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?
This could be a cryptic fusion transcript. May be better to send the diagnostic sample for RT-PCR once again and ask to look for rare fusions
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.
I work as a cytogeneticist and I would like to help from that field. I agree with my collegues that it is necessary to identify the BCR::ABL1 transcript in order to follow it so you may need to know if your molecular approach has ruled out all possibilities. Is it any chance that it would be p190 or p230?
Maybe it could be somehow informative to know about the pattern of FISH positive cells because the sensitivity of the technique depends on it since 1/200 is only useful if you use double fusion probes and the pattern is 1G1O2F (and the lab has validated that point). And in the case that there is a loss of one of the derivatives or the rearrangement is originated by insertion, sensitivity of FISH may be no more than a 10% (FISH pattern 1G1O1F) so you have to be aware of it.
If karyotype is normal it could be because of several points: pathologic cells are non-proliferative in culture, the chromosomal resolution is not good enough to study them, or the aberration is cytogenetically cryptic. Have you reached at least 20 metaphases?
BCR-ABL1 constituted with some isoforms including typical and rare atypical (e1a2, e2a2, e6a2, e19a2, e1a3, e13a3, and e14a3) that have been sporadically reported. At diagnosis, atypical transcripts may yield a false negative PCR using routine primer/probe sets in qualitative or quantitative reverse transcriptase PCR protocols. Therefore, FISH alongside RT-PCR should be done in patients with atypical BCR-ABL1 transcripts that cannot be measured by RT-quantitative PCR. In that case, FISH may also need in patients with atypical transcripts for diagnosis and atypical transcripts can be positive by FISH.
This may be a case of cryptic fusion transcript BCR/ABL1 CML?, PCR primers in RT-PCR may fail to detect BCR/ABL1 transcript e14a3 or e13a3 and therefore can be negative on RT-PCR but positive with FISH which is at the DNA level unlike the former which detects at RNA level. Similarly if the quantity of the ABL 1 gene on chr.9 is too small, insertion into the BCR gene on chr,22 may be missed and therefore resulting in normal karyotyping. Such difficult cases are better resolved by requesting for NGS which can give additional findings not obtainable by other methods.
My sincere gratitude to all the experts for their valuable opininion and scientific insight.
I should have said earlier that PCR for BCR-AB1 included e1a2, e13a2, e14a2 and e19a2.
My curiosity - molecular scientists like Prof. Branford, Dr. Gonzalez and Dr. Chanda - is that whether commercial primers for e1a2, e13a2, e14a2 and e19a2 can identify all types of transcript for those 4 types, or are there some cryptic types within those 4 that can be missed?
Can anyone mention any lab where translocation to exon 3 of ABL1 (a3) can be tested (if needed in future).
After this discussion, I'm convinced that I can monitor response by FISH, as at baseline it was positive in high level (75%). I would do it every 3 months, but after achieving FISH negative I may not jump rapidly to test every 6 months as FISH negativity is not equivalent to MMR but merely equivalent to slightly more than 2 log reduction. After 4 or 5 consecutive negative FISH in 3 month intervals thought, I may test every 6 month.
And probably cannot approach for TFR directly. But can I do that through dose de-escalation?
Thanks once again.
Going back in this chain of comments, I would like to note what Nick Cross said - is this actually CML? This case illustrates what many of our colleagues face in the resource restricted world and we often take this for granted.
Start with the first few comments on the diagnosis. A good differential did not seem to be available, so that even the "classical" left-shift was not identified. So, we have no differential, cytogenetics or pcr to confirm the diagnosis. Are these negative because they truly are negative or because of difficulties getting the test done in the technically difficult scenario described? The post imatinib differential appears normal, but normal from what? Is the case here CML or a rarely described imatinib sensitive example of a chronic neutrophilic leukemia with a p230 abnormality that might be picked up with the FISH probes used?
I think it is academically nice to suggest ways to monitor, but the bottom line is what is available to the treating physician, that is readily and reliably available, whether this is a local standard or can be finessed through an industry sponsorship. It would appear that this is FISH with all the limitations of not knowing a certain disease, breakpoint, etc. Assuming that this is correctly CML, FISH will give an approach that can be accurate, less sensitive than pcr or next generation, but more sensitive that looking at a CBC and differential. This will at least give some lead as to patient compliance or early relapse which can help with counseling or availability of other therapeutic options, limited by drug availability, ability to detect mutations or even allograft options. It will not allow a safe consideration of a TFR attempt.
Unless we can all have access to the same resources, we need to support our colleagues by recommending the best options for them from what they can access.