Robert Schoch

Which compartments are involved in Philadelphia-chromosome positive chronic myeloid leukaemia? An answer at the single cell level by combining May-Grünwald-Giemsa staining and fluorescence in situ hybridization techniques

Chronic myeloid leukaemia (CML) is believed to represent a stem cell disorder involving all three cell lineages. The typical chromosomal aberration, the Philadelphia chromosome, is the translocation (9;22)(q34;q11). Several studies with cytogenetics, fluorescence in situ hybridization (FISH), or polymerase chain reaction have investigated the presence of the t(9;22) in different cell compartments. However, questions still remain. In six cases of CML we combined the standard May‐Grünwald‐Giemsa staining with FISH at the single‐cell level and were able to demonstrate that not only all maturation stages of granulopoiesis, erythropoiesis, and megakaryocytes, but also plasma cells, eosinophils, basophils and monocytes carried the Philadelphia chromosome in 53–98% of samples. Using immunological identification of single cells we were able to demonstrate that the t(9;22) is detectable in 34% of CD3‐positive T lymphocytes, in 32% of CD19‐positive B lymphocytes, and in 82% of CD34‐positive precursor cells. The results give new insight into the biology of CML and may have implications for future therapeutic strategies.

Establishment and optimization of a flow cytometric method for evaluation of viability of CD34+ cells after cryopreservation and comparison with trypan blue exclusion staining.

BACKGROUND:  Trypan blue exclusion staining is probably the most frequently applied method (Method I) for assessment of viability in peripheral blood progenitor cell grafts after cryopreservation. Alternatively, a flow cytometry–based method (Method II) was established and optimized.

Cell lineage specific involvement in acute promyelocytic leukaemia (APL) using a combination of May-Grünwald-Giemsa staining and fluorescence in situ hybridization techniques for the detection of the translocation t(15;17)(q22;q12)

Acute promyelocytic leukaemia (APL) is strongly associated with the translocation t(15;17) which therefore provides a reliable marker to assess the potential involvement of different cell lineages. Six cases with morphologically, cytogenetically and molecularly proven APL were analysed at diagnosis or relapse by combining fluorescence in situ hybridization (FISH) with standard May‐Grünwald‐Giemsa (MGG) staining at the single cell level on bone marrow and blood smears. With the FICTION technique, combining immunophenotyping with FISH, haemopoietic precursor cells were identified using monoclonal antibodies against CD34, B‐ and T‐lymphocytes could be identified with CD19 and CD3. In addition, HLA‐DR‐positive cells were studied for the presence of t(15;17).

New insights into the biology of Philadelphia‐chromosome‐positive acute lymphoblastic leukaemia using a combination of May‐Grünwald‐Giemsa staining and fluorescence in situ hybridization techniques at the single cell level

It is sometimes difficult to discriminate chronic myeloid leukaemia (CML) in lymphatic blast crisis from Ph‐chromosome positive acute lymphoblastic leukaemia (ALL). Previous studies have suggested that ALL is restricted to the lymphatic lineage only, whereas CML involves all cell lineages.

Integration of amplified BCR/ABL fusion genes into the short arm of chromosome 17 as a novel mechanism of disease progression in chronic myeloid leukemia

We describe the cases of two patients with Philadelphia chromosome–positive chronic myeloid leukemia (CML), in whom the extramedullary blastic phase developed during disease progression. The similar clinical presentations of these patients was accompanied by gain of identical secondary chromosome abnormalities, that is, monosomies 9, 14, and 22, and by a clustered amplification of the BCR/ABL fusion gene. The additional copies of the BCR/ABL fusion gene were integrated into the short arm of structurally abnormal chromosomes 17 in both patients. The conformity of these genetic features in two patients with a rare disease manifestation leads us to the assumption that either the clustered amplification of the BCR/ABL fusion gene or the integration of this cluster into the short arm of chromosome 17 or both are associated with extramedullar disease progression in CML. Furthermore, the insertion of amplified BCR/ABL fusion genes into structurally abnormal chromosomes provides a novel mechanism of disease progression in BCR/ABL‐positive CML.

Amplification of ERBB2, RARA, and TOP2A genes in a myelodysplastic syndrome transforming to acute myeloid leukemia

A patient is described with myelodysplastic syndrome (MDS) progressing to acute myeloid leukemia (AML) FAB M4. Cytogenetic analysis revealed an unusual rearrangement between chromosomes 9 and 17, leading to a dicentric chromosome with an insertion of material of unknown origin between both chromosomes. By fluorescence in situ hybridization (FISH), the insertion was shown to be an amplification of part of 17q, involving ERBB2, RARA, and TOP2A genes. The median copy number of ERBB2, RARA, and TOP2A genes in the tumor cells was six (range: 4--10). Only one copy of the MPO gene at 17q21.3 was detected, suggesting a deletion of the telomeric part of 17q. To our knowledge, this is the first report of a 17q amplification in AML.

Glass slide smears are a suitable source for RT-PCR-based analysis of chromosomal aberrations in leukaemias

The polymerase chain reaction (PCR) is a well‐established method to detect cytogenetic abnormalities. The handling of fresh specimens is difficult, therefore a method to use smears of blood or bone marrow as a source would be advantageous. Furthermore, such a technique would give the opportunity to investigate retrospectively bone marrow smears in leukaemias without cytogenetic results. The aim of the present study was to investigate the influence of staining procedures and laboratory handling of smears. We chose CML cases as a model. We demonstrated that smears are a suitable source for PCR without loss of information caused by previous routine laboratory handling.

Dramatic hyperleukocytosis after treatment of myelodysplastic syndrome with pegfilgrastim and darbepoetin-alfa.

Dear Editor, We report a case of a 69-year-old Caucasian man with myelodysplastic syndrome (MDS), first diagnosed in January 2001 as refractory anaemia with ringed sideroblasts (WHO classification = RARS; IPSS score = low). The cellularity of the bone marrow was moderately increased, with a predominance of granulopoetic cells. The granulopoetic cells were frequently hypogranular; pseudo-Pelger cells were frequently seen. The erythropoesis was dysplastic as well with megaloblastoid changes, multinuclearity, and budding of the nucleus. The blast count was increased to 4%, and the iron-staining revealed 17% ringed sideroblasts. No typical aspects of myeloproliferative disease were present. Bone marrow histology showed no evidence for fibrosis. Cytogenetics of the bone marrow was normal. White blood cell count was 3.5×10/l, with neutropenia (1.3×10/l). The platelet count was 150×10/l, haemoglobin 10.0 g/dl. One year after initial diagnosis of MDS treatment was started with Epoetin-alpha (10,000 I.U.) subcutaneously three times a week because the patient was still anemic (9.9 g/dl), and clinical side effects of anaemia developed (e.g., dyspnoea). The endogenous Epoetin level was slightly increased with 69 U/l. Haemoglobin level increased up to 12.6 g/dl. Granulocytopenia (1.1–2.6×10/l) persisted. Since December 2003, haemoglobin levels gradually decreased again (8.9–9.4 g/dl). Because of patients’ convenience in August 2004, therapy with darbepoetin-alfa 500 μg weekly was initiated. No significant changes of haemoglobin levels were detected until August 2005 (8.1–9.0 g/dl). In August 2005, the patient received one dose of pegfilgrastim 6 mg, while the therapy with darbepoetin-alfa was continued. Only 10 h after pegfilgrastim administration, discomfort with pains in the abdomen, sternum and shoulders started. Four days later, the patient admitted himself to the hospital for persisting severe thoracic pain. In addition, dyspnoea under exercise occurred. Clinical examination was inconspicuous. Differential blood cell count revealed leukocytosis (111.0×10/l) with granulocytosis (98%), haemoglobin 7.2 g/dl and platelets 191×10/l. An electrocardiogram (ECG) showed a sinus rhythm, a preexisting incomplete right bundle branch block and a new depression of the ST segment in V3–V6. Laboratory findings for troponin were negative. Within the next 4 days, leukocytes normalized, and depression of the ST segment in the ECG, thoracic pain and dyspnoea disappeared. Darbepoetin-alfa was continued. Nevertheless, the patient needed infusions of erythrocyte concentrates in November 2005 and January 2006. Until now, haemoglobin levels and granulocytes remain below normal range. Pegfilgrastim was not applied again. Granulocyte colony-stimulating factors (G-CSF) are frequently used to abbreviate time of chemotherapyinduced granulocytopenia in patients under chemotherapy for solid tumours and hematologic malignancies. Pegfilgrastim is a pegylated form of G-CSF. Due to its neutrophil-mediated clearance, it has a longer half-life and must only be administered once per chemotherapy cycle [1]. In the same manner, erythropoetin (EPO) and darbepoetin-alfa are used for treatment of chemotherapyinduced anaemia. Darbepoetin-alfa has a longer half-life and needs to be administered only once a week [2]. Recent reports demonstrated an equivalent effect of darbepoetin-alfa instead of erythropoetin in the treatment of MDS [3]. Several studies reveal synergistic effects for the combination of non-pegylated G-CSF and EPO on the Ann Hematol (2008) 87:77–78 DOI 10.1007/s00277-007-0364-9

Semiquantitative reverse transcription polymerase chain reaction analysis for detection of bcr/abl rearrangement using RNA extracts from bone marrow aspirates compared with glass slide smears after 0, 2 and 4 d of storage

The polymerase chain reaction (PCR) is an established tool for the detection of specific chromosomal aberrations in different haematological malignancies. Owing to fast degradation of RNA, the immediate processing of samples is thought to have a major influence on the reliability of results. Any delay caused by transport may be an obstacle to reverse transcription PCR (RT–PCR)‐based methods in multicentre studies. However, as air‐dried bone marrow smears are usually available, we have improved a method to use smears as a source for routine RT–PCR investigations. We studied whether this source of RNA could overcome problems caused by delayed transport of samples. The aim of the present study was (i) to investigate the influence of a storage period of up to 4 d before processing of a specimen by nested bcr/abl RT–PCR, and (ii) to compare bone marrow aspirates with bone marrow smears stored at room temperature in parallel. Bone marrow aspirates and smears were taken from 11 patients with Ph‐positive chronic myeloid leukaemia (CML). PCR results were semiquantified using a limiting dilution assay. We observed a loss of sensitivity < 1 log in stored bone marrow aspirates, even after 96 h. Results obtained from air‐dried unstained glass slide smears were similar to investigations performed on approximately 1 × 105 cells of a bone marrow aspirate. We conclude that a storage period of up to 96 h has little influence on the detection of a bcr/abl fusion transcript in CML at diagnosis. Glass slide smears were equivalent to bone marrow aspirates in 8 out of 11 cases as a source for RT–PCR analysis when nested PCR was performed.

Routinely Prepared Cells for Cytogenetic Analysis Stored at −20 °C for Several Years can be Used for RT-PCR-Based Detection of Chromosomal Aberrations in Leukemias

Reverse transcriptase polymerase chain reaction (RT-PCR) is a well established method to detect chromosomal abnormalities in hematological malignancies. Because RT-PCR requires RNA of high quality, freshly obtained cells must be processed immediately or have to be frozen in liquid nitrogen or at −80 °C to prevent RNA degradation. However, in many cases no material has been stored for RNA extraction. Therefore, it would be of great advantage, if RTPCR could be applicable to methanol/acetic acid fixed material, stored in many cytogenetic laboratories. We have applied RT-PCR to methanol/acetic acid fixed cells from 28 patients with acute and chronic leukemias. Cytogenetically prepared bone marrow cells had been stored frozen in methanol/acetic acid for up to 5 years. RNA was extracted by a modified guanidinium thiocyanate phenol/chloroform protocol without prior manipulation of the cells. The patients had the following diagnosis: 23 patients (pts.) suffered from acute leukemia including 3 pts. with t(4;11), 9 pts. with t(8;21), 5 pts. with t(15;17) and 6 pts. with inv(16). Five pts. suffered from chronic myelogenous leukemia (CML) with t(9;22). RT-PCR was performed for the intact cABL gene to verify the integrity of the RNA and for the respective fusion gene using a nested or semi-nested primer system. In all cases the intact cABL transcript was amplified. In 3/3 cases with t(4;11), 8/9 cases with t(8;21), 3/5 cases with t(15;17),6/6 cases with inv(16) and 3/5 cases with t(9;22) the specific fusion genes could be detected (82.1%). Our results demonstrate that routinely stored cells in cytogenetic laboratories can be a useful source for RT-PCR based detection of chromosomal aberrations in many cases.