Cytogenomic features of Richter transformation.

BACKGROUND: Richter transformation (RT) is the development of aggressive lymphoma in patients with chronic lymphocytic leukemia (CLL) or small lymphocytic lymphoma (SLL). This rare disease is characterised by dismal prognosis. In recent years, there has been a deeper understanding of RT molecular pathogenesis, and disruptions of apoptosis (TP53) and proliferation (CDKN2A, MYC, NOTCH1) has been described as typical aberrations in RT. RESULTS: A single-institution cohort of 33 RT patients were investigated by karyotyping, fluorescence in situ hybridization and single nucleotide polymorphism/copy number (CN) arrays. Most of RTs were typically manifested by diffuse large B-cell lymphoma, not otherwise specified, among the remaining cases one was classified as high-grade B-cell lymphoma with 11q aberrations. The most frequent alterations (40-60% of cases) were represented by MYC rearrangement/gain, deletions of TP53 and CDKN2A, IGH rearrangement and 13q14 deletion. Several other frequent lesions included losses of 14q24.1-q32.33, 7q31.33-q36.3, and gain of 5q35.2. Analysis of 13 CLL/SLL-RT pairs showed that RT arised from the CLL/SLL by acquiring of 10 ~ 12 cytogenetic or CN lesions/case, but without acquisition of loss of heterozygosity regions. Our result affirmed the higher genetic complexity in RT than CLL/SLL and confirmed the linear features of RT clonal evolution as predominant. CONCLUSIONS: Cytogenomic profile was concordant with the literature data, however the role of IGH rearrangement, 14q deletion and 5q35.2 gain need to be explored. We anticipate that further characterization of RT lesions will probably facilitate better understanding of the RT clonal evolution.

Predictive role of NKCD56bright cells in monitoring the progression of chronic lymphocytic leukemia during treatment.

INTRODUCTION: Standard treatment for chronic lymphocytic leukemia (CLL) has experienced a dramatic change over the last few years. Until recently, CLL was treated using chemoimmunotherapy (CIT) with anti-CD20 monoclonal antibodies. Even though novel agents such as BTKi (Bruton Tyrosine Kinase inhibitor) and BCL2 inhibitors are the standard of care in most therapeutic settings, CIT still has its place in CLL treatment. Interestingly, little is known about its effects on the immune system of patients with CLL. Contrary to the reduction of the number of CLL cells during CIT administration, little attention has been paid to the cellular microenvironment, the evaluation of which during treatment may provide additional information about the course of the disease and prognosis and therefore was set as the aim of this study. MATERIAL AND METHODS: Flow cytometry was used to evaluate the phenotypes of different populations and subpopulations of lymphocytes in the peripheral blood (PB) of 20 patients with CLL before, during, and after CIT. RESULTS: During the CIT with R-FC (Rituximab, Fludarabine, and Cyclophosphamide) and R-B (Rituximab, Bendamustine) regimens, the sizes of the assessed populations and subpopulations of lymphocytes were dramatically reduced. Twenty-eight days after the first course of treatment, the exponential decrease of CLL cells was observed, and their number had declined to the median level of 10% of the numbers observed before the treatment. T cells, NK cells, NKCD56dim, NKT-like, and NKT-like CD56dim also decreased exponentially. After the second treatment course, a decline in the numbers of T, NK, NKCD56dim, NKT-like, and NKT-like CD56dim cells was observed, which were stable until the sixth treatment course. However, the number of NKT-like CD56bright cells decreased to the third course of treatment and then increased. The number of CLL cells in peripheral blood correlated with the number of NKCD56bright cells, influencing the treatment response. CONCLUSIONS: Upon CIT, the reduction of CLL cells is accompanied by shifts in immune cell populations, T, NK, and NKT-like cells. Monitoring changes of those cell populations in the peripheral blood may serve as an important predictive and prognostic indicator.

Cryptic MYC insertions in Burkitt lymphoma: New data and a review of the literature.

The occurrence of MYC-negative Burkitt lymphoma (BL) has been discussed for many years. The real frequency of the MYC insertion in MYC-negative BL is still unknown. Fine-needle aspiration biopsies of 108 consecutive patients with clinicopathologically suspected BL (suspBL) were evaluated by flow cytometry, classical cytogenetics, and fluorescence in situ hybridization (FISH). We found 12 cases (11%) without the MYC rearrangement by FISH with a MYC breakapart probe: two patients (1.9%) with cryptic MYC/IGH fusion (finally diagnosed as BL) and 10 patients (9.3%) with 11q gain/loss (finally diagnosed as Burkitt-like lymphoma with 11q aberration). The exact breakpoints of the cryptic MYC/IGH were investigated by next-generation sequencing. The MYC insertions' breakpoints were identified in PVT1 in the first case, and 42 kb upstream of 5'MYC in the second case. To date, a molecular characterization of the MYC insertion in BL has only been reported in one case. Detailed descriptions of our MYC insertions in a routinely and consecutively diagnosed suspBL cohort will contribute to resolving the issue of MYC negativity in BL. In our opinion, the presence of the MYC insertions in BL and other lymphomas might be underestimated, because routine genetic diagnostics are usually based on FISH only, without karyotyping.

Genetic progression of post-transplant Burkitt-like lymphoma case with 11q-Gain/Loss and MYC amplification.

"Burkitt-like lymphoma with 11q aberration" is a new provisional entity in the latest revision of lymphoma's World Health Organization classification described as carrying the specific 11q-gain/loss aberration and lacking MYC rearrangement. Morphologically, phenotypically and by gene and microRNA expression profiling these lymphomas resemble Burkitt lymphoma. The 11q-gain/loss was also found in post-transplant patients with molecular Burkitt lymphoma signature without MYC rearrangement. Recent reports describe aggressive lymphomas with coexistence of 11q-gain/loss and MYC rearrangement. In this report we describe post-transplant Burkitt-like lymphoma with 11q aberration and MYC amplification. Genetic studies were conducted at two time points: before therapy and during progression. In both cytogenetic examinations, peculiar 11q-gain/loss was detected. Percentage of cells carrying MYC amplification increased from 2% at initial diagnosis to 97% during progression. The MYC amplification can functionally correspond to MYC translocation and to MYC overexpression. The presence of MYC amplification seems to increase the aggressiveness of the reported disease course, so even a small clone with this change should be indicated in cytogenetic result.

A comprehensive flow-cytometry-based immunophenotypic characterization of Burkitt-like lymphoma with 11q aberration.

We previously described a subset of MYC translocation-negative aggressive B-cell lymphomas resembling Burkitt lymphoma, characterized by proximal gains and distal losses in chromosome 11. In the 2016 WHO classification, these MYC-negative lymphomas were recognized as a new provisional entity, 'Burkitt-like lymphoma with 11q aberration'. Here we present an immunophenotype analysis of Burkitt-like lymphomas with 11q aberration. Cells were acquired by fine needle aspiration biopsy from 10 young adult patients, 80% of whom presented recurrence-free 5-year survival. Twenty-three MYC-positive Burkitt lymphomas, including three carrying both MYC rearrangement and 11q aberration, served as controls. By immunohistochemistry, all Burkitt-like lymphomas with 11q aberration were CD20+/CD10+/BCL6+/BCL2-/MUM1-/MYC+/EBV-, usually LMO2+/CD44-/CD43- and sometimes CD56+, and showed high proliferation rate. By flow cytometry, Burkitt-like lymphoma with 11q aberration immunophenotypically resembled MYC-positive Burkitt lymphoma, except for significantly (adjusted P<0.001) more frequent CD38higher expression in Burkitt lymphoma (91% MYC-positive Burkitt lymphoma vs 10% Burkitt-like lymphoma with 11q aberration), more frequently diminished CD45 expression in Burkitt lymphoma (74% vs 10%), an exclusive CD16/CD56 and highly restricted CD8 expression in Burkitt-like lymphoma with 11q aberration (60% vs 0% and 40% vs 4%, respectively). We showed high diagnostic accuracy and effectiveness of flow cytometry in Burkitt lymphoma. CD16/CD56 expression without CD38higher and the lack of CD16/CD56 with CD38higher expression proves to be a reliable, fast, and cost-effective method for diagnosing 11q aberration and MYC rearrangements in CD10(+) aggressive lymphomas, respectively. In addition, we confirmed a pattern of an inverted duplication with telomeric loss of 11q, as a recurrent 11q abnormality, but one case presented alternative changes, possibly resulting in an equivalent molecular effect. Our findings reveal similarities along with subtle but essential differences in the immunophenotype of Burkitt-like lymphoma with 11q aberration and MYC-positive Burkitt lymphoma, important for the differential diagnosis, but also for understanding the pathogenesis of Burkitt-like lymphoma with 11q aberration.

The 11q-Gain/Loss Aberration Occurs Recurrently in MYC-Negative Burkitt-like Lymphoma With 11q Aberration, as Well as MYC-Positive Burkitt Lymphoma and MYC-Positive High-Grade B-Cell Lymphoma, NOS.

OBJECTIVES: The latest revision of lymphoma's World Health Organization classification describes the new provisional entity "Burkitt-like lymphoma with 11q aberration" (BLL, 11q) as lacking MYC rearrangement, but harboring the specific11q-gain/loss aberration. We report genetic characteristics of 11 lymphoma cases with this aberration. METHODS: Classical cytogenetics, fluorescence in situ hybridization (FISH), and single nucleotide polymorphism/array comparative genomic hybridization. RESULTS: The 11q aberrations were described as duplication, inversion, and deletion. Array comparative genomic hybridization showed two types of duplication: bigger than 50 megabase pairs (Mbp) and smaller than 20 Mbp, which were associated with bulky tumor larger than 20 cm and amplification of the 11q23.3 region, including KMT2A. Six cases revealed a normal FISH status of MYC and were diagnosed as BLL,11q. Five cases showed MYC rearrangement and were diagnosed as Burkitt lymphoma (BL) or high-grade B-cell lymphoma, not otherwise specified (HGBL, NOS). CONCLUSIONS: The 11q-gain/loss is not specific for BLL, 11q, but occurs recurrently in MYC-positive BL and MYC-positive HGBL.

Chromosome 1 amplification has similar prognostic value to del(17p13) and t(4;14)(p16;q32) in multiple myeloma patients: analysis of real-life data from the Polish Myeloma Study Group.

The study aimed to assess prognostic significance of del(13q14), del(17p13), t(4;14)(p16;q32), and amp(1q21) in newly diagnosed myeloma patients treated mostly with thalidomide-based therapies. All genetic abnormalities except del(13q14) were independent prognostic factors associated with shortened progression-free survival (PFS) and overall survival (OS). Patients with no abnormalities, one abnormality, and ≥2 abnormalities had a median PFS of 41.8, 17.0, and 10.0 months, respectively; a median OS was not reached, 48.0 and 23.3 months, respectively. According to the presence of amp(1q21), t(4;14)(p16;q32), and del(17p13) and the International Staging System (ISS), we stratified patients into low-risk, intermediate-risk and high-risk groups. A median PFS was 52.9, 25.6, and 10.0 months, respectively; a median OS was not reached, 64.0 and 25.0 months, respectively. In conclusion, our study confirmed the prognostic value of cytogenetic changes and showed that prognostic models based on ISS and cytogenetic studies should include not only del(17p13) and t(4;14)(p16;q32), but also amp(1q21).

A novel IGH@ gene rearrangement associated with CDKN2A/B deletion in young adult B-cell acute lymphoblastic leukemia.

Acquired copy number changes are common in acute leukemia. They are reported as recurrent amplifications or deletions (del), and may be indicative of involvement of oncogenes or tumor suppressor genes in acquired disease, as well as serving as potential biomarkers for prognosis or as targets for molecular therapy. The present study reported a gain of copy number of 14q13 to 14q32, leading to immunoglobulin heavy chain locus splitting in a young adult female. To the best of our knowledge, this rearrangement has not been previously reported in B-cell acute lymphoblastic leukemia (ALL). Low resolution banding cytogenetics performed at the time of diagnosis revealed a normal karyotype. However, retrospective application of fluorescence in situ hybridization (FISH) banding and locus-specific FISH probes, as well as multiplex ligation-dependent probe amplification and high resolution array-comparative genomic hybridization, revealed previously hidden aberrations. Overall, a karyotype of 46, XX, del(9) (p21.3 p21.3),derivative(14) (pter-> q32.33:: q32.33-> q13 ::q32.33-> qter) was determined. The patient was treated according to the Polish Adult Leukemia Group protocol and achieved complete remission. The results of the present study indicate that a favorable prognosis is associated with these aberrations when the aforementioned treatment is administered.

Monoallelic and biallelic deletions of 13q14 in a group of CLL/SLL patients investigated by CGH Haematological Cancer and SNP array (8x60K).

BACKGROUND: Deletion of 13q14 is the most common cytogenetic change in chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) and is detected in about 50 % of patients by fluorescence in situ hybridization (FISH), which can reveal presence of del(13)(q14) and mono- or biallelic deletion status without information about the size of the lost region. Array-comparative genomic hybridization (aCGH) and single nucleotide polymorphism (SNP) can detect submicroscopic copy number changes, loss of heterozygosity (LOH) and uniparental disomy (UPD) regions. The purpose of this study was detection of the size of del(13)(q14) deletion in our group of patients, comparing the size of the monoallelic and biallelic deletions, detection of LOH and UPD regions. RESULTS: We have investigated 40 CLL/SLL patients by karyotype, FISH and CGH and SNP array. Mutational status was of immunoglobulin heavy-chain variable-region (IGVH) was also examined. The size of deletion ranged from 348,12 Kb to 38.97 Mb. Detected minimal deleted region comprised genes: TRIM13, miR-3613, KCNRG, DLEU2, miR-16-1, miR-15a, DLEU1. The RB1 deletions were detected in 41 % of cases. The average size in monoallelic 13q14 deletion group was 7,2 Mb while in biallelic group was 4,8 Mb. In two cases 13q14 deletions were located in the bigger UPD regions. CONCLUSIONS: Our results indicate that bigger deletion including RB1 or presence of biallelic 13q14 deletion is not sufficient to be considered as adverse prognostic factor in CLL/SLL. CytoSure Haematological Cancer and SNP array (8x60k) can precisely detect recurrent copy number changes with known prognostic significance in CLL/SLL as well as other chromosomal imbalances. The big advantage of this array is simultaneous detection of LOH and UPD regions during the same test.

Long-term survival of a child with refractory anaplastic large cell lymphoma following therapy with an antisense oligonucleotide, topotecan, and vinblastine.

Anaplastic large cell lymphoma includes a subset of highly aggressive tumours and has a relapse rate of 30% at 2 years. Relapsed patients often have poor clinical outcome. The use of antisense oligonucleotides to down-regulate Bcl-2 protein can reverse chemotherapy resistance. The authors describe an 11-year-old boy with recurrent anaplastic large cell lymphoma who had received double high-dose chemotherapy followed by autologous haematopoietic stem-cell transplantation, had refractory disease and then had achieved long-term remission with the use of an antisense oligonucleotides in combination with vinblastine and topotecan.

Cytogenetic and flow cytometry evaluation of Richter syndrome reveals MYC, CDKN2A, IGH alterations with loss of CD52, CD62L and increase of CD71 antigen expression as the most frequent recurrent abnormalities.

OBJECTIVES: Richter syndrome (RS) is a transformation of chronic lymphocytic leukemia (CLL) or small lymphocytic lymphoma (SLL) into high-grade lymphoma. There are only limited data on flow cytometry (FCM) and cytogenetics in RS. METHODS: In this study, FCM, classic cytogenetics (CC), and fluorescence in situ hybridization (FISH) were performed in eight RS cases. RESULTS: Most cases of RS were characterized by a loss/decrease of CD52 and CD62L and increased CD71 expression. CC identified complex karyotypes, with losses of 9/9p and 17/17p as the most frequent in four of seven cases. Seven RS cases demonstrated MYC abnormalities. Disruptions of CDKN2A and IGH were identified in five of seven and four of seven RS cases, respectively. CONCLUSIONS: Newly diagnosed RS is an oncologic emergency, and a quick diagnostic decision is crucial in clinical practice. Therefore, in patients with CLL/SLL and rapidly enlarging asymmetric lymphadenopathy and/or extranodal tumors, we strongly advise FCM of fine-needle aspiration biopsy (FNAB) material, including CD62L, CD52, and CD71 analysis as well as assessment of karyotype and at least MYC abnormalities by FISH of the same FNAB material. Loss of CD52 expression in RS most likely predicts resistance to alemtuzumab therapy, which is frequently used in CLL.

Acute panmyelosis with myelofibrosis with EVI1 amplification.

EVI1 is located on chromosome 3q26 and is up-regulated mostly through an inv(3)(q21q26) or t(3;3)(q21;q26). Chromosomal aberrations involving 3q26 comprise 1-2% of all acute myeloid leukemia (AML). These changes result in overexpression of the EVI1 oncogene. EVI1 transcriptional activation has been reported in up to 10% of AML patients, even in the absence of 3q26 changes, and is an independent indicator of adverse prognosis. Rearrangements of the EVI1 locus are often associated with monosomy 7. We present a case of acute panmyelosis with myelofibrosis with a unique EVI1 amplification within a derivative 8 chromosome, characterized by karyotyping and fluorescence in situ hybridization, conventional high resolution comparative genomic hybridization, as well as by gene expression studies. We conclude that EVI1 overexpression as a consequence of EVI1 gene amplification causes similar biological effects to the changes caused by the typical 3q26 aberrations such as an inv(3)(q21q26) or t(3;3)(q21;q26) with EVI1 gene rearrangements.

Comparison of cytogenetic changes between primary and relapsed patients with borderline tumors of the ovary.

The aim of this work was to compare cytogenetic changes in primary and relapsed borderline tumors of the ovary. We analyzed 11 tumors (6 primary and 5 relapsed) by conventional GTG banding analysis and fluorescence in situ hybridization. The tumors studied were clinical stages I and III. Genomic imbalances were detected in both investigated groups. In the primary tumors group, only simple chromosome changes were detected. There were gains of chromosome 12, 7, and 8. The presence of additional copies of chromosomes 12 and 7 was independent of histologic subtype, whereas trisomy 8 appeared only in serous tumors. In the group of relapsed borderline tumors, besides trisomies 7 and 12, the structural aberrations of chromosomes 1, 6q, 7q, and 10q were revealed. Gains of tested oncogenes (CCND1 and MYC) have been demonstrated in both groups of investigated tumors. Gains of CCNC1 and MYC genes could be of prognostic value in borderline tumors, but this assumption requires further research.

Karyotype changes during long-term targeted therapy of chronic myeloid leukemia with imatinib.

The main risk factors during imatinib therapy of chronic myeloid leukemia are still subject to discussion. A group of 39 patients was cytogenetically examined and monitored before and during long-term treatment with imatinib. The cytogenetic response was investigated using karyotype analysis and fluorescence in situ hybridisation method. Different therapy effects were shown for three subgroups distinguished before the start of treatment: patients with the sole translocation t(9;22) with a typical pattern of BCR/ABL fusion vs. patients with submicroscopic deletion in the fusion region ABL/BCR of the sole t(9;22) vs. patients with aberrations additional to t(9;22) and without submicroscopic deletion. Of the two group with sole t(9;22) the group with deletion in the ABL/BCL region suffered a poorer treatment outcome than the group without deletion. The risk of progression of cytogenetic changes in group with deletion was more than nine times higher than in patients with sole t(9;22) without deletion (statistically significant).

Spontaneously immortalized T lymphocytes from Nijmegen Breakage Syndrome patients display phenotypes typical for lymphoma cells.

We found that the peripheral T lymphocytes from four of eight patients with the lymphoma predisposing Nijmegen Breakage Syndrome (NBS) acquired an unlimited growth potential following in vitro mitogen stimulation and subsequent interleukin-2-dependent propagation. The immortal T cell lines revealed morphological and other features typical for anaplastic large cell lymphoma (ALCL). In addition, multiple copies of ALK, but with no ALK gene rearrangements were found in a subpopulation of cells of one of the immortalized lines. These cell lines may be useful for the in vitro elucidation of mechanisms involved in the development of ALCL.

Frequent aberrations of chromosome 8 in aggressive B-cell non-Hodgkin lymphoma.

Translocations involving chromosome 8 are the most common aberrations in B-cell non-Hodgkin lymphoma (B-NHL). The presence of the typical t(8;14)(q24;q32) or its variants has been confirmed in all cases of Burkitt lymphoma (BL), in some cases of Burkitt-like lymphoma (BLL), and in diffuse large B-cell lymphoma (DLBCL). The alterations lead to deregulated expression of c-myc protein by a chromosomal translocation joining C-MYC gene with sequences from immunoglobulin (Ig) enhancers. The C-MYC gene rearrangement plays an essential role in leukemogenesis of BL and probably plays a part in other aggressive NHLs. The present study was undertaken to compare the cytogenetic features in cases of BL, BLL, and DLBCL. We detected chromosomal aberrations by G-banding and fluorescence in situ hybridization (FISH) painting in 10 cases of aggressive B-NHL and used FISH to visualize the C-MYC gene rearrangement. Chromosome 8 was most frequently involved in structural aberrations (8/10 cases), and 4 cases showed the typical t(8;14)(q24;q32). Only two of 5 patients suspected of having BL fulfilled all the criteria for this diagnosis; in the others, chromosome 8 was aberrant, but the absence of C-MYC rearrangement or the results of flow cytometry excluded the diagnosis of BL. All BLL cases showed C-MYC overexpression, but only one had a rearrangement of the C-MYC gene; the remaining cases showed other aberrations of chromosome 8. This study indicates that the mechanisms of C-MYC activation involved in BLL can be different from that for the BL.

Spontaneously immortalized human T lymphocytes develop gain of chromosomal region 2p13-24 as an early and common genetic event.

To gain further insight into the molecular events responsible for the extended life span and immortalization of human lymphoid cells, we analyzed a series of spontaneously immortalized, IL2-dependent human T-cell lines using molecular cytogenetic techniques. Two of the cell lines were derived from normal spleen and three from patients with Nijmegen breakage syndrome (NBS), a recessive disorder characterized by a high incidence of lymphoid malignancies. Here we show that spontaneous immortalization of the five T-cell lines was associated with the acquisition of copy number gains involving chromosomal region 2p13-24 as common early alterations. In addition, we found an amplification of 8q21-24 after prolonged propagations in all three NBS-derived cell lines as well as early development of near-tetraploidy in two of these lines. Gains involving the short arm of chromosome 2 recently were found in several lymphoid malignancies. Therefore, the cell lines described here can be used for identification and characterization of genes involved in the pathogenesis of lymphoid neoplasms and would also provide a useful tool for better understanding the mechanisms responsible for cell immortalization.

Significance of chromosomal markers in the diagnosis of mantle cell lymphoma (MCL).

According to the REAL/WHO classification, the diagnosis of mantle cell lymphoma (MCL) should be based on clinical, histopathological, immunological and cytogenetic or molecular data. This study is based on 13 cases, which were initially diagnosed as MCL with the use of conventional cytogenetic method and fluorescent in situ hybridization (FISH). MCL is associated with a specific cytogenetic aberration t(11;14)(q13;q32). The chromosomal analyses confirmed the MCL diagnosis in four cases. A neartetraploid cell line and two copies of t(11;14) were observed in three cases. These results correspond with a blastoid variant of MCL, accompanied by aggressive course and poor prognosis. The presence of karyotype with t(11;14) as the sole anomaly predicts an intermediate clinical outcome. Six patients had normal karyotypes, which is characteristic for the typical form of MCL, associated with a better prognosis. In this study we show that detection of chromosomal abnormalities is useful in diagnosis of MCL and has some prognostic significance.