of a triplet (both, insertions and deletions); V (2) of 2 triplets, and so on. Statistics: 2 test where possible and Fisher exact test in the other cases. The tumour line DNA showed no differences with germline DNA regarding the microsatellite instability under study. In terms of the total E2F4 altered alleles in the germline, 9% is observed in the CRC
夽 Please cite this article as: Ríos A, Rodríguez JM, Carbonel P, Parrilla P. E2F4 como factor de susceptibilidad en el cáncer colorrectal de inicio temprano. Med Clin (Barc). 2016;146:230–231.
˜ S.L.U. All rights reserved. 2387-0206/© 2015 Elsevier Espana,
group (8 out of 88 alleles) compared to 7% of the population group (48 out of 688) (p > 0.05) (Table 1). Regarding the distribution of the alleles in the population group, there were more insertions than deletions, while in the CRC group their numbers were similar. As shown in Table 1, there is an imbalance in the distribution of allele frequencies, being more frequent those representing larger variations in the CRC population. Thus, in the population group, 75% (36/48) of the variations correspond to insertions and/or deletions of one single triplet, while this variation only represents 12% in the CRC group (1/8) (p = 0.003). The assay of mRNA synthesized by different E2F4 alleles showed no differences in the expression or the half-life of the same. The in situ E2F4 protein assay by immunohistochemistry showed greater E2F4 protein concentration in the preparations derived from tumour tissue from individuals with altered E2F4 alleles. There is a gradation from high to low, which correlates with the greatest difference regarding 13. In humans, the AGC repetitive sequence of E2F4 is polymorphic in 4–6% of the population.9,10 Thus, in our study, no differences were observed in the frequency with which the E2F4 alleles were identified other than 13 repeats in the CRC group when compared to the population group (9 versus 7%). However, differences are observed in the distribution of the different alleles. Thus, in the population group, 75% of the variations (36/48) corresponded to expansions and contractions of a single triplet, whereas in the CRC group, these were only 12% (1/8). Furthermore, the distribution of alleles between the two populations is different for all major variations of a triplet, allowing speculation on the hypothesis that the alleles which move away from 13 in more than one triplet provide increased susceptibility to CRC development. The MRNA assay in individuals carrying different-sized alleles showed no difference in the expression of each one of them, nor in their half-life. Immunohistochemistry was used to assess the influence of this polymorphism in the distribution and concentration of E2F4 protein at cellular level, observing an increase in staining intensity when the E2F4 alleles were smaller in the germline. The staining was exclusively cytoplasmic, primarily affecting glandular cells, especially neoplastic cells. Likewise, the staining was strong in macrophages, in all cases, probably due to their phagocytic activity on tumour cells. The absence of nuclear staining is, possibly, a result of the inability of the antibody used in the immunohistochemical technique to recognize its epitope when E2F4 is dimerized with any of the DP proteins and bound to DNA. The regulatory activity of E2F4 is partly modulated by its cellular location (mainly nuclear in resting cells and cytoplasmic in dividing cells). Therefore, the presence of E2F4 in the cytoplasm of glandular cells could be related to the high proliferative activity of these cells, especially the neoplastic ones. The diversity of mechanisms by which the transcription factors of the E2F family are able to regulate gene expression makes interpreting the consequences of E2F4 accumulation difficult. In this case the activity of the protein seems to be closely linked to the active transport through the nuclear membrane, mediated by
Scientific letters / Med Clin (Barc). 2016;146(5):230–232 Table 1 Allelic frequencies for each of the variations in the general population and in colorectal carcinoma cases.
V (1) V (2) V (3) V (4) V (5) V (6) a
Control (n = 688)
Colorectal cancer (n = 88)a
36 (30i + 6d) 5 (41 + 1d) 3 (2i + 1d) 1 (1d) 3 (2i + 1d) 0
1 (1i) 2 (1i + 1d) 1 (1d) 0 3 (2i + 1d) 1 (1d)
This is twice the number of patients, as the analysis involves alleles.
members of the pRB family as a result of their accumulation, which may alter the cell cycle regulatory activity of other members of the E2F family. Using paraffin-embedded specimens has disadvantages, like the poor quality of the DNA extracted or the inability to analyze mRNA or proteins, except when immunohistochemistry techniques are applied. However, it allows to select the different tumour tissues areas in a more precise way, and in some cases, address the sequencing of not very extensive DNA fragments. In conclusion, there is no evidence supporting that the expansion or contraction in the number of triplets of the E2F4 gene have a trigger effect on the tumour development process of CRC, although there is insufficient data to suspect that the alleles that differ from the common of 13 repeats (AGC) in more than one triplet can provide some degree of susceptibility to the development of CRC, possibly as a result of the alteration in protein concentration. Conflict of interests The authors declare no conflict of interest. References 1. Xu G, Livingston DM, Krek W. Multiple members of the E2F transcription factor family are the products of oncogenes. Proc Natl Acad Sci U S A. 1995;92:1357–61.
Cyclosporin-induced thrombotic thrombocytopenic purpura. Report of three cases夽 Púrpura trombótica trombocitopénica inducida por ciclosporina. Estudio de 3 casos Dear Editor, Thrombotic thrombocytopenic purpura (TTP) was first described by Moschcowitz in 1925.1 TTP should be suspected when a patient presents with: microangiopathic haemolytic anaemia with peripheral blood schistocytes,2 haptoglobin reduction and increased LDH,3 thrombocytopenia,4 acute renal failure, neurological disorders and, in some cases, fever.5 Cyclosporine can cause renal thrombotic microangiopathy with clinical manifestations of TTP (reaction described in the summary of product characteristics as rare: ≥1/10,000, <1/1000).6 This form of TTP is often, but not always, reversible with treatment discontinuation.6,7 3 patients undergoing hematopoietic stem cell transplant who received cyclosporine as immunosuppressive therapy are reported.
夽 Please cite this article as: Selvi Sabater P, Espuny Miró A, Rizo Cerdá AM, de la Rubia Orti JE. Púrpura trombótica trombocitopénica inducida por ciclosporina. Estudio de 3 casos. Med Clin (Barc). 2016;146:231–232.
231
2. Lindeman GJ, Gaubatz S, Livingston DM, Ginsberg D. The subcellular localización of E2F4 is cell cycle dependent. Proc Natl Acad Sci U S A. 1997;94:5095–100. 3. Gaubatz S, Lees JA, Lindeman GJ, Livingston DM. E2F4 is exported from the nucleus in a CRM1-dependent manner. Mol Cell Biol. 2001;21:1384–92. 4. Paquin MC, Cagnol S, Carrier JC, Leblanc C, Rivard N. ERK-associated changes in E2F4 phosphorylation, localization and transcriptional activity during mitogenic stimulation in human intestinal epithelial crypt cells. BMC Cell Biol. 2013;14:33. 5. Yoshitaka T, Matsubara N, Ikeda M, Tanino M, Hanafusa H, Tanaka N, et al. Mutations of E2F4 trinucleotide repeats in colorectal cancer with microsatellite instability. Biochem Biophys Res Commun. 1996;227:553–7. 6. Paquin MC, Leblanc C, Lemieux E, Bian B, Rivard N. Functional impact of colorectal cancer-associated mutations in the transcription factor E2F4. Int J Oncol. 2013;43:2015–22. 7. Ikeda M, Orimo H, Moriyama H, Nakajima E, Matsubara N, Mibu R, et al. Close correlation between mutations of E2F4 and hMSH3 genes in colorectal cancers with microsatellite instability. Cancer Res. 1998;58:594–8. 8. Garneau H, Paquin MC, Carrier JC, Rivard N. E2F4 expression is required for cell cycle progression of normal intestinal crypt cells and colorectal cancer cells. J Cell Physiol. 2009;221:350–8. 9. Souza RF, Yin J, Smolinski KN, Zou TT, Wang S, Shi YQ, et al. Frequent mutation of the E2F-4 cell cycle gene in primary human gastrointestinal tumors. Cancer Res. 1997;57:2350–3. 10. Matsubara A, Yoshitaka T, Matsuno T, Ikeda M, Isozaki H, Tanaka N, et al. Multiple tumors and a novel E2F-4 mutation. A case report. Digestion. 2000;62:213–6.
Antonio Ríos a,b,c,∗ , José Manuel Rodríguez a,b,c , Pablo Carbonel d , Pascual Parrilla a,b,c a
Departamento de Cirugía, Universidad de Murcia, Murcia, Spain Servicio de Cirugía General y de Aparato Digestivo, Hospital Clínico Universitario Virgen de la Arrixaca, Servicio Murciano de Salud, El Palmar, Murcia, Spain c Instituto Murciano de Investigación Biosanitaria Virgen de la Arrixaca (IMIB), Murcia, Spain d Centro de Bioquímica y Genética Clínica, Instituto Murciano de Investigación Biosanitaria Virgen de la Arrixaca (IMIB), Hospital Clínico Universitario Virgen de la Arrixaca, Servicio Murciano de Salud, El Palmar, Murcia, Spain b
∗ Corresponding
author. E-mail address: [email protected] (A. Ríos).
Patient 1. Male, 61 years of age at the time of transplantation, diagnosed with stage IVB diffuse large B-cell non-Hodgkin lymphoma, who underwent allogeneic peripheral blood stem cell transplant from an unrelated donor, HLA-compatible and ABO. The patient started on day +8 with increased bilirubin, and after ruling out the hepatic veno-occlusive disease, intravascular haemolysis data were observed with increased LDH levels reaching 972 IU/l, decreased platelet count (9 × 109 /L), haemoglobin decrease (90 g/l), haptoglobine decrease (0.05 g/l) and presence of schistocytes in peripheral blood. In addition, there is evidence of renal involvement with increased creatinine levels in blood. Patient 2. Female, 14 years of age at the time of transplantation, diagnosed of acute lymphoblastic leukaemia with 11q abnormalities and skin infiltration. Compatible with the diagnosis of acute lymphoblastic leukaemia ALL-L2 (FAB), Pre-B acute leukaemia (EGIL B-III), Precursor B-cell ALL (WHO). She underwent allogeneic peripheral blood stem cell transplant from an unrelated donor, HLA-compatible and ABO-compatible. The patient began on day +30 with data which demonstrated haemolysis with increased LDH levels reaching 1368 IU/l, decreased platelet count (30 × 109 /L), haemoglobin decrease (75 g/l), haptoglobine decrease (0.01 g/l) and presence of schistocytes in blood smear. Patient 3. Male, 46 years of age at the time of the second transplant, diagnosed with acute lymphoblastic leukaemia. Compatible with the diagnosis of acute lymphoblastic leukaemia ALL-L2 (FAB),