11q23/MLL rearrangements in adult acute leukemia

Zotova O.V.*1, Lukianova A.S.2, Valchuk M.O.1, Karol Yu.S.3, Shalay O.O.1, Novak V.L.1, Loginsky V.E.1

Summary. Aim: To detect the frequency, diagnostic and prognostic significance of 11q23/MLL rearrangements and to determine the chromosomes that are most frequently involved in 11q23/MLL abnormalities in adult acute leukemia (AL). Materials and Methods: Cytogenetic investigations of bone marrow and/or peripheral blood cells from 140 patients with acute myeloid leukemia (AML) and 57 patients with acute lymphoblastic leukemia (ALL) were performed. The methods of conventional cytogenetics (GTG-banding) and fluorescence in situ hybridization were used. Results: Chromosomal abnormalities in leukemia cells were found by conventional cytogenetic methods in 80 (57%) and 37 (65%) adult patients with AML and ALL, respectively. 11q23/MLL rearrangements were found in 7 (5%) and 8 (14%) patients with AML and ALL, respectively. Among them, 8 (53.4%) patients had translocations, 2 (13.3%) — had deletions and 5 (33.3%) patients had trisomies or tetrasomies of chromosome 11. With respect to the distribution of partner chromosomes involved in 11q23/MLL translocations chromosome 4 was found to participate in 3 (37.5%) cases of 11q23/MLL translocations, 9 — in 2 (25%) cases and chromosomes 10, 14 and non-identified chromosome were involved in 1 (12.5%) case each. Nine patients (60%), besides abnormal ones, had 9–86% normal metaphases in their karyotypes. Of 15 patients with 11q23/MLL rearrangements, 5 (33%) patients had only 11q23/MLL rearrangements, whereas other 10 (67%) — had additional cytogenetic abnormalities, besides 11q23/MLL rearrangements. Conclusions: Chromosomal abnormalities of various kinds were found in 57% and 65% adult patients with AML and ALL, respectively. The frequency of 11q23/MLL rearrangements in patients with AML and ALL was 5% and 14%, respectively. Since AL patients with 11q23/MLL rearrangements are attributed to cytogenetic categories of AL with a poor or intermediate risk prognosis, cytogenetic methods should be included in the standard examination of AL patients for diagnosis, prognosis and selection of the optimal treatment strategy.

Submitted: February 25, 2021.
*Correspondence: E-mail: lnkzotova@gmail.com
Abbreviations used: AL — acute leukemia; ALL — acute lymphoblastic leukemia; AML — acute myeloid leukemia; BM — bone marrow; CK — complex karyotype; FAB — French-American-British classification; FISH — fluorescence in situ hybridization; MK — monosomal karyotype; MLL — mixed-lineage leukemia or myeloid-lymphoid leukemia; PB — peripheral blood.

DOI: 10.32471/exp-oncology.2312-8852.vol-43-no-3.16495

Acute leukemia (AL) is a heterogeneous disease characterized by diverse clinical course and varied sensitivity to therapy. A key event in the development of AL is the restructuring of the progenitor cell genome, causing the disruption in molecular control of cell cycle, transcription and translation of major protein regulators. Damage to the leukemic cells genome is mainly represented by chromosomal rearrangements of proto-oncogenes or suppressor genes (translocations, inversions, deletions, loss of chromosomes, extra copies of chromosomes, etc.). The cloning of the genes located at the breakpoints of chromosomal translocations has resulted in the identification of new genes involved in carcinogenesis [1–3]. Molecular studies of the breakpoint of several translocations involving chromosomal band 11q23 led to the cloning of the MLL gene (mixed-lineage leukemia or myeloid-lymphoid leukemia). Based on literature data, 11q23/MLL rearrangements were detected in 5% of acute myeloid leukemia (AML) and 10–20% of acute lymphoblastic leukemia (ALL). More than 100 different rearrangements involving band 11q23 have so far been identified, of which the most frequently observed are t(4;11)(q21;q23), t(9;11)(q22;q23), t(11;19)(q23;p13.1), t(11;19)(q23;p13.3) [4–6].

Despite the large variety of rearrangements involving the MLL gene, the presence of distinct 11q23/MLL rearrangements is an independent dismal prognostic factor, while very few 11q23/MLL rearrangements display an intermediate outcome. Therefore, detection of 11q23/MLL disruption or amplification is much needed for treatment decision. Studying the wide variety of fusion genes involving MLL could also lead to a better understanding of leukemogenesis.

The aim of the study was to detect the frequency, diagnostic and prognostic significance of 11q23/MLL rearrangements in adult AL and to determine the most common chromosomes involved.

MATERIALS AND METHODS

Cytogenetic investigations of bone marrow (BM) and/or peripheral blood (PB) cells from 197 adult patients with AL [age range, 18–85 years, 113 (57%) males and 84 (43%) females] were performed. AL was diagnosed according to the WHO definition of > 20% blasts in the BM or PB and based on French-American-British (FAB) classification and immunophenotype. 140 patients had AML and 57 patients had ALL. All patients were hospitalized at Hematology Departments of the Institute of Blood Pathology and Transfusion Medicine (Lviv, Ukraine) or Municipal Clinical Hospital № 5 (Lviv, Ukraine). Compliance of the study with bioethical standards was approved by the Ethics Committee of the State Institution “Institute of Blood Pathology and Transfusion Medicine” (Lviv, Ukraine), protocol № 02/01, February 25, 2021. All patients provided their consent to the participation in the study.

The cytogenetic analysis of BM/PB blast cells was performed after 24-h unstimulated culture. The methods of conventional cytogenetics (GTG-banding) and fluorescence in situ hybridization (FISH) were used. Conventional cytogenetic methods were carried out using standard techniques [7–10], which included exposure to colchicines, hypotonic treatment, fixation and samples preparation. The samples were analyzed at a ×1000 magnification under the light microscope Olympus BX41 (Olympus, Japan) using a system for chromosomal analysis CytoVision (Applied Imaging, UK) at the Laboratory of Immunology and Genetics of Blood Neoplasms. At least 20 metaphase plates were analyzed. The karyotypes were described according to the International System for Human Cytogenetic Nomenclature [11]. Only clonal abnormalities were considered as positive results. Abnormalities were considered clonal if ≥ 2 metaphases had the same aberration in the case of a structural abnormality or an extra chromosome, or if ≥ 3 metaphases shared the same aberration in the case of a monosomy. Complex karyotype (CK) was defined as 3 or more clonal abnormalities. Additionally FISH technique with appropriate probes was used. Samples preparation and hybridization procedure was carried out according to Pinkel et al. [12] taking into consideration the recommendations of the probe manufacturer under a microscope Olympus BX41 (Olympus, Japan) using a system for chromosomal analysis CytoVision (Applied Imaging, UK) at the Laboratory of Immunology and Genetics of Blood Neoplasms. At least 200 cells were analyzed. The results of the FISH analysis were described according to the International System for Human Cytogenetic Nomenclature [11].

RESULTS AND DISCUSSION

Chromosomal aberrations of various kinds were found in 80 (57%) and 37 (65%) cases of AML and ALL, respectively. The most common abnormalities were: monosomies of 5 and 7, –Y, trisomy of 8, deletions of 5q and 7q, rearrangements of 3q, 12p, 17p and 11q23, translocations t(8;21)(q22;q22), t(9;22)(q34;q11), t(15;17)(q22;q11-21), t(16;16)(p13;q22), inversion inv(16)(p13q22), marker and ring chromosomes, acentric structures, CK, monosomal karyotype (MK) for AML patients; trisomy of 8, rearrangements of 7q, 17p and 11q23, translocations t(4;11)(q21;q23), t(9;22)(q34;q11), marker chromosomes, acentric structures, hypodiploidy, hyperdiploidy, CK for ALL patients. Some genetic abnormalities (BCR/ABL, PML/RARA, AML/ETO, CBFβ/MYH11, MLL/AF4, MLL/AF10 fusion genes) were detected by FISH.

Among the detected chromosomal aberrations, 11q23/MLL rearrangements were found in 7 (5%) and 8 (14%) cases of AML and ALL, respectively. The presence of MLL/AF4 and MLL/AF10 fusion genes was confirmed by FISH in 2 cases (Table).

Table. Results of cytogenetic investigations of leukemic cells from patients with 11q23/MLL rearrangements
Case Sex Age Subtype of AL Karyotype FISH
1 F 23 B-ALL 46,XX,t(4;11)(q21;q23)[7]/46,XX[13]
2 F 33 B-ALL 46,XX,t(4;11)(q21;q23)[10]/46,XX[10] MLL/AF4
3 M 82 B-ALL 77,XXY,t(4;11)(q21;q23)×2,+13,+16,+17,+18,+19,+20,+21,+22[20]
4 M 37 AML M5 46,XY,t(9;11)(p21-22;q23)[21]
5 F 63 AML M2 46,XX,del(3)(q21q26),t(9;11)(p21-22;q23)[21]/46,XX[2]
6 F 39 AML M1 46,XX,t(10;11)(p11-15;q13-23)[14]/46,XX[8] MLL/AF10
7 M 33 B-ALL 46,XY,t(2;9)(q11.2;q34),t(11;14)(q23;q32)[17]/46,XY[3]
8 M 69 AML M4 46,XY,r(3)(p26q29),-5,t(11;?)(q23;?),+mar[17]/46,XY[7]
9 M 35 B-ALL 46,XY,del(11)(q23),add(14)(q32)[20]
10 F 51 AML M5 46,XX,del(11)(q23)[10]/46,XX[10]
11 F 29 B-ALL 57,XX,+X,+2,+4,+6,+8,t(9;22)(q34;q11),+11,+14,+17,+21,+21,+der(22)t(9;22)[20]
12 F 53 B-ALL 62~67,XX,+2,+3,+3,+4,+5,+6,+7,+10,+11,+11,+12,+12,+13,+14,+15,+16,+17,+18,+19,+19,+20,+20,+21,+22[cp3]/46,XX[18]
13 F 20 B-ALL 60~61,XX,+X,+1,+3,+4,+6,+8,+9,+10,+11,+14,+15,+17,+18,+21,+mar[cp3]/46,XX[18]
14 F 46 AML M5 43~46,XX,-5,-8,-10,+11,-16,-17,-17,+1~4mar[cp20]
15 M 71 AML M5 46~57,XY,+Y,+1,-5,+6,+8,+9,+11,+12,+1~2mar1,+mar2,+mar3,+mar4[cp20]

Of 15 AL patients with 11q23/MLL rearrangements, 8 (53.4%) patients had translocations (cases 1–8) (Fig. 1, 2), 2 (13.3%) — had deletions (cases 9, 10) (Fig. 3) and 5 (33.3%) patients had trisomies or tetrasomies of chromosome 11 (cases 11-15). With respect to the distribution of partner chromosomes involved in 11q23/MLL translocations: chromosome 4 was found to participate in 3 (37.5%) cases of 11q23/MLL translocations (cases 1–3) (Fig. 1), 9 — in 2 (25%) cases (4, 5) (Fig. 2) and chromosomes 10, 14 and non-identified chromosome were involved — in 1 (12.5%) case each (cases 6, 7, 8, respectively). Nine patients (60%), besides abnormal ones, had 9–86% normal metaphases in their karyotypes. Of 15 patients with 11q23/MLL rearrangements, 5 (33%) had only 11q23/MLL rearrangements, whereas others 10 (67%) — had additional cytogenetic abnormalities, besides 11q23/MLL rearrangements. Among them, the presence of one additional karyotype abnormality was established in 3 (30%) cases, multiple structural and/or numerical changes (≥ 3) — in 7 (70%) cases. Spectrum of additional chromosomal aberrations associated with 11q23/MLL rearrangements was as follows: t(2;9)(q11.2;q34), del(3)(q21q26), r(3)(p26q29), t(9;22)(q34;q11), add(14)(q32), +der(22)t(9;22)(q34;q11), -5, marker chromosomes (mar), hypodiploidy, hyperdiploidy (Table).

 11q23/<i>MLL</i> rearrangements in adult acute leukemia
Fig. 1. Karyotype of leukemic cell from ALL patient with 11q23/MLL rearrangements (Table, case 1) — 46,XX,t(4;11)(q21;q23)
 11q23/<i>MLL</i> rearrangements in adult acute leukemia
Fig. 2. Karyotype of leukemic cell from AML patient with 11q23/MLL rearrangements (Table, case 4) — 46,XY,t(9;11)(p21-22;q23)
 11q23/<i>MLL</i> rearrangements in adult acute leukemia
Fig 3. Karyotype of leukemic cell from AML patient with 11q23/MLL rearrangements (Table, case 10) — 46,XX,del(11)(q23)

According to the literature data, 11q23/MLL rearrangements are detected in 5% of AML and ~10–20% of ALL. Differences in the frequency and the distribution of translocations were noted according to the type of AL and age of the patients [2, 4, 13]. In our study, the frequency of 11q23/MLL rearrangements was 5% for AML and 14% for ALL, which is comparable to the data reported in the literature. Of the cases with 11q23/MLL rearrangements, 33% patients had only 11q23/MLL rearrangements, whereas other 67% patients — had also additional cytogenetic abnormalities of various kinds, besides 11q23/MLL rearrangements. In our investigation of 15 cases with 11q23/MLL rearrangements, 40% patients had only abnormal metaphases in their karyotypes, whereas other 60% — had also 9–86% normal metaphases, besides abnormal ones.

11q23/MLL abnormalities can be divided in two categories. The first category consists of MLL rearrangements, usually as translocations or insertions, some of them cryptic, leading to fusion genes with a large number of partners. In addition, self-fusion of two parts of MLL within the breakpoint cluster region, leading to internal rearrangements called partial tandem duplication, have also been described in several cases. A second category of abnormalities is the amplification of the 11q23 region resulting in multiple copies of MLL gene, located either intrachromosomally as homogeneously staining region, or extrachromosomally in double minutes (dmin). The numerical abnormalities of chromosome 11, such as trisomies or tetrasomies, also result in additional copies of MLL gene [4–6].

MLL is known to be required for the expression of posterior HOXA (homeobox A cluster) genes in the hematopoietic cell lineage. The expression of the latter is highest at the immature progenitor stage, such as multipotent progenitors, but then gradually declines. Therefore, MLL is required for the proliferation of immature hematopoietic progenitors while MLL fusion proteins constitutively activate genes that promote self-renewal of hematopoietic stem cells [14, 15].

The differences in the frequency and the distribution of translocations were noted according to the form of AL and age of the patients. The prognostic effect of 11q23/MLL aberrations may depend on MLL partner genes. Many studies have shown that the translocations t(6;11)(q27;q23), t(10;11)(p12;q23) in AML and translocation t(4;11)(q21;q23) in ALL are associated with an unfavorable prognosis; however, the t(9;11)(p22;q23) translocation is accompanied with a significantly longer patient survival rate in AML. However, none of the 11q23/MLL aberrations has a favorable prognostic value [4–6].

All patients with 11q23/MLL rearrangements are usually classified into cytogenetic categories of AL with a poor or intermediate risk prognosis. Distribution of patients into risk groups according to the identified cytogenetic prognostic markers allows us to choose the most appropriate treatment approach for them, namely the intensity of therapy, the necessity of BM transplantation at the first remission, the necessity of the prescription of tyrosine kinase or all-trans retinoic acid [16, 17]. Thus, the modern diagnosis of AL should include the study of cytogenetic and molecular genetic features of blast cells together with cytomorphological, cytochemical and immunophenotype peculiarities of these cells. The combination of these techniques allows not only individualizing treatment according to the prognostic risk factors, but also it is the basis for understanding the pathogenesis and biological nature of AL.

In our study, chromosomal abnormalities in leukemia cells were found by conventional cytogenetic methods in 57% and 65% adult patients with AML and ALL, respectively and 11q23/MLL rearrangements were established in 5% and 14% adult patients with AML and ALL, respectively. Among the cases with 11q23/MLL rearrangements, 33% patients had only 11q23/MLL rearrangements, whereas other 67% — had also additional cytogenetic abnormalities, besides 11q23/MLL rearrangements.

Since AL patients with 11q23/MLL rearrangements are referred to cytogenetic categories of AL with a poor or intermediate risk prognosis, cytogenetic investigations should be included in the standard examination of these patients for diagnosis, prognosis and selection the optimal treatment strategy.

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ПЕРЕБУДОВИ 11q23/MLL ПРИ ГОСТРИХ ЛЕЙКЕМІЯХ У ДОРОСЛИХ

O.В. Зотова1, *, A.С. Лук’янова2, M.O. Вальчук1, Ю.С. Кароль3, O.O. Шалай1, В.Л. Новак1, В.Є. Логінський1

1ДУ “Інститут патології крові та трансфузійної медицини НАМН України”, Львів 79057, Україна
2Медико-біологічний центрГеном”, Київ 04216, Україна
3Комунальне некомерційне підприємство “Муніципальна клінічна лікарня № 5”, Львів 79057, Україна

Резюме. Мета: Визначити частоту, діагностичну та прогностичну значущість перебудов 11q23/MLL, а також визначити, які хромосоми найчастіше залучені до аномалій 11q23/MLL у дорослих хворих на гостру лейкемію. Матеріали та методи: Проводили цитогенетичне дослідження клітин кісткового мозку та/або периферичної крові у 140 хворих на гостру мієлоїдну лейкемію (ГМЛ) та 57 пацієнтів з гострою лімфобластною лейкемією (ГЛЛ) із застосуванням традиційного методу диференційного забарвлення (GTG), а також флуоресцентної гібридизації in situ. Результати: Хромосомні аномалії в лейкемічних клітинах за допомогою традиційного методу цитогенетичного аналізу виявили у 80 (57%) та 37 (65%) хворих на ГМЛ та ГЛЛ відповідно. Перебудову 11q23/MLL виявили у 7 (5%) та 8 (14%) хворих на ГМЛ та ГЛЛ відповідно. Серед них у 8 (53,4%) хворих визначали транслокації, у 2 (13,3%) — делеції і у 5 (33,3%) — трисомію або тетрасомію за хромосомою 11. Стосовно розподілу хромосом-партнерів, залучених до транслокацій 11q23/MLL, у 3 (37,5%) випадках це була хромосома 4, у 2 (25%) — хромосома 9; було виявлено ще по одному випадку залучення хромосом 10 та 14, в одному випадку хромосому-партнера не було ідентифіковано. У 9 (60%) хворих поруч з аномальними метафазними пластинками виявляли від 9 до 86% метафаз з нормальним каріотипом. Серед 15 хворих з перебудовами 11q23/MLL у 10 (67%), окрім перебудов 11q23/MLL, виявляли також додаткові цитогенетичні аномалії. Висновки: Різноманітні хромосомні аномалії виявляли у 80 (57%) та 37 (65%) дорослих хворих на ГМЛ та ГЛЛ відповідно. Частота перебудови 11q23/MLL у дорослих хворих на ГМЛ та ГЛЛ станови відповідно 5 та 14%. Оскільки хворі на гостру лейкемію з перебудовами 11q23/MLL належать до категорії високого ризику з несприятливим або проміжним прогнозом, цитогенетичні методи дослідження мають бути обов’язково включені до стандартного дослідження хворих на гостру лейкемію з метою точної діагностики, прогнозування та вибору оптимальної стратегії лікування.

Ключові слова: гостра лейкемія, каріотип, цитогенетичні аномалії, перебудови 11q23/MLL, діагностика, прогноз.

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