Fulminant onset of acute leukemia from normal hematopoiesis within 3 months of follow up for multiple myeloma treated with total therapy protocols.

Assiduous surveillance for genetic aberrations is necessary in patients on cytotoxic therapies to detect therapy-related myeloid neoplasms (t-MN). Current modalities include metaphase cytogenetics and FISH. Since t-MN may develop abruptly in cytogenetically normal patients, a discussion exploring additional methods such as SNP-array and targeted-deep-sequencing to detect subchromosomal abnormalities is needed.

Introduction

The development of secondary malignancies following high-dose chemo-radiotherapy (HDT) in multiple myeloma (MM) is well established 1–3; recent reports have described an association with IMiD therapy as well 4,5. Based on a large population-based study 6 therapy-related myeloid neoplasms (t-MN) are the most common second malignancy in MM patients on therapy, with a standardized incidence ratio of 11.51 (CI 8.19–15.74). We and others, have previously shown that clonal, myelodysplasia-associated cytogenetic abnormalities (MDS-CA) may be transiently observed during the course of chemotherapy, and do not always correlate with presence of morphological dysplasia, or require specific therapeutic intervention. 5,7,8 However, most patients who have persistent or progressive cytopenia, and eventually develop t-MN, harbor persistent cytogenetic abnormalities 1–3. In a recent paper, 5 we described a number of pretreatment host-, and treatment-related variables that could be linked to development of MDS-CA, such as advanced age, longer duration of pretransplantation chemotherapy, low CD34 yield, and more leukapheresis sessions to obtain the desired quantity of hematopoietic progenitor cells. In this study, we draw attention to fulminant onset of acute leukemia (FOAL) in patients who did not have any previously identified significant morphological dysplasia or MDS-CA, and had a morphologically and karyotypically normal staging bone marrow (BM) within the previous 3 months. We searched for FOAL-defining features in the context of the more common therapy-related acute leukemia (t-AL) arising with prodromal clinical features or MDS-CA.

Patients and Methods

Patient cohort

Our MM database was scrutinized for all patients who had received hematopoietic progenitor cell-supported HDT at our institution since 1989. Only patients who had received at least one HDT regimen and an autologous hematopoietic stem-cell transplantation (auto-HSCT), and had informative cytogenetic data, defined as at having least one evaluable karyotype before, and one at a minimum of 28 days after HDT, are included in the study. All participating patients had signed an initial informed consent in keeping with institutional, federal, and international guidelines. A subset of the patients in this cohort (patients on TT2, TT3a, and TT3b protocols) was included in a recent publication on risk factors for myelodysplasia 5. This study includes all qualifying MM patients who received any of the following: Total therapy protocols (TT-P) for newly diagnosed MM 9–12 non-TT protocol, or off-protocol therapies. BM examinations and cytogenetic studies were performed for all patients at diagnosis, at 3-month intervals during the first year of maintenance, every 6 months for 3 years, and annually thereafter.

Definitions

CA commonly observed in myelodysplasia or acute leukemia 13 are referred to as MDS-CA in this study. We define MDS-CA as “persistent” when the same clonal chromosomal abnormality was observed in three successive metaphase cytogenetic analyses, and as “transient” when the clonal abnormalities were observed in less than three successive analyses, or when the abnormalities observed in the successive evaluations were dissimilar 8. Since chemotherapy per say can result in morphological dyspoiesis, to avoid confounding bias, we identify a patient as having developed t-MN only when associated with persistent MDS-CA. Patients who required management of cytopenias with disease-modifying therapies such as hypomethylating agents including decitabine and 5-azacytidine, or cytotoxic chemotherapy, which is typically a combination of doxorubicin, Ara-C, and melphalen (DAM), or HSCT with BEAM conditioning, are designated as clinical MDS (C-MDS). Patients who received transfusion support, erythropoiesis-stimulating agents, or myeloid growth factors are excluded from the C-MDS because these management modalities are frequently used to facilitate recovery from cytopenia following HDT or HSCT. The FOAL group has already been defined. Acute leukemia developing following HDT is referred to as t-AL (AML/ALL). Patients with t-MN who had less than 20% blasts in the BM or peripheral blood are termed as t-MDS in this study. Complete remission is defined by the international myeloma working group (IMWG) criteria. 14.

Morphology, flow cytometry, and cytogenetics

A diagnosis of FOAL pertained to eight patients who, within 3 months of their last thorough evaluation at our institution, qualified as having no clinical, morphological, or genetic evidence indicative of MDS. All BM specimens within the one-year period prior to FOAL were reevaluated. Morphological evaluation of myelodysplasia was based on previously described parameters 15,16 and performed by three experienced hematopathologists (Z. S., G. P., D. A.). Flow cytometry was performed on BM samples for quantitation and immunophenotypic characterization of the blasts. Metaphase cytogenetic analysis by GTG banding was performed on unstimulated BM specimens using standard techniques. At least 20 metaphases were examined from each specimen. The cytogenetic nomenclature followed standard ISCN criteria 17. Interphase FISH was performed using probes to detect abnormalities commonly observed in MDS and AML such as −5/del(5q), −7/del(7q), del(20)(q11.2), +8, and −13. Additional probes were used in some patients at the onset of FOAL to detect translocations involving 11q23/MLL gene, t(8;21), t(15;17), inv(16) or t(16;16), t(9;22), and t(8;14).

Results

Of the 3941 patients undergoing mainly melphalan-based HDT, 391 patients developed MDS-CA (Fig.1). Since the emphasis of this work is on t-MN, the 56 patients who exhibited MDS-CA prior to first transplant were excluded from further analysis. Of the 335 remaining patients, 213 showed transient MDS-CA and 122 had persistent MDS-CA. Fifty patients with t-MN requiring disease-modifying therapies qualified as C-MDS. Of these 50 patients, 11 presented with t-AL (t-AML (10); t-ALL(1)) and 39 with t-MDS. Fifteen of the t-MDS patients subsequently progressed to t-AL. Of the 3550 patients who did not have MDS-CA, nine developed t-AL: eight of them qualified for a diagnosis of FOAL as previously defined; one patient who had persistent, clinically significant cytopenia prior to onset of leukemia, was not included in the FOAL group.

Figure 1

Study cohort showing the distribution of patients according to presence of MDS-CA, the development of t-MN (t-MDS or t-AL [AML or ALL]), and of FOAL. MDS-CA, myelodysplasia-associated cytogenetic abnormalities; t-MN, therapy-related myeloid neoplasms; t-AL, therapy-related acute leukemia; FOAL, fulminant onset of acute leukemia.

Characteristics and treatment of FOAL patients

The baseline MM parameters of FOAL patients are outlined in Table1. Their age ranged from 41 years to 66 years, six were males and two females. The immunoglobulin distribution of the MM was representative of our referral population. The time from MM diagnosis ranged from 4 to 12 years. Myeloma-associated CA (MM-CA) were present in three of the eight patients at diagnosis of MM. Gene expression profiling (GEP)-defined molecular subgroups 18 were representative of newly diagnosed patients. Both GEP-70 and GEP-80 risk scores 19 were low in seven patients, while one had high-risk features in both models. Treatment regimes included TT2 without thalidomide in 1, TT2 with thalidomide 9 in 2, TT3a (maintenance with bortezomib, thalidomide, and dexamethasone) 10 in 3, TT3b (maintenance with bortezomib, lenalidomide, and dexamethasone) 11 in 1, and TT4 (similar to TT3b for GEP-defined low-risk MM) 12 in one patient. Table2 summarizes the clinical, cytogenetic, morphological, and immunophenotypic features of patients with FOAL at onset of t-AL. The complete blood count (CBC) values over the last 12–18 months (at 3- to 6-month intervals) were as follows: hemoglobin 12.8 g/dL (range 7.8–13.6 g/dL; mean 12.0 g/dL), WBC 3.52 K/μL (range 1.17–9.7 K/μL; mean 4.38 K/μL), and platelet count 147 K/μL (range 60–280 K/μL; mean 157 K/μL). Latency periods to FOAL diagnosis from treatment initiation, and from first HDT, ranged from 11 to 134 months and 6–118 months, respectively (Table2). The MM status at onset of FOAL was stringent complete response (sCR) in seven patients and complete response (CR) in one patient. Seven of the eight FOAL patients were classified as t-AML and 1 as t-precursor B-ALL. The morphology and immunophenotype from representative t-AML and t-ALL cases are illustrated in Figures2A–C and 3A–C, respectively.

Table 1

Baseline myeloma parameters in FOAL patients

Patient no.	1	2	3	4	5	6	7	8
Parameters
Age/Sex	41/M	44/M	59/M	54/M	61/M	66/F	48/F	66/M
Race	AA	W	W	W	AA	W
MM Ig subtype	IgG K	IgG L	K light chain	Nonsecretory K light chain	K light chain	IgA K	IgG K	IgG K
Date of diagnosis	09/2005	08/2009	01/2001	05/2005	03/2001	09/2005	12/2001	09/2006
Risk-score at Diagnosis	70-gene: Low 80-gene: Low	70-gene: Low 80-gene: Low	70-gene: Low 80-gene: Low	70-gene: High 80-gene: High	70-gene: Low 80-gene: Low	70-gene: Low 80-gene: Low	70-gene: NA 80-gene: NA	70-gene: Low 80-gene: Low
Molecular subtype	CD2	LB	Hyperdiploidy	Proliferation subset with LB	LB	MS	HY	NA
Karyotype at diagnosis of MM	46,XY20	46,XY20	46,XY20	38∽40,X, −Y,+1, add(1)(p11), der(1;16)(q10;p10), −4,−6, t(8;14) (q24.1;q32),−8,−12,−13,−16 [cp4]/ 70∽75,idemx2, inc[cp3]/ 46,XY[cp13]	46,XY20	42∽44,X,−X, add(6)(q?22), der(12)t(1;12) (q11∽12;q24.3),-13 [cp9]/46, XX[cp11]	53,XX,+3,+5,+7,+9,+9,+11,+15, +19,−221/ 46,XX12	46,XY20
Treatment protocol	2003-33 (TT3) randomized to VTD arm	2008-01 (TT4) randomized to lite arm	98-026 (TT2) randomized to T-arm	2003-33 (TT3) randomized to VTD arm	98-026 (TT2) randomized to T-arm	2003-33 (TT3) randomized to VTD arm	98-026 (TT2) randomized to non-T-arm	2006-66 (TT3)
Lenalidomide: Yes/No, duration	Yes, 7 months	No	No	Yes, 30 months	No	Yes, 4 months	No	Yes, 6 months
No. of HSCT	2	2	2	2	2	2	3	3
Preparatory regimen	Mel 200 Mel 200	Mel 200 Mel 200	Mel 200 Mel 200	Mel 200 Mel 200	Mel 200 Mel 200	Mel 200 Mel 200	Mel 200 BEAM Mel 200, Gemcitabine	Mel 200 Mel 200
Stem cells collected >3 × 106	No	No	Yes	No	No	No	No	No
>2 apheresis	No	No	Yes	No	No	No	No	No

Table 2

Clinical, cytogenetic, morphological, and immunophenotypic characteristics of the FOAL group 3 months prior to, and at the onset of acute leukemia

Patient no.	1	2	3	4	5	6	7	8
Parameters
Latency period from start of chemotherapy to FOAL	66 months	11 months	134 months	75 months	127 months	77 months	68 months	65 months
Latency period from last transplant to FOAL	52 months	6 months	118 months	71 months	116 months	68 months	25 months	60 months
MM status at FOAL	sCR	sCR	CR	sCR	sCR	sCR	sCR	sCR
Karyotype within 3 months prior to FOAL	46,XY20	46,XY20	46,XY20	46,XY20	46,XY20	46,XX20	46,XX20	46,XX//XY19/46,XY,del(20)(q11.2)1
FISH within 3 months prior to FOAL	ND	Normal	Normal	ND	ND	ND	Normal	Normal
BM within 3 months prior to FOAL	Normocellular, No dysplasia, no residual MM	Normocellular, No dysplasia, no residual MM	Normocellular, No dysplasia, no residual MM	Normocellular, No dysplasia, no residual MM	Normocellular, No dysplasia, no residual MM	Normocellular, No dysplasia, hematogones 10%, no residual MM	Normocellular, No dysplasia, no residual MM	Mildly hypocellular with rare hypolobate megakaryocytes
Karyotype at time of FOAL	46,XY20	46,XY20	43∽44,XY,+3,add(3)(p13),del(3)(q12),−5,−7, ?t(8;10) (q22;q26),−16,−17,−18,inc[cp15] /46,XY22	39∽44,XY,−5,−7, der(12)t(12;?17) (p13;q21), −16, add(17)(p11.2),−18,−19, +2mar[cp40]	42∽43,XY, der (3;12)(q10;q10), del(4)(q31.3),ucode>−5,−6, der(14)t(11;14) (q12∽13.1;q32) del(11)(q23), inv(16)(p?13.1q?22), −17,+mar[cp11]/ 42∽43, idem, del(1)(q21), ?inv(7)(p21q11.1) [cp9]/46,XY1	46,XX,del(9)(p12), del(20)(q11.2q13.1) 2/46,XX[cp38]	43∽52,XX,der(1;16)(q10;p10),add(10)(q26), del(11)(p11.1),add(?13) (q34),del(13)(q12q22),del(16)(q22),add(17)(p13), del(20)(q11.2), +1∽3mar,2∽3dmin, inc[cp14]/46,XX6	46,XY,t(3;8)(q27;q24.1), −7,+mar4/45,idem,-mar6/ 46,XY10
FISH	ND	Normal	del(5q31),−7, del(7q)	−5, del(5q31), −7, +21q22, +17q21, del (16q22), +9q34, −13	del(5q31)	del(20q)	Normal	−7,del(7q)
Other molecular tests	ND	ND	ND	Negative for JAK2 V617F	Negative for the FLT3 Internal Tandem Duplication, FLT3 D835 variant; and NPM1 mutation	ND	ND	ND
FOAL subtype	AML	AML	AML	AML	AML	Precursor B-ALL	AML	AML
BM at FOAL	BM not reviewed at our institution. Details NA	Hypercellular, blasts 75%;Decreased megakaryopoiesis and erythropoiesis	Normocellular (35%), blasts 20%, No significant dyspoiesis	Hypercellular, blasts 32%, marked trilineage dysplasia	Hypercellular, blasts 85–90%, markedly decreased Erythropoiesis and megakaryopoiesis, marked dysgranulopoiesis on peripheral blood	Normocellular, blasts 35%; trilineage dysplasia	Hypercellular, blasts 40%; erythroid and Megakaryocytic dysplasia, mild granulocytic dysplasia.	Hypercellular, blasts 65%; dysmegakaryopoiesis
Immunophenotype	Details not available	Positive: CD33, CD15, CD13 (variable), HLA-DR, CD14, CD11b, CD56 (subset), and CD4(dim)	Positive: CD34, CD117, CD13, CD33, CD7, CD5, and MPO, and CD4 (partially, weak)	Positive: CD45 (dim), CD34, CD117, CD15, CD33, CD13, CD56, and HLA DR	Positive: CD45 (dim), CD34, CD117, CD33, CD13, CD56 (dim), and HLA-DR	Positive: CD34, TdT, CD19, CD10, CD20, CD99, CD44	Positive: HLA-DR, CD117, CD33, CD13	Positive: CD34, CD117, HLA-DR, CD33, CD13, CD56, CD5, and CD7
		Negative: CD34, CD117, cMPO	Negative: HLA-DR, TdT, CD14, CD56, CD15, glycophorin-A, and all lymphoid antigens.	Negative: CD3, CD19, MPO, TdT, and other tested myeloid, and lymphoid antigens	Negative: CD3, CD19, CD15, CD11b, all other myeloid antigens, and lymphoid antigens.	Negative: CD117, CD33, CD13, CD15, MPO, all T-lymphoid antigenss	Negative: CD34, and all myeloid and lymphoid antigens	Negative: cMPO,, all other myeloid and lymphoid antigens (except D5 and CD7)
Acute leukemia therapy	Clofarabine, Idarubicin, Cytarabine	Idarubicin, ara-C, etoposide, and fludarabine	Decitabine, followed by Decitabine + Azacytidine + Melphalen	Decitabine, Allogenic HSCT	7 + 3 induction with idarubicin, ara-C; Decitabine + Azacytidine + Melphalen	Hyper-CVAD with Rituxumab and inotuzumab	ara-C, VP-16, Mylotarg	Idarubicin, ara-C, and dexamethasone
Response	Refractory AML	In CR for t-AML and MM for 3 months	In CR for t-AML and MM for 2 months	CR t-AML and MM	In CR for 5 months, then relapsed	In CR for t-ALL and MM for 3 months	In CR for t-ALL and MM	In CR for t-ALL and MM for 2 months
Survival in months after FOAL	11	17	6	11	5	9	38	2
Outcome	Death	Death	Alive	Death	Alive	Alive	Death	Alive

The bold text highlights the cytogenetic abnormalities commonly associated with myelodysplasia.

Figure 2

(A) BM aspirate smear from patient no.4, 3 months prior to FOAL showing normal trilineage hematopoiesis without dyspoiesis (Wright-Giemsa, 200X). (B) BM aspirate smear from same patient at onset of FOAL (t-AML). Red arrows point to leukemic blasts (Wright-Giemsa, 200X). (C) Flow cytometry of BM sample from same patient at FOAL with the gated dim CD45+ blasts (C) blasts coexpress CD34, CD13, and CD56 (D and E). FOAL, fulminant onset of acute leukemia; BM, bone marrow; t-AL, therapy-related acute leukemia.

Figure 3

(A) BM aspirate smear from patient no.6, within 3 months prior to FOAL showing normal trilineage hematopoiesis without dyspoiesis (Wright-Giemsa, 200X). (B) BM aspirate smear from same patient at onset of FOAL (t-precursor B-ALL). Red arrows point to leukemic blasts (Wright-Giemsa, 400X). (C) Flow cytometry of BM sample from same patient at FOAL with the gated dim CD45+ blasts (C) blasts coexpress CD34, CD19, and CD10 (D and E). FOAL, fulminant onset of acute leukemia; BM, bone marrow; t-AL, therapy-related acute leukemia.

None of the patients had MM-related karyotypic abnormalities in the year preceding the onset of FOAL, or any MDS-CA by conventional metaphase cytogenetic study or FISH, at any time prior to the development of FOAL. FISH data available in four patients during the 3-month-period prior to FOAL, did not identify MDS-CA with the employed probes. In one patient, del(20)(q21) was identified transiently in 1 of 20 metaphases, which does not qualify as clonal. At onset of FOAL, the karyotype in this patient did not show the del(20)(q21) abnormality (Table2). At the onset of FOAL, clonal MDS-CA were present in six of eight patients, in five of whom the karyotype was highly complex. Complete or partial deletions of chromosomes 5 or 7 were observed in three and three patients, respectively, and abnormalities of both 5 and 7 were present in two patients. The MDS-CA identified by FISH in four of the seven cases examined matched those observed by metaphase karyotyping.

Treatment of FOAL was individualized and was based on the patients’ comorbidities and performance status (Table2). Seven of the eight patients achieved CR, which was short-lived, ranging from 2 to 17 months.

Discussion

The development of secondary malignancies following HDT in multiple myeloma (MM) is well established 1–3; recent reports have described an association with IMiD therapy as well 4,5. In our population of 3941 patients who received HDT, 8.5% developed MDS-CA, which were transient in the majority (two-thirds) of the patients. A subset of patients with MDS-CA required therapy (C-MDS); of these patients, 39 fulfilled diagnostic criteria for t-MDS and 11 were classified as t-AL (group C). Of those patients with t-MDS, 15 transformed to t-AML (group B-indicate). In contrast, FOAL was observed in eight patients without antecedent MDS-CA, significant morphologic dysplasia or clinically significant cytopenias (group A). The classic form of t-MN that develops following heavy prior treatment with alkylating agents, is usually associated with a prior myelodysplastic phase marked by cytopenia, and significant morphological dysplasia in the BM, often involving all three cell lines 1–3. In this respect the FOAL patients, all of whom were heavily pretreated with melphalen, an alkylating agent, had an unusual presentation.

We attempted to identify predictive features for FOAL vis-à-vis patients with MDS-CA who presented as t-AL (11 patients; group C) or developed t-AL from antecedent t-MDS (15 patients; group B). There were no differences among the three groups with respect to age, sex number of transplants received or latency phase to t-AL. In our previous study on a large cohort of patients 5, and also from a recent paper by Pemmaraju et al. from M.D. Anderson cancer center 20, data highlights advanced age, longer duration of pretransplantation chemotherapy, low CD34 yield, and more leukapheresis sessions to obtain the desired quantity of hematopoietic progenitor cells as predictive of t-MN. Interestingly, 3 (6.4%) patients described by Pemmarju and others developed therapy-related chronic myelomonocytic leukemia, not a commonly described t-MN. Forty-three of the 46 patients (93%) who developed t-MN in their study had cytogenetic abnormalities, with high-risk abnormalities in 78% patients, but no patients are described to have an abrupt onset of acute leukemia without prior t-MDS or CA as observed by us. In fact, in our review of literature no specific mention of such a catastrophic development of t-AL has been previously made. This is especially remarkable because although the FOAL patients did not have any prior MDS-CA, interestingly, the proportion of patients with high-risk MDS-CA including −7, del(17)(p13)/TP53 and complex karyotypes (>3 abnormalities), were higher in this group (FOAL) of patients at the onset of t-AL compared with the two other groups in our study. There was no difference in survival from onset of t-AL among the patients groups (medians of 14, 9, and 10 months, respectively), nor did the latency duration affect survival in any of the groups of t-AL (data not shown). Notably, all FOAL patients had received TT protocols compared with five of 11 of group B (non-TT-P- 1; off-P, 5), and seven of 15 of group C patients (non-TT-P, 4; off-P, 4). Of all the parameters evaluated, we noted that patients with prior exposure to lenalidomide had a higher incidence of developing FOAL; four of eight patients in the FOAL group had prior lenalidomide exposure, which is higher in incidence than the two of 15 of group B, and 0 of 11 of group C patients (P = 0.018). This observation is interesting with respect to recent reports linking lenalidomide to secondary malignancies in MM 4,5. The sample size in our analysis is small to derive definite conclusions.

Cytotoxic therapies cause BM damage by formation of highly mutagenic methylated-base-lesions, inter- and intrastrand crosslinks in DNA, DNA double strand-breaks, and impaired DNA repair 21–23. Progressive accumulation of DNA-damaging mutations results in emergence of abnormal clones with a proliferative and survival advantage 21–23. Li et al. 24 found differences in expression levels of genes related to mitochondrial function, metabolism, DNA repair, and cell cycle regulation in CD34+ hematopoietic stem and progenitor cells between patients with Hodgkin or non-Hodgkin lymphoma, who developed t-MDS/AML following autologous HSCT, and those (matched controls) who did not have such an outcome. Barletta et al. 25 reported detection of RAS mutations in the peripheral blood of patients with chemotherapy exposure, prior to onset of hematological abnormalities (cytopenia). These studies provide evidence that cytotoxic exposure makes the cells genetically unstable and vulnerable to subsequent insults. The exact host factors, or the type of, or dosage of chemo- or radiotherapy that can tip the balance and trigger development of t-MN are poorly understood, and may also involve the BM microenvironment 25. Prior studies by Bejar et al. 26 and Thol et al. 27 demonstrated that point mutations in TP53, EZH2, ETV6, RUNX1, ASXL1, 26 and in genes involved in the splicing machinery 27 are significantly associated with progression from MDS to AML. By sequencing of MDS genomes, scientists have hypothesized that early driver mutations, typically involving RNA splicing, direct evolution of the disease, and also influence the clinical phenotype including BM morphological features, blast count, hemoglobin level, and clinical presentation 26–29. Although mutational analysis for these genes was not performed in this study, it is very likely that one or more such genetic events, not detected at the resolution level of metaphase cytogenetics or FISH were the trigger for leukemic transformation in these patients.

Conclusion

The FOAL in the index group of heavily pretreated patients suggests that hematopoietic dysregulation leading to this clinical event may be the result of genetic alterations at the DNA level that cannot be detected by conventional cytogenetics or FISH. Studies in our institution are in progress to determine whether MM- and stroma-related genetic features can be identified as contributors to clinical t-AL development. Our group is also working on a GEP-based model, which will help in identifying patients at increased risk of developing t-MDS after chemotherapy. We, and others 25,30 envision that data from studies based on GEP and massively parallel, whole-genome sequence analysis will help to select specific, frequently occurring genomic aberrations that can reliably predict a t-MN event, and be used to develop targeted genetic tests for surveillance of patients receiving cytotoxic therapy. A collaborative approach to develop cost-effective clinical tests for submicroscopic genomic aberrations in this target population is clearly warranted.

Conflict of Interest

None declared.

Ethics

We confirm that we have given due consideration to the protection of intellectual property associated with this work and that there are no impediments to publication, including the timing of publication, with respect to intellectual property. In so doing we confirm that we have followed the regulations of our institutions concerning intellectual property.