12.7: Chronic Myelogenous Leukemia (CML) - Biology

12.7: Chronic Myelogenous Leukemia (CML) - Biology

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Leukemia is an uncontrolled proliferation of one kind of white blood cell (or leukocyte). Like all cancers (probably), all the leukemic cells are descended from a single cell that lost the ability to maintain normal control over the cell cycle. There are a number of types of leukemia, as you would expect from the number of types of white blood cells (5) and the number of stages they pass through as they mature. One of the most common is chronic myelogenous leukemia or CML.

Chronic Myelogenous Leukemia (CML) arises in a bone marrow stem cell that is the precursor to all the types of blood cells. However, it usually affects the so-called myeloid lineage (hence the name) that produces granulocytes and macrophages. As the name suggests, the disease often exists for years with only moderately elevated numbers of leukemic cells (descended from the stem cells) and few symptoms. At some point, however, the patient goes through a "blast crisis" when the leukemic granulocyte-macrophage progenitors begin to divide by themselves — increasing their numbers enormously while failing to continue their differentiation.

The Philadelphia Chromosome (Ph1)

In most cases of CML, the leukemic cells share a chromosome abnormality not found in any nonleukemic white blood cells, nor in any other cells of the patient's body. This abnormality is a reciprocal translocation between one chromosome 9 and one chromosome 22. This translocation is designated t(9;22). It results in one chromosome 9 longer than normal and one chromosome 22 shorter than normal. The latter is called the Philadelphia chromosome and designated (Ph^1).

The DNA removed from chromosome 9 contains most of the proto-oncogene designated c-ABL. The break in chromosome 22 occurs in the middle of a gene designated BCR. The resulting Philadelphia chromosome has the 5' section of BCR fused with most of c-ABL.

The micrograph in Figure (PageIndex{2}) uses fluorescence in situ hybridization (FISH) to reveal the ABL DNA (red) and the BCR DNA (green) in the interphase nuclei of the leukemic cells of a patient with CML. The red dot at left center reveals the location of ABL on the normal chromosome 9; the green dot (top center) shows BCR on the normal chromosome 22. The combined dots (red + green = yellow) at the lower right reveal the fused BCR-ABL gene on the Philadelphia chromosome. Figure 12.7.3 is a schematic which can help you interpret the micrograph.

Transcription and translation of the hybrid BCR-ABL gene produces an abnormal ("fusion") protein that activates constitutively (all the time) a number of cell activities that normally are turned on only when the cell is stimulated by a growth factor, such as platelet-derived growth factor (PDGF).

This unrestrained activation increases the rate of mitosis and protects the cell from apoptosis. The outcome is an increase in the number of Ph1-containing cells. During the chronic phase of the disease, these are still able to exit the cell cycle and to differentiate into mature cells that perform their normal functions. At some point, however, another mutation in a proto-oncogene (RAS, for example) or in a tumor-suppressor gene (p53, for example), will occur in one of these cells. The additional mutation causes the rate of mitosis in that cell and its descendants to rise sharply. The daughter cells fail to differentiate and the patient enters the crisis phase of the disease.

A Promising Treatment

Until recently, the only successful treatment of CML was to destroy the patient's bone marrow and then restore blood-cell production by infusing stem cells from the bone marrow of a healthy donor. But now treatment with the drug imatinib mesylate (Gleevec® also known STI571) appears to be able to cure the disease. This molecule fits into the active site of the ABL protein preventing ATP from binding there. Without ATP as a phosphate donor, the ABL protein cannot phosphorylate its substrate(s). A phase 2 study, found that almost 90% of the CML patients treated with the drug showed no further progression of their disease.

Gleevec also shows promise against one type of stomach cancer (gastrointestinal stromal tumors = GIST), which is a life-threatening excessive production of eosinophils. In this disease, Gleevec inhibits a different overactive tyrosine kinase. This one also results from the fusion of parts two different genes (because of the deletion of the DNA between them):

  • the first 233 codons of a gene designated FIP1L1 fused to
  • the final 523 codons of the gene (PDGFRα) encoding the tyrosine kinase domain of a receptor for platelet-derived growth factor. The fusion protein produced, like BCR-ABL, is hyperactive.


Blast crisis is the terminal phase of chronic myeloid leukemia (CML) with a short median survival of approximately six months. At present, little is known about molecular mechanisms underlying disease progression. We hypothesized that mutations occurring in other myeloid and lymphatic malignancies are acquired during disease progression from chronic phase to blast crisis. Here, in total 40 blast crisis CML cases (n=25 myeloid, n=10 lymphoid, n=5 not specified) were analyzed, all diagnosed between 9/2005 and 7/2009. First, all cases were investigated for IKZF1 deletions by PCR using specific primer pairs for the common intragenic deletions spanning from exon 2–7, or exon 4–7 as published by Iacobucci et al. (Blood, 114:2159-67, 2009). In total, in 17.5% (7/40) of cases intragenic IKZF1 deletions were detected. Secondly, next-generation deep-sequencing (454 Life Sciences, Branford, CT) was used to investigate 11 candidate genes in all 40 patients for a broad molecular screening. Known hotspot regions were sequenced for CBL (exons 8 and 9), NRAS (exons 2 and 3), KRAS (exons 2 and 3), IDH1 (exon 4), IDH2 (exon 4), and NPM1 (exon 12). Complete coding regions were analyzed for RUNX1, TET2, WT1, and TP53. To perform this comprehensive study, amplicon-based deep-sequencing was applied using the small volume Titanium chemistry assay. To cope with the great number of amplicons, in total 59, 48.48 Access Arrays were applied (Fluidigm, South San Francisco, CA), amplifying and barcode-tagging 48 amplicons across 48 samples in one single array (2,304 reactions). In median, 430 reads per amplicon were obtained, thus yielding sufficient coverage for detection of mutations with high sensitivity. Further, ASXL1 exon 12 aberrations were investigated by Sanger sequencing. In summary, after excluding known polymorphisms and silent mutations in 33/40 patients 53 mutations were identified: RUNX1 (16/40 40.0%), ASXL1 (12/40 30.0%), WT1 (6/40 15.0%), NRAS (2/40 5.0%), KRAS (2/40 5.0%), TET2 (3/40 7.5%), CBL (1/40 2.5%), TP53 (1/40 2.5%), IDH1 (3/40 7.5%), IDH2 (0/40), and NPM1 (0/40). Thus, 82.5% of blast crisis CML patients harbored at least one molecular aberration. In median, one affected gene per patient was observed (range 1–5). In detail, RUNX1 was associated with additional mutations in other genes, i.e. 9/16 cases were harboring additional mutations in combination with RUNX1. Similarly, in 8/12 patients with ASXL1 mutations additional aberrations were detected. With respect to myeloid or lymphoid features ASXL1 mutations (n=11) were exclusively observed in patients with myeloid blast crisis (n=1 not specified), in contrast 5/7 IKZF1 cases were detected in cases with lymphoid features (n=1 myeloid, n=1 not specified). Interestingly, besides IKZF1 (n=5) and RUNX1 (n=3) alterations there was no other mutated gene occurring in lymphoid blast crisis CML. In addition, no aberration was detected in NPM1, and in contrast to published data, in our cohort only one patient harbored a mutation in TP53. Moreover, for 8 patients with mutations in IKZF1 (n=3), RUNX1 (n=3), ASXL1 (n=1), WT1 (n=2), and IDH1 (n=2), matched DNA from the initial diagnosis at chronic state was available. In these specimens respective IKZF1 deletions, RUNX1, and ASXL1 mutations were not detectable indicating that IKZF1, RUNX1, and ASXL1 mutations had been developed during disease progression and act as driver mutations in these cases. WT1 and IDH1 mutations occurred at first diagnosis in one case each, indicating these genes would constitute passenger mutations. In conclusion, this comprehensive study on 12 molecular markers enabled to characterize for the first time that 82.5% of blast crisis CML cases harbor specific molecular mutations. IKZF1 and RUNX1 alterations were identified as important markers of disease progression from chronic state to blast crisis. Moreover, technically, a novel combination of a high-throughput sample preparation assay for targeted PCR-based next-generation deep-sequencing was developed and allowed to broaden our molecular understanding in blast crisis CML.


Grossmann: MLL Munich Leukemia Laboratory: Employment. Eder: MLL Munich Leukemia Laboratory: Employment. Schindela: MLL Munich Leukemia Laboratory: Employment. Kohlmann: MLL Munich Leukemia Laboratory: Employment. Wille: MLL Munich Leukemia Laboratory: Employment. Schnittger: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership, Research Funding.

Efficacy of a Medication for Chronic Myeloid Leukemia

This activity guides the analysis of a published scientific figure from a study that tested a new treatment for chronic myeloid leukemia (CML), a cancer of white blood cells.

In CML, white blood cells divide uncontrollably due to an overactive mutant tyrosine kinase protein called BCR-ABL. In this study, scientists developed and tested a drug, STI571 (trade name Gleevec), that blocks the activity of BCR-ABL. The figure shows the white blood cell counts for six CML patients, each represented by a different line, who were treated with STI571 for 150 days. The dotted line represents the upper limit of a healthy white blood cell count. The “Educator Materials” document includes a captioned figure, background information, graph interpretation, and discussion questions. The “Student Handout” includes a captioned figure and background information.

Student Learning Targets
  • Analyze and interpret data from a scientific figure.
  • Use patient data to evaluate the efficacy of a medical treatment.

cancer, cell division, chromosomal translocation, Gleevec, health care, line graph, medicine, oncogene, tyrosine kinase inhibitor (TKI)

Druker, Brian J., Moshe Talpaz, Debra J. Resta, Bin Peng, Elisabeth Buchdunger, John M. Ford, Nicholas B. Lydon, et al. “Efficacy and safety of a specific inhibitor of the BCR-ABL tyrosine kinase in chronic myeloid leukemia.” New England Journal of Medicine 344, 14 (2001): 1031–1037.

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Blood Tests for Leukemia

If you are experiencing any symptoms of CML, talk to your doctor. They will perform several lab tests to confirm a diagnosis. These include blood tests and a bone marrow biopsy.

A complete blood count (CBC) measures the levels of different blood cells, including red and white blood cells and platelets. A blood smear is also done, where a sample of blood is put on a glass slide and looked at under a microscope. In some cases, people with CML will have too many immature cells known as blasts or myeloblasts.

Leukemia begins in the bone marrow, so it is vital to check for cancer cells there. A thin, hollow needle is inserted into the bone and the marrow inside, and samples are taken to be examined under a microscope.

If you’re diagnosed with leukemia and begin treatment, you may also notice side effects that may seem like symptoms.

Chronic myelogenous leukemia

Chronic myelogenous leukemia (CML), also known as chronic myeloid leukemia, is a cancer of the white blood cells. It is a form of leukemia characterized by the increased and unregulated growth of myeloid cells in the bone marrow and the accumulation of these cells in the blood. CML is a clonal bone marrow stem cell disorder in which a proliferation of mature granulocytes (neutrophils, eosinophils and basophils) and their precursors is found. It is a type of myeloproliferative neoplasm associated with a characteristic chromosomal translocation called the Philadelphia chromosome.

CML is largely treated with targeted drugs called tyrosine-kinase inhibitors (TKIs) which have led to dramatically improved long-term survival rates since 2001. These drugs have revolutionized treatment of this disease and allow most patients to have a good quality of life when compared to the former chemotherapy drugs. In Western countries, CML accounts for 15–25% of all adult leukemias and 14% of leukemias overall (including the pediatric population, where CML is less common). [3]

YouTube Encyclopedic


- Chronic Myelogenous Leukemia or sometimes known as Chronic Myeloid Leukemia is a type of cancer where white blood cells are mass produced in the blood. These cells tend to be of the myeloid stem cell lineage and because this is a chronic leukemia, the cells we see in the blood stream tend to be the more maturer type of blood cells as opposed to an acute leukemia where you have more immature cells in the blood stream. This type of cancer occurs when a very specific mutation happens where a gene that sits on chromosome 22, that's called the BCR gene. The BCR gene is shuffled so that it sits next to a gene on chromosome nine that's called the ABL or the ABL gene. This defect is known as a translocations, so we write translocation of a piece of chromosome nine onto chromosome 22 that for the purposes of drawing here will look something like this where the BCR gene sits on the ABL gene and they're attached together. This new protein is a type of receptor tyrosine kinase that's constantly on, telling cells that they should keep on dividing, which is the whole premise of cancer. You're making more cells than you need. In fact, this translocation that puts a piece of chromosome nine on a chromosome 22 produces a new type of chromosome altogether that's called the Philadelphia chromosome. Named for the university it was discovered at. But the problem here is that now we're going to be producing a whole bunch of white blood cells we don't need. So I'm drawing here the bone marrow, and as you might recall, one of the first things that's produced here is something that's called a pleuripotent. Meaning that something that has the potency or the ability to make plural or many things hematopoietic, meaning related to the blood stem cell. Pleuripotent hematopoietic stem cell. This can produce two different lineages. I'll draw them out this way and we're not focusing so much on this lymphoid stem cell lineage here, so I'll just draw it off to the side. But let's pay better attention here where we have the myeloid stem cell. As you know there are a variety of things that could be produced here, so I'm just gonna draw them out here, but not name all of them. But recall it's these types of cells right here, these white blood cells that we'll see being produced excessively in Chronic Myelogenous Leukemia. Because the defect is really high up, like around here is where the mutation occurs, we'll tend to see an irregular or an unusual amount of platelets or even red blood cells as well. That translates into a couple of key signs and symptoms. Now Chronic Myelogenous Leukemia or CML as it's sometimes referred to has three distinct phases that have different signs and symptoms. First, there's the chronic phase. In the chronic phase, about 90% of patients are asymptomatic, so without any symptoms. Asymptomatic when they're diagnosed, but later in this phase you can have some signs and symptoms such as abdominal fullness, due to the fact that excess platelets or other white blood cells that are being made have to go to your spleen to get processed or even destroyed if there's too many defective ones causing the spleen to become big or for you to have something that's called splenomegaly. Splenomegaly, and the same thing can happen to your liver as you have a higher basal metabolism because of all the extra cells you have in your blood stream. When your liver gets big, we call that hepatomegaly, so in total you could have Hepato-splenomegaly. Other than the abdominal fullness, a common symptom is having a fever and this is mainly due to having an increased number or a white blood cell count because as I mentioned, that means that you're going to have an increased basal metabolism, which is just your metabolism at rest. The chronic phase will progress, and the next step would be what's called the accelerated phase. The accelerated phase and it's called the accelerated phase because you are more rapidly making cells and often times these are defected cells. Those that don't actually work correctly and this is well illustrated when you consider the platelet because these patients can have bleeding and it's because platelets are supposed to clot and make sure you don't bleed when you're cut, but if you're platelets are not working, you will bleed more and these can manifest as petechiae, which are just small dots that you might see, which is bleeding from your vessels or you can see what are called ecchymoses. Ecchymoses, which are just bruising or bruises that occur when you even just bump into things very lightly and so you'll have an accumulation of blood under the skin that'll look like you hit it there much more intensely than you actually had. In the accelerated phase, you could also get a fever, but this time, it's more likely due to opportunistic infections, which just means that microbes like bacteria, or fungi, or viruses see an opportunity to infect a human being or host because the white blood cells are not functioning correctly in the accelerated phase. Finally, the most advanced phase someone can be in is what's referred to as a, or the blast phase, or even the blast crisis. This is characterized by rapid immature cell production which can cause you to have some pretty significant bone pain. Bone pain related to increased production of myeloblasts in the bone marrow. You can also have a fever and this is for the reasons we've mentioned above, either from an opportunistic infection or from having too many of these cells in your blood stream. Now these are just some things you would see on phyiscal exam, but how would we make a more definitive diagnosis? Well, one of the simplest things you can do is get a complete blood count, which may show you that you have elevated white blood cells in the blood stream on the order of something like 50 to 200,000, whereas you should have less than 12,000. You can also take some of the blood and look at it under a microscope and you'd see on this peripheral blood smear that there are a lot of leukocytes, so you've got what's called leukocytosis, which would look like this where you've got a whole bunch of these white blood cells and you can see they're very different from these guys, which are your red blood cells in your blood stream. Let's minimize this guy and go back up to this list right here. One of the other things that you can see that is considered a pretty slam dunk diagnosis is a positive Fluorescent In Situ Hybridization test or a FISH that will light up to show you that there is a Philadelphia chromosome present. Now one thing I should mention at this point, the Philadelphia chromosome, it's present in about 95% of cases of CML, so it's pretty good, but there are some cases that you won't have it. The translocation of taking the BCR gene and putting it close to the ABL gene now that's present in 100% of CML cases, so you don't have to have the full chromosome translocation just the gene to get CML, but we check for this because it's very commonly present with CML. 95% is a pretty good odds. It would look like this right here. This is a positive FISH, where you've got, for example, green, perhaps lighting up the BCR gene. Red lighting up the ABL gene and you can see very interestingly here there's a green attached to a red so that's the 9/22 translocation we're talking about. Finally, the last thing or the thing we try to avoid to do is what's called a bone marrow aspiration where we inject a big needle into the bone marrow and suck out some of the marrow and look at it under a microscope where you will see an increased number of myeloblasts. Okay, so what do we do now to treat this? Well, if you were with us for our conversation on polycythemia vera or essential thrombocythemia, you might recall some of these cytotoxic drugs like Hydroxyurea or Interferon Alpha and all they do is they make it difficult to mass produce the white blood cell, but perhaps the most important treatment to know for CML is the use of a drug that's called Imatinib, which is one of the greatest successes of science. Long ago, scientists realized that the BCR-ABL translocation produced this unusual receptor tyrosine kinase that kept on telling cells, myeloid cells, to keep on dividing and the idea was if we made a drug that could block this receptor, something that would come in here and sort of bind the receptor and make it impossible for it to communicate with the other types of enzymes or proteins in a cell that signal that signal that it's time to keep on dividing, you could somehow prevent production of myeloblasts and that's exactly what Imatinib does. It prevents this unusual protein from working and as a result, you can actually cure cancer, cure CML from employing this drug and making it so that you don't produce all these extra white blood cells. This was an amazing accomplishment because before Imatinib, the prognosis for CML was pretty poor. Most would be dead in three to five years without this drug, but now when you use it, so with Imatinib, 90% are alive at five years. Finally, coupled to Imatinib because you're gonna block off production of this protein that is actually necessary for division from time to time, you would have to give this patient what's called an allogeneic stem cell transplantation to make sure they have the cells or the protein machinery to continue dividing and producing white cells correctly. CML's a pretty interesting disease and I think what's great about it is that it really illustrates the success of science, kind of looking into what is the protein produced from a defect and what we can do to generate something to essentially cure cancer.


CML-N was described in 1996 as a clinical entity characterized by primary, chronic, nonprogressive leukocytosis. 7 The original description required the following criteria: 1) moderate neutrophilic leukocytosis 2) rare, circulating, immature myeloid cells without a myelocyte peak 3) excess bone marrow mature myeloid cells and 4) absent or minimal splenomegaly. 7 The Ph carrying e19/a2 BCR/ABL gene fusion abnormality added a molecular diagnostic confirmation of the CML-N entity. Although the cytogenetically visible abnormality is the same as in classic CML, the different molecular lesion may be responsible for the more benign clinical course.

In this report, we describe six new patients and update (with molecular analysis) five previously reported patients with Ph positive myeloproliferative disorders and the e19/a2 BCR/ABL abnormality, resulting in p230 mRNA production. A review of all 23 patients with Ph positive p230 CML (Table 1) showed that their presentation was somewhat atypical compared with classic CML: Sixteen patients were females, 16 patients had no palpable splenomegaly, and only 3 patients had WBC counts > 100 × 10 9 /L. Five patients presented with very high plt counts (> 1000 × 10 9 /L). Although some authors have suggested that these patients had Ph positive essential thrombocytosis, 9 , 11 the diagnostic criteria for that disorder specifically exclude patients with BCR/ABL gene rearrangement. 24 Most patients described previously with Ph positive essential thrombocytosis, when studied by RT-PCR analysis, had the classic p210 junction. 25 Among patients with CML and thrombocytosis, at least four different BCR/ABL junction variants (including e19/a2) have been detected, albeit with various frequencies. 26

There is some confusion regarding the relation between CML-N and chronic neutrophilic leukemia. In chronic neutrophilic leukemia, clonality of the cells has not been reported consistently thus, there is uncertainty about whether all reported patients had leukemia. 27 You and Weisbrot 28 reported that the syndrome of chronic neutrophilic leukemia was comprised of severe, sustained, mature, neutrophilic leukocytosis hepatosplenomegaly elevated leukocyte alkaline phosphatase elevated serum vitamin B12 elevated serum uric acid the absence of an underlying disease provoking a reactive leukemoid reaction anatomic evidence of organ infiltration by granulocytes and myeloid metaplasia. Although the clinical criteria for the diagnosis of chronic neutrophilic leukemia and CML-N overlap to some extent, CML-N is defined by Ph and the presence of the p230 BCR/ABL fusion gene. 2

In their original report, Pane et al. 7 suggested that patients with CML-N may have a more benign clinical course than patients with classic CML. This was questioned later as new patients with p230 disease and an adverse clinical course were described. 9-18 A review of all reported patients (Table 1) showed that 10 patients were well and alive ≥ 3 years after the diagnosis (the longest follow-up is > 16 years). Six patients have died: Patient 8 was treated with busulphan, achieved a hematologic response, and died of an unrelated cause 10 years after the diagnosis Patient 12 was never treated for CML-N and died of myocardial infarction 3 years after the diagnosis Patient 17 died of complications 3 months after undergoing bone marrow transplantation (BMT) while in complete remission and Patients 9, 15, 20, and 23 died in the blastic phase of CML. Three of four patients who died of leukemia presented with extrachromosomal abnormalities in addition to Ph. Overall, eight patients presented with additional chromosomal abnormalities. Patient 5 presented with accelerated-phase CML and has failed induction chemotherapy. Patient 14 had no response to IFNα treatment and was awaiting BMT. Patient 17 (death from complications of BMT) had failed IFNα treatment and was in the accelerated phase of CML prior to BMT. Patient 19 presented with significant basophilia and a high proportion of circulating, immature granulocytes. Patient 21 had failed IFNα treatment and was in the accelerated phase of CML prior to BMT. Thus, as suggested recently, 17 chromosomal abnormalities in addition to Ph appear to be associated with a more malignant course of the disease.

The presence of Ph (which has the p230 fusion gene) as a single abnormality, however, does not necessarily confer an indolent course, as evidenced by Patient 9, who died in the blastic phase of CML. Still, the reason for the association of p230 disease with a milder leukemia phenotype in the majority of patients with CML-N (those without additional cytogenetic abnormalities) has not been clear to date. It has been reported that the three forms of BCR/ABL have different intrinsic leukemogenic activity when expressed in a hematopoietic progenitor cell. 21 , 29 The characterization of the tyrosine kinase activity of the three proteins revealed that p230 was less potent than p190 or p210. 29 Recent in vitro experiments have shown that p230 had transforming ability similar to that of p190 or p210 when expressed in 32D myeloid cells or Rat1 fibroblasts. 30 However, in mouse primary bone marrow cultures, p230 was less transforming, so that the cells required exogenous hematopoietic growth factors for optimal growth, whereas cells that expressed p190 and p210 grew independently of growth factors. 21 Persistence of a pool of Ph negative progenitors in some patients with CML-N years after diagnosis, and in the absence of treatment (Patients 6 and 7), may suggest a limited proliferative advantage of the Ph-abnormal clone.

Attempts by our group 7 , 8 and by others 9 to verify the expression of p230 protein in patients bearing the e19/a2 type BCR/ABL gene have been unsuccessful. Patient 6 had undetectable p230 protein in both peripheral blood and bone marrow 12 years after diagnosis and 6 years after discontinuation of IFNα. In the only reported patient with detectable p230 protein (Patient 20 in Table 1), most of the leukemic cells showed a duplication of the Ph. 15 This may suggest that a greater degree of p230 protein production may have conferred a more malignant course, although other chromosomal abnormalities were found in addition to Ph. In this report, using two different techniques, we have shown that the transcription of the p230 gene in the great majority of patients with CML-N was extremely low, resulting in undetectable levels of p230 protein. Samples from Patient 6 contained minimal numbers of molecules of p230 transcripts per total RNA. This may explain the milder leukemic phenotype in most patients with CML-N. Although the level of p210 BCR/ABL transcription in newly diagnosed patients with classic CML is in the range of ≈35,000 p210 molecules per μg total RNA, the order of magnitude of p230 BCR/ABL transcription in most patients with untreated CML-N is similar to the order of magnitude of p210 BCR/ABL transcription in patients with classic CML patients who achieved complete cytogenetic remission after IFNα treatment. 31 , 32 Although transcription of the p230 gene in most patients with CML-N was very low, we did find patients with very high p230 mRNA expression. Those patients presented with more aggressive disease: Patient 9 died in blastic phase CML, whereas Patient 11 had a poor response to IFNα and cytarabine therapy.

Why there is a difference in individual p230 gene expression remains unknown. The transcription of p230 gene may be low because of the presence of at least 3 transcription silencer elements within the intronic sequences at the 3′ of exon 14 of the M-BCR region. 33 These elements are lost in p190 disease, are partly retained in p210 disease, and are fully retained in p230 disease. Sequence analysis also revealed that at least one of the GAP-homology domains of the BCR gene was retained within the p230 gene. 7 In addition, p230 expression may be low because of the occurrence of multiple alternative splicing of the original messenger RNA responsible for the production of out-of-frame transcripts that produce truncated p230 protein. 10

It is interesting to note that, in three patients with newly diagnosed CML-N (Patients 3, 4, and 5), we detected p210 transcripts in addition to p230 transcripts. Similar to the classic p210-associated CML, in which p190 transcripts are detectable along with p210 transcripts, 4-6 this finding is likely due to the alternative splicing of primary p230 mRNA. Thus, resultant numbers of both p230 and p210 transcripts are minimal. Unlike classic CML, however, in which p190 protein is detectable along with p210 protein 4 and may or may not have clinical significance, 5 , 6 , 34 in our three patients with CML-N, neither p230 nor p210 proteins were detectable.

It should be emphasized that patients with p230 positive CML (CML-N) can be overlooked easily if appropriate molecular studies are not performed. In standard practice, molecular studies are not requested if cytogenetic studies already have shown t(922). The detection of p230 positive patients occurs when simultaneous cytogenetic and molecular studies are done (e.g., in a research setting), which may show a paradoxical situation of the presence of t(922) but absence of M-BCR and m-BCR rearrangements by conventional Southern blot analysis. In addition, RT-PCR positivity for P230 BCR/ABL mRNA may be overlooked unless primers are used that cover the e19/a2 breakpoint. The presence of t(922) and RT-PCR negativity for M-BCR and m-BCR then stimulates additional studies to detect p230 disease at the mRNA level.

In conclusion, CML-N may have an indolent course in the absence of chromosomal abnormalities other than Ph because of the low expression of p230. This supports the need to conduct additional molecular studies, even if cytogenetic studies already have shown t(922), because of the prognostic importance of the molecular findings.


Study population

Previously diagnosed patients with CML were recruited from January 2019 and followed up through October 2020 at the CML day clinic at ORCI in Tanzania. Diagnosis of CML was established by examination of peripheral blood and bone marrow and confirmed by PCR for the BCR-ABL fusion gene. The PCR test was carried out in all patients to fulfill a requirement for access to free imatinib under the GIPAP. Eligible patients were adults aged ≥18 years who had been receiving imatinib treatment for at least 3 months. Excluded were patients with newly diagnosed disease receiving imatinib treatment for <3 months, or receiving TKIs other than imatinib, or undergoing cytoreduction with hydroxyurea.

Data collection

Data from eligible consenting patients was collected at 3 time points: (1) at diagnosis traced retrospectively from the patient’s clinical files, (2) at a follow-up visit at which patients were recruited into this study, and (3) for 22 months after recruitment for assessment of survival. Retrospective data at the time of diagnosis from the patient’s clinical files included presenting symptoms, signs, and laboratory parameters, such as full blood cell count and blast count from bone marrow reports. The Sokal risk score at baseline was calculated according to each patient’s clinical and laboratory parameters. Baseline BCR-ABL was assumed to be 100% as per International Standard (IS) recommendations. Retrospective, systematic complete full blood count results at 3, 6, and 12 months were not available, primarily because patients’ clinic visits depend on the financial ability to travel to the prescribing center.

Data collected at the follow-up visit during the recruitment into the study included socio-demographic characteristics (age, sex, residence, marital status, living arrangement, education level, and employment status) and information on imatinib toxicity and adherence. A physical examination was performed for signs of imatinib toxicity or disease regression or progression, including spleen size (if palpable). After the interview, blood samples were collected in EDTA tubes to run a full blood panel on a hematology analyzer (Dymind DH 76) and PCR for BCR-ABL transcripts on the Gene Xpert diagnostic system, version 4.4a. Data collected during the 22-month follow-up included time and cause of death.

Variables and measurements

CML disease phases were assessed according to 2016 World Health Organization criteria as chronic phase: bone marrow blasts <10% accelerated phase: blasts 10% to 19% or peripheral blood basophils ≥20% and blast crisis: blasts ≥ 20%. 14

CHR was defined as WBC <10 × 10 9 /L with no immature granulocytes on peripheral blood, <5% basophils on differential, platelet count <450 × 10 9 /L, and no palpable spleen. 15

Molecular response was defined as magnitude of reduction of BCR-ABL transcripts from the original value of 100%. Molecular response was categorized into 3 groups as per ELN 2020 guidelines. 15 (1) Optimal molecular response: at 3 months, BCR-ABL ≤10% at 6 months, BCR-ABL ≤1% and at ≥12 months, BCR-ABL ≤0.1%. (2) Warning: at 3 months, BCR-ABL >10% at 6 months, BCR-ABL >1% to 10% and at ≥12 months, BCR-ABL >0.1% to 1%. (3) Failure: at 3 months, BCR-ABL >10% if confirmed within 1 to 3 months at 6 months, BCR-ABL >10% and at 12 months and anytime BCR-ABL is >1%.

Adherence was defined as the extent to which a patient followed the clinician’s instructions on daily imatinib ingestion and was assessed with the 8-item Morisky Medication Adherence Scale (MMAS-8), as previously described. 16,17 Adherence was categorized into 3 groups: low adherence, score 0 to <6 medium adherence, score 6 to <8 and high adherence, score 8.

Imatinib-induced myelotoxicity was defined per the National Cancer Institute Common Terminology Criteria for Adverse Events (NCI-CTCAE) version 5.0 18 : neutropenia grade 1, absolute neutrophils <2.0 × 10 9 /L to 1.5 × 10 9 /L grade 2, <1.5 × 10 9 /L to 1.0 × 10 9 /L grade 3, <1.0 × 10 9 /L to 0.5 × 10 9 /L and grade 4, <0.5 × 10 9 L. Thrombocytopenia: grade 1, <150 × 10 9 /L to 75 × 10 9 /L grade 2, <75 × 10 9 /L to 50 × 10 9 /L grade 3, <50 × 10 9 /L to 25 × 10 9 /L and grade 4, <25 × 10 9 /L. Anemia: grade 1, <12 to 10 g/dL grade 2, <10 to 8 g/dL and grade 3, <8 g/dL.

Statistical analysis

Descriptive statistics were used to summarize sociodemographic data and clinical and treatment-related characteristics. Bivariate multinomial logistic regression was conducted to determine the association of response to imatinib treatment with independent variables. All variables with P <0.25 in bivariate multinomial logistic regression were included in the multivariate, multinomial logistic regression. Survival probabilities were estimated using the Kaplan-Meier method and the log-rank test was used to estimate the statistical significance of survival functions among the 3 ELN treatment response categories: optimal, warning, and failure. Values with P < .05 were considered statistically significant.

Ethical considerations

The ethical clearance for the study was obtained from the Senate Research and Publication Committee of Muhimbili University of Health and Allied Sciences (MUHAS). Permission to conduct the study was obtained from the authorities and the administration of ORCI. Written informed consents were obtained from the patients before recruitment, in accordance with the Declaration of Helsinki.


The only curative treatment for CML is a bone marrow transplant or an allogeneic stem cell transplant. [15] Other than this there are four major mainstays of treatment in CML: treatment with tyrosine kinase inhibitors, myelosuppressive or leukopheresis therapy (to counteract the leucocytosis during early treatment), splenectomy and interferon alfa-2b treatment. [15]

Chronic phase

In the past, antimetabolites (e.g., cytarabine, hydroxyurea), alkylating agents, interferon alfa 2b, and steroids were used as treatments of CML in the chronic phase, but since the 2000s have been replaced by Bcr-Abl tyrosine-kinase inhibitors [16] drugs that specifically target BCR-ABL, the constitutively activated tyrosine kinase fusion protein caused by the Philadelphia chromosome translocation. Despite the move to replacing cytotoxic antineoplastics (standard anticancer drugs) with tyrosine kinase inhibitors sometimes hydroxyurea is still used to counteract the high WBCs encountered during treatment with tyrosine kinase inhibitors like imatinib in these situations it may be the preferred myelosuppressive agent due to its relative lack of leukemogenic effects and hence the relative lack of potential for secondary haematologic malignancies to result from treatment. [17] IRIS, an international study that compared interferon/cytarabine combination and the first of these new drugs imatinib, with long-term follow up, demonstrated the clear superiority of tyrosine-kinase-targeted inhibition over existing treatments. [18]


The first of this new class of drugs was imatinib mesylate (marketed as Gleevec or Glivec), approved by the U.S. Food and Drug Administration (FDA) in 2001. Imatinib was found to inhibit the progression of CML in the majority of patients (65–75%) sufficiently to achieve regrowth of their normal bone marrow stem cell population (a cytogenetic response) with stable proportions of maturing white blood cells. Because some leukemic cells (as evaluated by RT-PCR) persist in nearly all patients, the treatment has to be continued indefinitely. Since the advent of imatinib, CML has become the first cancer in which a standard medical treatment may give to the patient a normal life expectancy. [19]

Dasatinib, nilotinib and radotinib

To overcome imatinib resistance and to increase responsiveness to TK inhibitors, three novel agents were later developed. The first, dasatinib, blocks several further oncogenic proteins, in addition to more potent inhibition of the BCR-ABL protein, and was initially approved in 2007 by the US FDA to treat CML in patients who were either resistant to or intolerant of imatinib. A second new TK inhibitor, nilotinib, was also approved by the FDA for the same indication. In 2012, Radotinib joined the class of novel agents in the inhibition of the BCR-ABL protein and was approved in South Korea for patients resistant to or intolerant of imatinib. In 2010, nilotinib and dasatinib were also approved for first-line therapy, making three drugs in this class available for treatment of newly diagnosed CML.

Treatment-resistant CML

While capable of producing significantly improved responses compared with the action of imatinib, neither dasatinib nor nilotinib could overcome drug resistance caused by one particular mutation found to occur in the structure of BCR-ABL known as the T315I mutation. Two approaches were developed to the treatment of CML as a result.

In 2007, Chemgenex released results of an open-label Phase 2/3 study (CGX-635-CML-202) that investigated the use of a non BCR-ABL targeted agent omacetaxine, administered subcutaneously (under the skin) in patients who had failed with imatinib and exhibited T315I kinase domain mutation. [20] [21] This is a study which is ongoing through 2014. [22] In September 2012, the FDA approved omacetaxine for the treatment of CML in the case of resistance to other chemotherapeutic agents. [23] [24]

Independently, ARIAD pharmaceuticals, adapting the chemical structures from first and second-generation TK inhibitors, arrived at a new pan-BCR-ABL inhibitor which showed (for the first time) efficacy against T315I, as well as all other known mutations of the oncoprotein. The drug, Ponatinib, gained FDA approval in December 2012 for treatment of patients with resistant or intolerant CML. Just as with second generation TK inhibitors, early approval is being sought to extend the use of Ponatinib to newly diagnosed CML also. [ citation needed ]

Due to the high median age of patients with CML it is relatively rare for CML to be seen in pregnant women, despite this, however, chronic myelogenous leukemia can be treated with relative safety at any time during pregnancy with Interferon-alpha hormones. [25]


In 2005, encouraging but mixed results of vaccination were reported with the BCR/abl p210 fusion protein in patients with stable disease, with GM-CSF as an adjuvant. [26]


The advent of tyrosine kinase inhibitors redefined the treatment of chronic myeloid leukemia (CML) [ 1 ]. Complete cytogenetic response rates can now be seen in up to 90% of newly diagnosed patients in chronic phase (CP) [ 2 ]. However, these “targeted” therapies have yet to prove curative. In fact, relapse is common after discontinuation of imatinib, even for patients in complete molecular remission at the time of cessation [ 3 , 4 ]. This appears to be due to resistance of CML stem cells to the pro-apoptotic effects of imatinib [ 5 , 6 ] and even newer tyrosine kinase inhibitors, such as dasatinib [ 7 ]. Indeed, primitive leukemic progenitors still can be readily detected in CML patients who have achieved complete cytogenetic remission on imatinib [ 8 ]. Blast crisis (BC) CML presents an even greater challenge, where in contrast to CP CML, tyrosine kinase inhibition rarely results in durable remission [ 9 ]. Hence, there has been a search for other therapeutic targets in CML, particularly on the leukemia stem cell (LSC), which in theory must be eradicated to achieve cure [ 10-12 ].

A number of candidates have emerged as potential therapeutic targets in leukemia. Among the more promising are: the Wilm's tumor gene (WT1), SURVIVIN, the preferentially expressed antigen of melanoma (PRAME), PROTEINASE 3 (PR3), and hTERT (the enzymatic component of telomerase). All of these are immunogenic, and each is over-expressed to varying degrees in many cancers, including CML [ 13 ]. Many of these candidate targets have been implicated in therapeutic resistance, including inhibition of apoptosis, and appear to correlate with prognosis [ 13-15 ]. There are ongoing vaccine trials targeting many of these antigens [ 13 , 16 ], as well as early phase clinical trials of pharmacologic inhibitors of telomerase [ 17 ] and SURVIVIN [ 18 ]. Presumably, any such new therapies will have curative potential only if their targets are actually expressed by the LSC. However, the expression of these putative targets in CML stem cells is largely unknown.

Indeed, existing data are limited regarding the precise characterization of CML stem cells and expression of a gene by the differentiated leukemic bulk does not necessarily guarantee expression by the LSC. In fact, in many respects LSC more closely resemble normal hematopoietic stem cells (HSC) than their own differentiated leukemic progeny [ 19-21 ]. Nonetheless, it is expected that qualitative or quantitative expression of some genes must distinguish LSC from their normal counterparts. Accordingly, an optimal therapeutic target would not only be highly expressed by the LSC (and ideally their progeny as well) but it would also be absent or only minimally expressed in normal HSC to avoid unacceptable toxicity [ 13 ].

LSC research to date has been impeded by the relative rarity of these cells, as well as the lack of a consensus on their exact phenotype. LSC are often phenotypically defined as simply the CD34 + leukemia cells or, occasionally, the more enriched CD34 + CD38 − subset but even the CD34 + CD38 − cells are a heterogeneous population, of which the LSC constitute only a fraction [ 19 , 22-24 ]. The CD34 + CD38 − population can be further refined for stem cells based on low side scatter and high aldehyde dehydrogenase (ALDH) activity [ 23 ]. ALDH, specifically the ALDH1A1 isoenzyme, mediates the biosynthesis of all-trans-retinoic acid, as well as the detoxification of a variety of compounds such as ethanol and active metabolites of cyclophosphamide [ 25 ] and it is typically present at higher levels in adult stem cells, than in their differentiated progeny [ 22-27 ]. The fluorescently labeled ALDH1A1 substrate, Aldefluor, permits the isolation of viable normal and cancer stem cells [ 22-24 , 26-28 ].

Here we report that CML stem cells are characterized by high ALDH expression, and that these cells have a unique expression profile of putative targets as compared to both the more differentiated CML progenitors and normal HSC.


Previous studies that describe putative binding partners for Bcr have provided relatively little information about its cytoplasmic function. The NH2-terminus of Bcr interacts with and phosphorylates at least five isoforms of the 14-3-3 proteins ( 44). Because different members of the 14-3-3 family have been implicated in a variety of cytoplasmic activities, including apoptosis, signal transduction, trafficking, and secretion, the meaning of these interactions remains unclear. Bcr contains a COOH-terminal PDZ-binding domain through which it is reported to interact with several cytoplasmic proteins, including AF-6, PDZK1, and Mint3 ( 45, 46). AF-6 has been colocalized with tight junctions and adhesion junctions and is thought to mediate the interaction between the plasma membrane and the actin cytoskeleton ( 46). PDZ-K1 is also a plasma membrane–associated protein that is predominantly found in association with apical membrane proteins in polarized epithelia ( 45). Although these associations are consistent with the presence of Rho-modulating domains within Bcr, most Bcr immunoreactivity is found on intracellular membranes and none is readily observed on the plasma membrane. This suggests that these associations are either transient or highly cell specific. Interestingly, the binding partner for Bcr that has the most similar cellular distribution is Mint3 ( 45). Mint3 is found primarily in the Golgi compartment where it has been implicated in protein processing and vesicular trafficking in the distal secretory pathway. Mint3 has a punctate cytoplasmic staining that partially overlaps with Bcr and it has been previously proposed that Bcr may have a role in cellular trafficking ( 45).

In the current study, we have identified two structurally unrelated subunits of the endosomal sorting machinery as binding partners for Bcr. Bcr interacts with TSG101 and Vps28 in HeLa and 293T cells, and Bcr and TSG101 exhibit a similar subcellular distribution in these cell types. The association of Bcr with both TSG101 and Vps28 suggests a very specific role for Bcr in the endosomal pathway. In eukaryotic cells, transmembrane proteins can be removed from the cell surface by endocytosis, followed by lysosome-mediated degradation. On the limiting membrane of the multivesicular body, proteins that are being transported in this pathway are sorted into vesicles that first bud into the lumen of the compartment and then are delivered to the lysosome ( 34). Although the mechanism by which cargo is sorted as it passes through the multivesicular body is poorly understood, there is evidence to suggest that monoubiquitination is used as the flag that targets proteins into this pathway ( 28). The formation of the multivesicular body and the sorting of ubiquitinated proteins is controlled by a large collection of proteins that was originally identified in yeast as the class E Vps (reviewed in ref. 47). Because both TSG101 and Vps28 are class E Vps proteins in yeast and mammalian cells, we propose that Bcr may also be a member of this class.

Vps proteins are organized into three discrete complexes, termed ESCRT-I, ESCRT-II, and ESCRT-III, that are recruited from the cytoplasm to act sequentially at the surface of the multivesicular body ( 28, 39, 48). ESCRT-I is thought to recognize ubiquitinated proteins and trigger the assembly and activation of ESCRT-II. ESCRT-II, in turn, is required for the recruitment of ESCRT-III whose role is to concentrate the multivesicular body cargo for internalization and remove the ubquitination tag. Once sorting is completed, Vps4 binds to ESCRT-III and disassembles the complex ( 37, 38). The Vps proteins are highly conserved from yeast to mammals, and mutations in the mammalian orthologues exhibit phenotypes consistent with defects in endosomal trafficking. For most components of the ESCRT complexes, one or more mammalian counterparts have now been identified. Because the mammalian counterpart of the ESCRT-I complex contains both TSG101 and Vps28 (reviewed in ref. 34), our binding data suggests that Bcr is either a component or a regulator of this complex. Whether Bcr also binds directly to Vps28 in this complex is unclear. Because it has been shown previously that TSG101 interacts with mammalian Vps28 in HeLa cells, our immunoprecipitations with the Vps28 antibody may be detecting Bcr indirectly through its association with TSG101 ( 36). To resolve this issue, in vitro studies are currently under way to examine the ability of Bcr to directly interact with the various components of the mammalian ESCRT complexes. However, regardless of whether the interaction with Vps28 is direct, the existence of a complex containing Bcr, TSG101, and Vps28 in mammalian cells strongly suggests that Bcr is a bona fide component of the mammalian endosomal sorting machinery.

Support for a role for Bcr in endosomal sorting also comes from our functional studies. In mammalian cells, the ESCRT-I complex is required for the down-regulation of growth factor receptors ( 27, 35, 42, 43). The complex is recruited from the cytoplasm to the multivesicular body in response to growth factor stimulation where it identifies and routes the internalized receptors to the lysosome. TSG101 binds directly to ubiquitinated proteins and thus may serve to recognize the targeted receptors ( 28, 35). Because the interaction between TSG101 and Bcr does not require ubiquitination, it is unlikely that Bcr is simply a preferred endosomal cargo in this pathway that is being recognized by TSG101. Once the receptor internalization is complete, the ESCRT complexes are disassembled from the limiting membrane of the multivesicular body in response to the action of the Vps4 ATPase. Interference of TSG101 through siRNA or Vps28 by neutralizing antibodies causes an accumulation of the EGFR on the cell surface in response to treatment with EGF ( 35, 49). Similarly, we have observed that siRNAs directed against both TSG101 and Bcr cause equivalent accumulations of EGFR on the cell surface. Thus, the association of Bcr with a complex that includes TSG101 and Vps28 seems to have functional relevance with regard to the efficient turnover of growth factor receptors.

Despite the fact that our binding studies suggest that Bcr interacts with ESCRT complexes, we do not observe a good convergence of signal between either TSG101 or Bcr, and a marker for the late endosomal membrane (LAMP-I). Because ESCRT complexes cycle between the cytoplasm and the limiting membrane of the late endosome, we assume that the majority of the TSG101-Bcr complexes in HeLa cells are in the soluble, resting state. Our observation that the dominant-inhibitory Vps4 mutant causes both TSG101 and Bcr to accumulate on densely staining cytoplasmic foci suggests that at least some of these complexes are being cycled onto membranes and can be trapped by the action of the mutant before membrane dissociation.

Our observation that Bcr interacts with subunits of the ESCRT complexes raises the possibility that endosomal trafficking may be impaired in CML. Although the interaction between Bcr and TSG101 is still observed in K562 cells that express p210 Bcr-Abl, we have also observed that incomplete suppression of either Bcr or TSG101 by siRNA is sufficient to impair growth factor receptor turnover in HeLa cells. Thus, the reduced dosage of Bcr that occurs in Philadelphia chromosome–positive cells may in fact be sufficient to cause impairment in trafficking. Alternatively, p210 Bcr-Abl may exhibit a dominant-inhibitory effect with respect to the normal endosomal function of Bcr. Although the TSG101 docking site is lost in p210 Bcr-Abl, the NH2 terminus of Bcr may interact with additional components of the sorting complex, such as Vps28. Thus, p210 Bcr-Abl may be recruited to the endosome and interfere with the normal function of Bcr. Both of these possibilities are currently under investigation.

Watch the video: Chronic Myeloid Leukemia CML. A Myeloproliferative Neoplasm MPN. Philadelphia Chromosome (January 2023).