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RECOMMANDATIONS FOR CYTOGENETICS AND FISH FOR CML

AND MYELOPROLIFERATIVE DISORDERS

INDICATION

- At diagnosis of any myeloproliferative neoplasm

- at the time of progression of any myeloproliferative neoplasm

- during follow-up of CML under tyrosine-kinase inhibitor therapy. If t(9;22) was present at diagnosis and the patient is treated with TKI, systematic cytogenetic follow up (first-year 1/6mo; later 1/y) is required until cytogenetic remission is reached). Expert opinions differ on the recommended frequency once cytogenetic remission is reached (1x /1 y; 1x/2y; or even less also depending on the duration of the cytogenetic remission). Indication for continued sampling after reaching Ph(-) status could be the detection of clonal abnormalities in Ph(-) marrow. However, the emergence of these abnormalities does not seem to have a negative impact on survival, and therefore karyotyping for this purpose is not supported by all. A similar cytogenetic sampling strategy can be adopted for other MPN that have a specific cytogenetic marker and are treated with TKI.

TYPE OF SAMPLE

In general, for myeloproliferative neoplasms, bone marrow aspirates are preferred over peripheral blood, at diagnosis and certainly at follow-up.

At diagnosis, cytogenetic examination of peripheral blood may also yield mitoses in case of CML, because of the presence of circulating myeloid precursor cells in peripheral blood. Yet, marrow is preferred because of the higher yield, and the fact that the marrow will be used as pretherapeutic baseline.

For PV and essential thrombocytosis, bone marrow samples are preferred at diagnosis. In general, the yield of cytogenetic aberrations in bone marrow is low in these disorders, more so in essential thrombocythemia than in polycytemia.

For chronic idiopathic myelofibrosis, blood samples can be accepted as well, as there is usually a low percentage of circulating blasts, and as marrow is usually difficult to obtain in sufficient quantity (dry tap).

During follow-up of CML, it is not useful to perform karyotyping on peripheral blood, if a patient achieves haematological and cytogenetic remission with TKI therapy, as there will be no mitoses.

It can be useful however in cases of CML with primary or secondary haematological resistance when circulating precursors persist or reappear, or in leukemic evolution of PV, essential thrombocytosis or CIMF.

CULTURE CONDITIONS

Cultures are recommended using a cell concentration of 1-2 x106 cells/ml media.

Recommended culture time is 48 h (synchronized), but 24 h (non-synchronized) -72h (non-synchronized) are also acceptable if more convenient.

Different modalities of synchronisation (FRDU/thymidine; MTX/BRDU; MTX/thymidine) are used in different labs.

CHROMOSOME ANALYSIS

- Diagnostic specimens

- If a normal karyotype is obtained at least 20 metaphases should be analysed in order to exclude a clonal abnormality.

- If an abnormal karyotype is obtained a sufficient number of cells (at least 10) should be analysed to establish clonality and identify clonal evolution.If a specific chromosomal alteration is detected, at least 2 metaphases are necessary to ascertain the clonality if there are structural aberrations or trisomies, and 3 in case of monosomies.

In newly diagnosed CML, the bone marrow usually uniformly yields Ph(+) mitoses. If this is not the case, it may be wise to do 20 metaphases to precisely establish a pretherapeutic baseline.

- Follow up specimens

For quantitation of the cytogenetic response under TKI therapy, at least 20 mitoses (GFCH recommends to do 30 or even more if possible) should be examined.

For PV, ET, CIMF regardless the karyotype at diagnosis, follow-up cytogenetics is indicated in case of progressive disease (see “diagnostic specimens”).

FISH ANALYSIS

- Diagnostic specimens

BCR-ABL1 FISH is recommended in every case of newly diagnosed Ph(+) CML,notasmuch to establish the diagnosis but to examine deletions of der(9q+) and establish the diagnostic pattern for later follow-up. Deletions centromeric of the breakpoint on der(9) have been associated with a less favourable prognosis before imatinib – their significance under imatinib therapy remains to be definitively established. Therefore probes that include these regions are to be used preferentially.

FISH is also indicated in CML with variant translocations, in case of Ph(-) CML with a positive BCR-ABL1 RQ/RT-PCR (cryptic translocations).

In situations with missing data (PCR or cytogenetics), the indication for FISH may depend on the degree of clinical or cytological suspicion. Ph(-), BCR-ABL1(-) disease can be an indication for FISH if the suspicion of an MPN is high.

For PV, ET, CIMF, FISH can be indicated at diagnosis to establish or refute the clonal nature of a metaphase abnormality observed in cytogenetics. Given the low a priori frequency of cytogenetic aberrations in theses diseases, it is not useful to FISH blindly for aberrations (trisomy 9, del20q-, …) in case of normal karyotype or failed cytogenetics.

For idiopathic hypereosinophilic syndrome / CEL, the cytogenetically cryptic del(4q12) should be explored by FISH. Commercial probes have become available recently, that will also pick up translocations of PDGFRA. In case of a normal karyotype, a systematic search by FISH of PDGFRB rearrangements does not appear to be indicated, as they are extremely rare and can be detected by standard cytogenetics.

- Follow-up specimens

During follow-up, BCRABL1 FISH on bone marrow is indicated for:

- follow-up of cytogenetically cryptic Ph-translocations

- examination of Ph(+) disease at follow-up in case of failed cytogenetics or insufficient mitoses.

The % of FISH positive cells on bone marrow can give an estimate of the type of cytogenetic remission but does not translate directly into types of cytogenetic responses (except in case of 0% positives). The use of FISH in the follow-up of peripheral blood samples is not validated and therefore not recommended.

REPORTING

The cytogenetic report should include the type of culture, the number of mitoses examined, and the karyotype according the ISCN 2005.

FISH results can be reported in a descriptive way, or according to ISCN 2005.

The conclusion indicates the significance of the cytogenetic findings in the clinical context, (e;g. diagnostic of a WHO MPN-entity; indicating clonal hematopoiesis, associated with MPN, associated with less favourable prognosis, …)

In cytogenetic reports for follow-up of CML, the type of response should be classified as:

- no cytogenetic response (Ph > 95%)

- minimal cytogenetic response (Ph > 66-95%)

- minor cytogenetic response (Ph 36-65%)

- major cytogenetic response (1-35 % Ph in BM)

- complete cytogenetic response (no Ph in BM).

If information is available on the type of treatment, it could be considered to include information as to whether the response is optimal, suboptimal, failing at the specific time point of the treatment.
A loss of cytogenetic response must also be reported if present.

29/09/2018