Clinical Case Discussion Forum
To share and enhance best practice management of CML, experts and interested clinicians can discuss difficult or interesting CML cases here. Physicians submit a brief history of the patient and the case for discussion using this forum.
Monitoring CML, BCR-ABL1 negative in PCR, positive in FISH and normal karyotype
46-year-old gentleman presented with low grade fever and malaise for 2-3 weeks. Initial CBC revealed total leukocyte count 225K/cmm with inappropriate documentation of differential.
Hydroxyurea 2 gram daily was started after sending blood sample for PCR for BCR-ABL1 which came negative. After a week of hydroxyurea treatment his spleen was still 2 cm from left costal margin; leukocyte count was 85K/cmm with 8% basophil, 8% myelocyte and 1% blast; platelet count 825K/cmm and hemoglobin was 11.2 gm/dl.
Then bone marrow was sent to a lab in neighboring country for conventional cytogenetics which came negative. To be noted the no lab in Bangladesh do conventional cytogenetics and we are compelled to send sample to neighboring country through informal channel where sample transportation can be an issue. FISH for BCR-ABL1 was sent to same lab thereafter and it came positive. Hydroxyurea was not given beyond 1st 7 days and imatinib was initiated. He achieved CHR in 6 weeks.
Please suggest what would be the best approach for laboratory monitoring of this gentleman considering conventional cytogenetics is challenging in terms of availability and quality.
Question from Professor Sue Branford (Australia) (followed by exchange and then advice):
The patient was both Ph chromosome negative and BCR::ABL1 negative by PCR. I assume the PCR would only detect typical BCR::ABL1 transcripts, e14a2 and e13a2. Rare to be negative for both tests. But the patient may be a rare patient with an atypical rearrangement and an atypical BCR::ABL1 transcript.
But the FISH test was positive. My question is whether BCR::ABL1 was detected at a high or low level? I assume the FISH test would indicate a high level in a patient recently diagnosed with CML, and FISH analysis may be the best way to monitor the patient. However, if FISH was low positive for BCR::ABL1 then I think further advice from a clinician on the diagnosis may be warranted.
Response from Dr Biswas:
His BCR-ABL1 in FISH was positive in 150 out of 200 signal (75%). I'm not sure about that cut-off for high level.
If it's high, what should be the frequency of FISH testing provided it's sensitivity is limited to 1/200?
As it cannot suggest deep MR, or even MMR, should I continue 3-monthly indefinitely? Or, I can make it every 3-6 months after a certain number of negative FISH?
Should I assume possible resistance if FISH come positive again after certain period of negativity?
Can KD mutation test be still applicable for this patient provided BCR-ABL1 in PCR is negative?
Advice from Professor Branford:
The FISH result does suggest a high level of leukaemia and regular monitoring will provide an indication of treatment response. The usual recommendation would be 3 monthly, particularly in the first year after diagnosis when most patients experience a rapid decline in BCR::ABL1, indicating an optimal response. 3 to 6 monthly monitoring could continue if the FISH value becomes undetectable. A rising FISH value could indicate relapse or non-adherence.
In the case of relapse, PCR amplification of the BCR::ABL1 kinase domain could be attempted but may only be successful if the BCR::ABL1 fusion includes BCR exon 13, which is the most common location of the PCR primers that are used for mutation testing. Failure to amplify in the standard kinase domain mutation assay likely indicates the inclusion of a non-standard BCR exon in the fusion. Without full characterization of the atypical BCR::ABL1 fusion from the diagnosis sample it is unknown how to proceed for successful kinase domain mutation testing.
It is challenging, indeed. In such a patient, who is likely to carry some atypical transcript, FISH should be the method of choice for monitoring, but given its non-availability locally, and the logistical (and economical, I suppose) hurdles to sample shipment to a lab in another country, routine monitoring by FISH is not feasible.
I would try to go back to the qualitative PCR that was initially performed (that was done in Bangladesh, right?), to check whether it is a multiplex enabling to detect atypical transcripts and whether it may have missed something. If any atypical transcript could be identified, then I assume that monitoring by qualitative PCR could be performed?
It is not clear if this is really a case of CML or not. When you say FISH positive, what exactly do you mean? How many cells were tested and what proportion were deemed positive? If truly positive then the patient probably expresses an atypical BCR::ABL1 mRNA which would ideally need to be confirmed in an international reference lab (but this would need adequate quality pretreatment material which may not be available).
If truly positive then FISH monitoring would probably be the easiest approach, but the sensitivity is limited. If an atypical fusion is confirmed then monitoring by bespoke RT-qPCR would be the most sensitive approach but may be very challenging logistically.