Cytogenetic Profile Of Acute Lymphoblastic Leukaemia in South India: A Series Of 1819 Patients From A Single Centre.

The management of patients with leukaemia: the role of cytogenetics in this molecular era.

Prognostic and biological importance of chromosome findings in acute lymphoblastic leukemia

To, The Editor, Leukemia is the most common childhood cancer worldwide [1]. Acute lymphoblastic leukemia (ALL) usually manifests with pallor, hepatosplenomegaly, lymphadenopathy, fever, bone pain, and bleeding [2]. Presence of frank osteolytic lesions with hypercalcemia is infrequent [3].We describes here a case of paediatric ALL who presented with multiple lytic lesions, hypercalcemia and absence of blasts in the peripheral blood film. A 10-year-old boy presents with generalised weakness, pain in right hypochondriac region which was non radiating, aggravated by coughing, associated with vomiting which was non bilious and non projectile for last 15 days. On examination, mild pallor was present with no icterus and lymphadenopathy. Liver was palpable 2 cm below the right costal margin. On investigations, complete hemogram showed Hb of 9.2g/ dl, WBC count of 8.0×109 /L with neutrophils 54%, lymphocyte 36%, monocyte 7%, eosinophil 2%, myelocyte 1% and platelet count 271 ×109 /L. Peripheral blood film showed mild anisocytosis with microcytes and hypochromia. His serum biochemistry studies were as follows: sodium: 134.8 mmol/L, potassium: 4.2 mmol/L, Crealinium: 0.93mg/dl, Calcium: 14.2 mg/dl, Phosphorous 6.4 mg/dl, uric acid: 7.6 mg/dl, LDH: 654 IU/L, Alkaline phosphatase: 110U/L, Albumin: 3.6g/dl. His ESR was 100 mm/h. Serum vitamin D3 level was 16.3 (15-30ng/ml). His serum parathyroid hormone (PTH) level was 7.2 (13.7-77.2 pg/ml). His abdominal ultrasound was normal except for hepatomegaly. The skeletal survey showed multiple lytic lesions in skull [Table/Fig-1] and ill defined translucent areas in pelvic bone. In view of anemia and multiple lytic lesions, bone marrow examination was done. Bone marrow examination revealed hypercellular marrow with 55% blasts which were positive for CD 10, CD 19, CD 22 and negative for myeloperoxidase confirming to diagnosis of precursor B acute lymphoblastic leukemia. Cytogenetic analysis of bone marrow showed 46XY karyotype. Molecular analysis for t (9; 22), t (12; 21), t (1; 19), and t (4; 11) using PCR method were negative. Patient was started on aggressive hydration, bisphosphonate and frusemide with monitoring of tumour lysis markers. For ALL, he was started with chemotherapy as per BFM 2002 protocol. His serum calcium levels decreased to 11.0 mg/dl after 48 hrs. Currently he is undergoing chemotherapy and his Day +15 marrow was hypocellular with 3% blasts (M1 marrow). Hypercalcemia is a rare finding in paediatric ALL ranging in frequency from 0.6- 4.8% [4]. Hypercalcemia of malignancy can occur due to two mechanisms. First is localized bone destruction by invasive cancer cells and second mechanism involves osteoclastic bone resorption after the release of humoral derived factors from tumour cells. Although hypercalcemia is assumed to be linked with high tumour bulk, a series of 22 patients showed no difference in event free survival in children with hypercalcemia at presentation. It has been reported that t (17;19), which is associated with poor prognosis, is frequently seen in patients with hypercalcemia due to humoral factors [5]. In our patient, hypercalcemia was due to direct bone invasion and cytogenetics was normal. The presence of multiple lytic lesions and hypercalcemia with no blasts in the peripheral blood are uncommon findings in ALL, which has prompted us to report this case. [Table/Fig-1]: X-ray Skull showing multiple lytic lesions

The role of cytogenetic analysis in the diagnosis and management of haematological malignancies is undisputed.The accuracy of cytogenetic diagnosis has improved steadily over the past 20 years, primarily due to a series of technical developments. However, despite improvements in high-resolution banding and culture methods to detect the chromosomally abnormal cells, many haematological malignancies are retractable to conventional cytogenetic analysis. This may be due to the presence of multiple abnormal clones, complex rearrangements, a low mitotic index, or poor chromosome morphology. Since the late 1980s a range of techniques based around fluorescence in situ hybridization (FISH) have greatly enhanced cytogenetic analysis. These use a variety of nucleic acid sequences as probes to cellular DNA targets and serve to bridge the gap between molecular genetic and conventional cytogenetic methods. Virtually any genomic DNA can now be used as a probe with which to investigate a wide variety of DNA targets, from metaphase chromosomes to mechanically stretched DNA fibres. The simultaneous detection of multiple target regions is also possible, using differentially labelled probes detected by different colours. In research, FISH has played a pivotal role in the identification of non-random translocations and deletions, pinpointing regions which contain genes involved in leukaemogenesis. Now, at the cutting edge, a new set of resources and technical innovations herald a new era for molecular cytogenetics, with colour karyotyping, comparative genomic hybridization (CGH) microarrays and mutation detection using padlock probes providing the promise of the future. The number of applications for FISH is almost unlimited (see Table I for some pertinent examples). This review will concentrate on the most recent developments in FISH which have had a considerable impact on the cytogenetic diagnosis and study of haematological malignancies, with some insight into the possible future roles for this flexible technology. The application of FISH to metaphase chromosomes provides unequivocal evidence of chromosome rearrangements. There are many different types of cloned or uncloned DNA which can be used for as a probe for FISH (reviewed in Buckle & Kearney, 1994). However, the most commonly used probes in cytogenetic analysis of haematological malignancy are: (i) repetitive sequence centromeric probes, (ii) whole chromosome paints, and (iii) locus-specific probes. Chromosome-specific centromeric probes which target tandemly repeated alpha (or beta) satellite sequences present in the heterochromatin of the chromosome centromeres are used to detect numerical chromosome abnormalities. Centromeric probes are commercially available for all human chromosomes and these provide a rapid and simple way of enumerating specific chromosome pairs, both in metaphase and interphase. This type of analysis is useful in many types of leukaemia where the chromosome morphology is poor and banding indistinct, such as in hyperdiploid acute lymphoblastic leukaemia (ALL). However, centromeric probes only give information on the number of centromeres of a particular type present; they cannot tell whether the chromosome is structurally abnormal. Whole chromosome painting probes are complex mixtures of sequences from the entire length of a specific chromosome. These are also available for all human chromosomes, and can be used to delineate chromosome pairs (Cremer et al, 1988; Pinkel et al, 1988). Whole chromosome painting probes (paints) can be derived from chromosome-specific libraries, PCR amplification of flow-sorted chromosome fractions, or microdissected DNA specific for each chromosome (Collins et al, 1991; Telenius et al, 1992; Vooijs et al, 1993; Guan et al, 1996). Chromosome paints are most useful for identifying the components of highly rearranged and marker chromosomes, where the banding pattern cannot be relied upon. However, their usefulness is limited to metaphase analysis, as the extended chromosome domains in interphase are often diffuse and difficult to quantitate. In addition, chromosome painting is a relatively insensitive technique and cannot detect small interstitial deletions, duplications or inversions. The resolution for the detection of small telomeric translocations is also limited. Single locus probes detect specific sequences present in only one copy. When using these probes the efficiency of hybridization needs to be considered; the larger the target sequence the more efficient the hybridization. Single-copy probes cloned in cosmid, YAC, P1, PAC and BAC vectors all give reliable FISH signals, with a fluorescent signal on both chromosome homologues in >90% of metaphases. Structural rearrangements detected using this type of probe include translocations, inversions and specific deletions (Dauwerse et al, 1990; Tkachuk et al, 1992; Sacchi et al, 1995; Jaju et al, 1998). The use of specific gene probes for chromosomal translocations has simplified the process of identifying known translocations, especially in complex or masked versions of the translocation (e.g. BCR/ABL, PML/RARα fusions), and has particular applications for interphase analysis. One of the greatest advances in cytogenetic analysis facilitated by FISH has been the ability to use non-dividing cells as DNA targets, referred to as interphase FISH (Cremer et al, 1986). This enables the screening of large numbers of cells and provides access to a variety of sources of haemopoietic cells including blood and bone marrow smears and haemopoietic progenitor cells from colony assays (Bentz et al, 1993; Poddighe et al, 1993; Mühlmann et al, 1998). This has considerable advantages for some haemopoietic malignancies, where the proliferative activity is low, or when the mitotic cells do not represent the neoplastic clone, for example chronic lymphoblastic leukaemia (CLL), Hodgkin's disease, multiple myeloma. Interphase FISH permits the identification of both numerical and structural chromosome abnormalities both as an aid to cyto-genetic diagnosis and for monitoring disease progression. Interphase FISH has had a major impact on the cytogenetic analysis of B-CLL, revealing a much higher incidence of trisomy 12 than found by conventional cytogenetic analysis (Anastasi et al, 1992; Garcia-Marco et al, 1997). An examination of the relationship between clinical stage and trisomy 12 showed an association with atypical morphology, advanced stage of disease and low proliferative activity. In addition, immunophenotyping and FISH showed that the +12 is present in only a proportion of clonal B cells (Garcia-Marco et al, 1997). All of this data suggests that trisomy 12 is a secondary event in the development of CLL. For chromosome deletions, specific locus or region-specific probes have been used to demonstrate a high frequency of mono-allelic deletions of the RB1 and p53 genes in B-cell malignancies (Stilgenbauer et al, 1993, 1995; Döhner et al, 1995; Cano et al, 1996). Interphase FISH was also instrumental in identifying the critical region of deletion on 11q13 associated with B-cell lymphoid malignancy, which consequently identified mutations of the ATM gene in T-prolymphocytic leukaemia (PLL) (Stilgenbauer et al, 1997). DNA probes for the fusion genes involved most specific chromosomal translocations and inversions in leukaemia are now commercially available. The differential labelling and detection of these probes in different colours enables a direct visualization of the fusion gene. The simplest scheme is to use two probes (one from each of the fusion genes), differentially labelled and detected with two different-coloured fluorochromes (see Fig 1A). An interphase cell positive for the translocation will exhibit a red–green fusion signal representing the translocation, and a single red and green signal corresponding to the normal chromosome homologues. However, the false positive rate using this approach is quite high (approximately 5%). In addition, the presence of variant translocations or translocations in which the breakpoints are spread over a large distance (e.g. Burkitt's lymphoma), means that the false negative rate can also be quite high. There are several more complex strategies to overcome this (see Figs 1B and 1C). Firstly, if a series of probes spanning both translocation breakpoints are used, this will result in splitting of both fluorescent signals, and the presence of two red–green fusions. Another, more complex, strategy is to employ three or even four different colours, so that the incidence of false positives and false negatives is reduced (Ried et al, 1993; Sinclair et al, 1997). However, the more complicated the colour scheme, the more difficult and complex the analysis. At present, this analysis is done manually, so this is a serious consideration. . Schematic representation of the detection, by FISH, of the Philadelphia translocation in interphase nuclei. In each case the left-hand panel shows the location of the FISH signals on metaphase chromosomes (partial karyotype), and the right-hand panel the interphase FISH signals. In (A) two probes from the flanking regions of the BCR and ABL genes are labelled and detected in different colours: BCR in red and ABL in green. The BCR/ABL fusion results in co-localization of the red and green signals on the der(22) (Philadelphia) chromosome, with a single red and green signal separated, corresponding to the normal chromosomes 22 and 9, respectively. A BCR/ABL negative cell would show two separate red and two green signals. The scheme in (B) uses two probes, this time spanning both the BCR and ABL breakpoint regions. In this case, two red/green fusion signals are formed: one corresponding to the der(9), and the second to the der(22). A positive cell would therefore exhibit one red, one green and two red/green fusions (from Dewald et al, 1998). In (C), a third probe from the region just proximal to ABL on 9q34 is used, labelled in a different colour (represented here in yellow). A translocation positive cell exhibits one green/yellow doublet, one red/green and a single red and yellow signal (from Sinclair et al, 1997). The possibility of using interphase FISH as screening test for specific abnormalities found in acute myeloid leukaemia (AML) subtypes was recently described by Fischer et al (1996). This study used 23 different probes and six to eight hybridizations per patient. They found that interphase FISH was more sensitive for the detection of t(8;21), inv(16), +8q, +11q, +21q, +22q and −Y, and obtained a cytogenetic result in a proportion of cases with no evaluable metaphases. However, this kind of analysis may eventually be replaced by disease-specific DNA chips (see Matrix-CGH below). The detection of residual Philadelphia-positive cells is important after allogeneic bone marrow transplant or interferon (IFN) treatment. In particular, the degree of response to IFN treatment has been shown to be an independent prognostic indicator. The sensitivity of conventional cytogenetics is around 5%, and may be difficult due to low mitotic rate of cells after treatment. RT-PCR is the most sensitive method for detection of BCR-ABL (approximately 10−6) but quantification is difficult. Interphase FISH offers the prospect of using peripheral blood samples, reducing the need for frequent bone marrow aspirates. However, 'in house' cut-off levels must be established for each probe set. Conventional FISH probes for the detection of BCR-ABL gene fusion in interphase cells have suffered from a high false positive rate (Tkachuk et al, 1992). The development of three-colour/three-probe FISH protocols for BCR-ABL detection has significantly lowered the false positive rate, and also increased the sensitivity of detection (Sinclair et al, 1997; Dewald et al, 1998). Sinclair et al (1997) used a third probe (for the ASS gene) 200 kb proximal to ABL, such that when a true BCR-ABL fusion was present, there was one co-localization for BCR-ABL, and a separate ASS signal corresponding to the der(9). In cells where the BCR and ABL signals co-localized due to chance, the ASS signal co-localized with the red ABL signal on both chromosomes 9 (see Fig 1C). This three-colour approach resulted in a low false positive and false negative rate. Dewald et al (1998) used a similar strategy, with probes spanning both the BCR and ABL breakpoints. This resulted in two different co-localizations: one representing the der(22) and the other the der(9) chromosomes (see Fig 1B). Strict scoring criteria, experienced operators and scoring of >3000 cells all enabled the detection of residual disease in 0.079% of cells. This skilled and time-consuming approach was also successful in detecting variant translocations. Although the sensitivity of dual-colour interphase FISH is less than for RT-PCR, PCR is not a possibility in a number of cases, for example for the detection of deletions, monosomy or trisomy. Interphase FISH has been used for the detection of residual disease after allogeneic bone marrow transplantation (Anastasi et al, 1991; Wessman et al, 1993). Kasprzyk & Secker-Walker (1997) studied hyperdiploid karyotypes in ALL to detect minimal residual disease. Using three-colour interphase FISH, targeting three chromosomes simultaneously, they were able to achieve a sensitivity of 10−4, and predict relapse in a number of cases. The ability to combine interphase FISH analysis with immunological staining for cell surface antigens provides a powerful method to combine cell by cell analysis with morphology or immunophenotype. Simultaneous immunophenotyping and FISH analysis has been used to investigate lineage involvement in myelodysplastic syndrome (MDS), chronic myeloid leukaemia (CML) and other myeloproliferative syndromes (Price et al, 1992; Nylund et al, 1993; Torlakovic et al, 1994; Soenen et al, 1995; Haferlach et al, 1997, reviewed in Knuutila, 1997). Concurrent immunophenotype and FISH analysis has also been used to demonstrate that the leukaemia which emerged 5 years after sex-mismatched allogeneic bone marrow transplant occurred in donor cells (Katz et al, 1993). In CML, three-colour detection of the Philadelphia translocation and immunophenotype enabled the identification of the translocation in CD20-positive B cells (Torlakovic et al, 1994) and more recently CD3-positive T cells and CD34-positive precursor cells (Haferlach et al, 1997). This supports the belief that CML is a disorder of an early progenitor cell, capable of differentiating into myeloid and some lymphoid lineages (reviewed in Knuutila, 1997). There are also reports of the clonal involvement of B cells in MDS, using del(20q) and monosomy 7 as clonal markers (White et al, 1994; van Lom et al, 1995). In Hodgkin's disease the low percentage of Hodgkin and Reed-Sternberg (HRS) cells means that even interphase FISH may not detect clonal abnormalities. In a recent study the combination of CD30+ staining and FISH with pairs of centromeric probes revealed numerical abnormalities in 100% of HRS cells (Weber-Matthiesen et al, 1995). Surprisingly, clonal abnormalities found in metaphase analysis were not consistent with the interphase FISH analysis, indicating that metaphase analysis of Hodgkin's disease may not be informative. FISH has proved an invaluable aid in the mapping of translocation breakpoints, resulting in the identification of many fusion genes (reviewed in Rabbitts, 1994). A recent addition to the repertoire of FISH techniques now provides significant advantages over other molecular methods for mapping breakpoints which are dispersed over large distances. The term Fibre-FISH is used to describe a collection of methods for performing FISH to extended DNA stretched out on a glass slide (Wiegant et al, 1992; Parra & Windle, 1993; Bensimon et al, 1994; reviewed in Raap, 1998). vandraager et al (1996) have demonstrated the usefulness of this technique for mapping breakpoints of the cyclin D1 gene in mantle cell lymphomas. Using a series of overlapping probes from the 11q13 breakpoint region labelled in alternating red and green fluorochromes creates a colour bar code for the region. Translocations are recognized by the disruption of this bar code into its two complementary parts. The advantages of this method over Southern blotting or pulsed field gel electrophoresis are its simplicity and speed: only a few images need to be examined, and chromosomal breaks over a distance of 250 kb can be visualized. However, the parameters underlying the technique are poorly understood, and at present it remains a research rather than diagnostic tool, confined to a few specialist laboratories. The strength of conventional (G-banded) cytogenetic analysis has always been the ability to survey the entire genome for clues to pathogenesis. However, the poor chromosome morphology and low mitotic index of many leukaemias and lymphomas means that conventional cytogenetic analysis is often limited. In addition, the analysis of banding pattern in highly rearranged karyotypes is difficult and unreliable. One of the remaining challenges for the new FISH techniques is to identify cryptic rearrangements, particularly involving telomeric regions, in apparently normal karyotypes. A significant proportion (15–20%) of bone marrow karyotypes in leukaemia are reported as normal by conventional (G-banded) cytogenetic analysis. Despite significant improvements in the quality of leukaemic metaphase preparations over the past decade, the abnormality rate has not improved. The t(12;21)(p13;q22) remained undetected until 1994, despite the fact that it accounts for 25% of childhood B-cell ALL cases (Romana et al, 1994). This translocation still remains undetectable by conventional cytogenetic analysis. The difficulty in detecting chromosome abnormalities such as this in the fact that there is a of staining regions of a similar The recent development of whole chromosome painting provides the promise of identifying cryptic chromosome rearrangements, a of all chromosome abnormalities in a single FISH using the method of probe labelling was described by et al In this probes are labelled with mixtures of fluorochromes such that no two probes have the The number of targets which can be in this is where number of fluorochromes available. FISH with to different colours has been available for a number of years, using probes labelled with three fluorochromes (Dauwerse et al, 1992; et al, 1992). the number of fluorochromes to enables the identification of all pairs of human The has been due in to the of new fluorochromes in the and and to two detection methods to mixtures of fluorochromes et al, et al, 1996). of these used a set of whole chromosome paints, labelled with different mixtures of The detection FISH relied on separate images for each of using et al, 1996). The labelling combination for each chromosome was and in using The second used a single of the and a combination of and et al, 1996). An was used to the at each of the of these techniques have demonstrated chromosome rearrangements in complex karyotypes in cell and in haematological malignancies et al, et al, 1997; also Fig However, the sensitivity of both or remains to be The of this are the on metaphase analysis, and the resolution of painting probes. All of the available whole chromosome paints are in some of the particularly the telomeric regions. that the sensitivity of painting for the detection of translocations involving regions may be as low as also Fig In addition, whole chromosome painting will not detect deletions, duplications or inversions. In both and still to the and a combination of FISH is still to identify all abnormalities in complex karyotypes. . FISH to the analysis of a complex in the myeloid cell (A) after analysis. The structural abnormalities identified are: A cryptic was also present, but difficult to identify by analysis. (B) A metaphase after analysis. of amplification are in green and deletions in of the genome which are regions were identified the entire chromosome of and A deletion of due to the of an The analysis identified the of a marker chromosome, as as revealing several cryptic translocations in The of the analysis was to the of the with large deletions translocations in most cases. et al (1997) have recently described an approach which use of the regions of between different to approach a of colour of The of a colour for each chromosome was described by et al using a series of from the length of the chromosome, labelled differentially and detected in a different banding on the of between and has been useful in comparative of regions (reviewed in et al, 1997). The by and have now a set of paints derived from cell Chromosome-specific painting probes were derived from and by chromosome and When used for FISH to human metaphase chromosomes, this resulted in the of each chromosome into between two and six labelling using three fluorochromes resulted in a colour banding pattern for each chromosome. In with specific an colour can be Although at present the number of colour is the of this approach is with the to identify chromosomal inversions and colour banding has been used to identify cryptic translocations in CML A set of chromosome-specific probes which identify the of all human chromosomes the of the is now available for FISH of and of 1996). These contain DNA sequences cloned in cosmid, and PAC clones, the of which have been to between and kb from human chromosome These probes have been in a FISH for rearrangements on a series of with cryptic chromosome rearrangements et al, 1997). This is dual-colour an of all chromosome regions on a single However, the approach a high mitotic index and is most for the analysis of which on peripheral blood or where a cell is available. In the of these probes for leukaemic karyotypes has been to identify the specific region in rearrangements found by painting (see Fig An a FISH would the of all chromosome regions in a single However, the of labelling and multiple colour detection methods for cosmid, or even and PAC a series of developments. Firstly, the simultaneous analysis of all chromosome in a different colour the number of targets with fluorochromes is the of such an would on it is not whether the targets of such small probes labelled with several different fluorochromes can be due to of resolution of the is that the development of fluorochromes will this type of analysis . The use of chromosome-specific probes to identify the of chromosome on two In each case dual-colour hybridization was out with the probe labelled in and detected in red fluorescent and the probe labelled with and detected with fluorescent In (A) probes for and identified the on the as derived from In (B) the on was identified as from Although these abnormalities were detected by painting no information of the chromosomal region is also important to that the abnormality in (A) was described by as The of all of the new is that they still metaphase The advantages of are that it the need for cells and not any of the chromosome The is genomic and DNA are labelled with different in and normal metaphase The in number between the normal and is by in red and green fluorescence the length of the chromosome (see Fig the 5 years its et al, has a of identifying new regions of amplification and deletion in a wide variety of types (reviewed in et al, 1997). The use of for haematological malignancies is more limited (Bentz et al, et al, et al, et al, 1997). The of for haematological malignancies are the to detect rearrangements, and the for cells with the clonal However, and some lymphomas have from the application of (Bentz et al, et al, et al, 1996). One study of identified and not identified or not detected by clonal were identified in six out of cases with a normal (Bentz et al, The for results between and were a complex the or a of metaphases. This study that banding analysis may abnormalities and may important chromosome have not been identified in CLL. In a study of myeloid leukaemias found a between and results (Bentz et al, The only were a of to detect and The major of is its due to the on metaphase For deletions, the resolution of has been at (Bentz et al, 1998). the most future for in to cloned DNA (see below). This to overcome the of using metaphase chromosomes as a target for by metaphase chromosomes with cloned DNA in small and to the surface of a glass et al (1997) used for the detection of high number amplification using as For low number larger cloned probes or were For deletions, a resolution of it not between and The other of metaphase also at of clonal cells and will not detect translocations. One types of (i) disease-specific probe (ii) or (iii) DNA for specific regions, at over the whole probes are DNA in which the has been replaced by The rapid and of with complementary DNA sequences for a number of including FISH probes have been for the human telomeric the fluorescent detection of all in a single These signals to be a for fluorescence than conventional FISH signals, an of length et al, 1996). This may also be extended to other sequences such as centromeric may also be to combine telomeric probes with the chromosome-specific DNA probes to provide a of and specific chromosome This new the promise of detecting single in cells. probes of two different each 20 by a When the probe sequence the the and of the probe are and the probe is et al (1997) used two different probes, each labelled with a different to detect single in a centromeric The sensitivity of this may be improved by the of new sensitive labelling techniques such as the use of fluorescent signal et al, 1995; et al, 1995). to the sensitivity is amplification of the This to the would fluorescent detection of mutations in nuclei. amplification has been with some using extended DNA from but at this stage not or on et al, 1998). In the relatively time its FISH has had a major impact on cytogenetic analysis, due to the sensitivity and of its Although some of the applications will research the and probes for most cytogenetic abnormalities are now the of most clinical laboratories. However, conventional FISH can only provide to the specific and some of the The recent of FISH to the visualization of the entire human genome in different colours has the of and The of this approach is the ability to the whole genome in a single hybridization the screening of cytogenetics with the accuracy of molecular The belief that cytogenetics is more an than a has been from the the aid of new colour techniques and cytogenetic analysis now a molecular of The major impact of this development in field of haematological is to be the identification of new and non-random chromosome rearrangements and clinical of the most recent innovations to The most of fluorescent metaphase is now by and and will not only the of such but the sensitivity of interphase FISH analysis. many of the of cytogenetic analysis by the future for cytogenetics has The all of the of the particularly for the and analysis of the cell of the described here was by the and the

There have been published reports on cytogenetic, immunophenotypic, and molecular changes at relapse in childhood acute lymphoblastic leukemia (ALL) including lineage switch and secondary leukemia. There are limited data, however, on the cytogenetic, immunophenotypic, and molecular parameters of adult ALL at relapse. Because, as in children, the cytogenetic and/or immunophenotypic changes observed in adult ALL at relapse may have prognostic significance, the authors investigated the significance of such changes. Fifty-three patients with relapsed adult ALL for whom cytogenetic, immunophenotypic, and/or molecular analyses were performed at diagnosis and at relapse were studied. Changes in any of the parameters at relapse were correlated with total survival and survival from the time of relapse. Of the 32 patients for whom cytogenetic studies were performed at relapse, 21 (66%) showed clonal cytogenetic changes, 40% of which were clonal evolution. None of these cases, however, showed two entirely different abnormal karyotypes at diagnosis and at relapse. The immunophenotypes showed occasional gain or loss of one or two surface markers, and the molecular genetic configurations for JH, JK, and the T-cell receptor beta were stable throughout the evolution of the disease. Patients with clonal evolution had a shorter overall survival than the rest of the group (P = 0.02). This difference, however, was not significant with respect to survival measured from the time of relapse. The most frequent changes in the biologic profile of adult ALL at relapse are shifts in the karyotype, with or without clonal evolution. Clonal evolution detected at relapse is associated with a higher frequency of unfavorable karyotypes at diagnosis and with a worse overall prognosis. However, survival from the time of relapse is similar in patients with and without clonal evolution.

Classical and Molecular Cytogenetic Abnormalities in 124 Pediatric Patients with Acute Lymphocyte Leukemia.

Acute Lymphoblastic Leukemia

The Role of Cytotoxic Therapy with Hematopoietic Stem Cell Transplantation in the Treatment of Adult Acute Lymphoblastic Leukemia: Update of the 2006 Evidence-Based Review

Plerixafor (AMD3100) induces prolonged mobilization of acute lymphoblastic leukemia cells and increases the proportion of cycling cells in the blood in mice

Epigenetic Regulation of Human γ-Glutamyl Hydrolase Activity in Acute Lymphoblastic Leukemia Cells

Chromosomal translocations involving chromosome 12p are rarely detected by conventional cytogenetic analysis in patients with acute lymphoblastic leukemia (ALL). Recently, fluorescence in situ hybridization (FISH) and molecular methods revealed that the t(12;21)(p13;q22) translocation is the most common genetic aberration in pediatric ALL. We established a reverse transcriptase-polymerase chain reaction (RT-PCR) protocol for the detection of TEL/AML1 fusion mRNA resulting from this translo cation. We analyzed bone marrow and/or peripheral blood from 54 pediatric patients with B-lineage ALL at diagnosis or relapse (pre-pre-B, n=3; common ALL, n=32; pre-B, n=18; B, n=1). Cytogenetic analyses did not reveal the t(12;21)(p13;g22) translocation in any of the patients. Patients with T-cell immunophenotype and those with RT-PCR proven hybrid genes BCR/ABL and MLL/AF4 were excluded. For the detection of TEL/AML1 fusion transcript, we performed two-round RT-PCR using primers in exon 5 of the TEL gene and exon 3 of the AML1 gene. Two different PCR products were identifiable — either a 464 or 425-bp product in the first round, thus implying the presence or absence of the 39-bp-long exon 2 of the AML1 gene. The one step amplification of ABL exon a2/a3 was used as a quality control. Out of 32 patients with common ALL, 12 were positive for TEL/AML1 rearrangement (34.4%). In addition, we identifled this genetic lesion in 4 out of 18 (22.2%) pre-B ALL patients. All pre-pre-B and B-cell leukemic samples were negative. We conclude that TEL/AML1 fusion can be demonstrated in one fourth of the children with a B lineage ALL and is associated with CD10+ immunophenotype. The high frequency of the t(12;21) provides a new tool for the study of minimal residual disease.

Oligo-based aCGH analysis reveals cryptic unbalanced der(6)t(X;6) in pediatric t(12;21)-positive acute lymphoblastic leukemia

Multicolor flow cytometry (MFC) has highly reliable and flexible algorithms for diagnosis and monitoring of acute lymphoblastic leukemia (ALL). However, MFC analysis can be affected by poor sample quality or novel therapeutic options (e.g., targeted therapies and immunotherapy). Therefore, an additional confirmation of MFC data may be needed. We propose a simple approach for validation of MFC findings in ALL by sorting questionable cells and analyzing immunoglobulin/T-cell receptor (IG/TR) gene rearrangements via EuroClonality-based multiplex PCR. We obtained questionable MFC results for 38 biological samples from 37 patients. In total, 42 cell populations were isolated by flow cell sorting for downstream multiplex PCR. Most of the patients (n=29) had B-cell precursor ALL and were investigated for measurable residual disease (MRD); 79% of them received CD19-directed therapy (blinatumomab or CAR-T). We established the clonal nature of 40 cell populations (95.2%). By using this technique, we confirmed very low MRD levels (<0.01% MFC-MRD). We also applied it to several ambiguous findings for diagnostic samples, including those with mixed-phenotype acute leukemia, and the results obtained impacted the final diagnosis. We have demonstrated possibilities of a combined approach (cell sorting and PCR-based clonality assessment) to validate MFC findings in ALL. The technique is easy to implement in diagnostic and monitoring workflows, as it does not require isolation of a large number of cells and knowledge of individual clonal rearrangements. We believe it provides important information for further treatment.

Objective To detect expression of TEL-AML1 fusion genes in pediatric cases with acute lymphoblastic leukemia(ALL) and discuss the role of reverse transcriptase polymerase chain reaction(RT-PCR)and fluorescence in situ hybridization(FISH) in detection of t(12 ;21) and the clinical significance. Methods TEL-AML1 fusion gene was identified in bone marrow munonuclear cells from 31 newly diagnosed childhood ALL patients by NRT-PCR, FISH and conventional cytogenetic analysis (CCA). Results TEL-AML1 fusion gene was found in 7 out of 31 cases, accounting for 22.6 % in pediatric ALL, and 7 out of 31 cases accounting for 25.9 % in B-ALL Seven cases were found with t (12;21) by FISH and NRT-PCR. The incidence of the t(12;21) was 22.6 % in newly diagnosed pediatric ALLs. Conclusion It is concluded that TEL-AML1 rearrangement is a frequent molecular abnormality in childhood ALL. t(12;21) is the most common cytogenetic translocations in Chinese pediatric ALLs, but it is always difficult to identify by routine CCA.Other molecular methods, e.g. NRT-PCR and FISH are powerful in detecting such a critical genetic translocation. Key words: Leukemia; lymphoblastic; acute; TEL-AML1 fusion gene; Polymerase chain reaction; In situ hybridization; fluorescence

The analysis of immunophenotype of the leukaemic cells has been of great importance for the diagnosis, classification and prognosis of acute lymphoblastic leukaemia (ALL). One hundred and thirteen Chinese patients with ALL were immunophenotyped by fl ow cytometry and 74 cases were also subjected to karyotype analysis by G-banding technology. Of the 113 Chinese ALL patients, 14.2% were identified as T-ALL and 85.8% as B-ALL. Myeloid antigen (MyAg) expression was documented in 34.9% of the cases analysed and CD13 was most commonly expressed MyAg in ALL patients (23.6%). MyAg positivity was higher in adult with ALL (47.6%) than in children with ALL (26.6%). Abnormal karyotypes were detected in 39 out of 74 (52.7%) cases. The clinical and biological characteristics of ALL patients between MyAg+ and MyAg- groups showed that increased white blood count (WBC) (>50 x 109/L), higher CD34 positivity and higher percentage of adult patients were found to be correlated with MyAg+ ALL. Our results indicate that the immunophenotype did have relevance to the abnormal cytogenetic changes and clinical features in ALL. Flow cytometry immunophenotype has become the most important method for diagnosis and typing of ALL.