APML: laboratory diagnosis

Question 1

Overview of APML and lab diagnosis

Answer 1

• Intro to APML
• BG info of APML
• Lab tests of APML (I)
• Lab tests of APML (II)
• Genetics and pathophysiology of APML
• Treatment of APML
• Conclusion on APML

Question 2

Intro to APML

Answer 2

• Acute leukaemia is the unregulated, clonal expansion of immature cells of leucocyte lineage.
• Acute promyelocytic leukaemia (APL) is a sub-type of acute leukaemia involving myeloid cells at a specific point of maturation. It is defined by its specific pathophysiology and clinical picture (Also referred to as AML M3).
• The objective of this report is to outline and explain the role of laboratory testing in the diagnosis of APL and to elucidate the rationale of its treatment.
• This report will not be addressing Acute myeloid leukaemia in general but this specific sub-type.

Question 3

-BG info of APML

Answer 3

• APL is uncommon in children, but increases in incidence into early adulthood
• APL is strongly associated with an increased risk of Disseminated intravascular coagulation (DIC).
• Therefore, APL can present as DIC, where it is treated as a life-threatening emergency. The clinical features of which mainly include excessive bleeding from minor wounds, generalized bleeding from the gastrointestinal tract and various other sites, and microthrombi that lead to skin lesions
• Otherwise, the clinical features of APL are similar to acute leukaemias in general
• Some clinical features include general signs of malignancy: fever, sweats, weight loss
• Furthermore, there may be signs of anaemia, infection, bruising and bleeding caused by bone marrow failure.
• Shortness of breath and hepatomegaly may also be present, as a result of tissue infiltration by leukaemic cells.
• APL is defined by the mutation that causes it. This being the t(15;17) translocation, also known as the PML-RARA fusion gene.

Question 4

-Lab tests of APML (I)

Answer 4

• There are numerous lab tests involved in the diagnosis of APL:
• In the case of non-urgent, usual leukaemic symptoms, the diagnostic protocol is as follows:
• Firstly, a full blood count may be taken, which will most likely show an anaemia, neutropenia and thrombocytopenia due to bone marrow failure
• The next investigation that may be performed is a bone marrow aspirate, which shows blast cells with granules and multiple aur rods.
• The laboratory test most significant in diagnosing APL would be a cytogenetic analysis of a bone marrow biopsy. Visualising the chromosomes would show the exclusive causative mutation, t(15;17)/PML-RARA, in the case of APL. This approach to diagnosis is definitive, but relatively time consuming.
• Another way to demonstrate t(15;17), and therefore diagnose APL, is to use florescent in situ hybridisation (FISH), where a specific probe can detect the mutation much more time effectively than standard cytogenetic analysis.
• There are additional lab investigations that may be performed when investigating leukaemia. Sampling the leukaemic cells for flow cytometry is another technique that may be employed to diagnose APL, where CD33 and CD13 positive samples, indicate myeloid lineage. However, this is not a high priority test for diagnosis.

Question 5

-Lab tests of APML (II)

Answer 5

• Molecular genetic techniques are not typically used in clinical practice with APL, since the discreet mutation causing the disease is detectable by the more practical means already mentioned. However, it is possible to use real-time PCR to test bone marrow cells for the PML-RARA fusion gene
• However, in the clinical case of DIC, the diagnostic approach has to be much more time efficient, where as soon as there is abnormal clotting and a pancytopenia on blood count, the most time –effective diagnosis can be achieved through FISH.
• The overall diagnosis of APL, in the absence of DIC, is usually a full blood count showing pancytopenia, followed by a cytogenetic analysis of bone marrow cells, which would show the t(15;17)/PML-RARA translocation.
• However, in the case of emergency, where patients present with DIC, the technique of FISH would provide an accurate diagnosis in much less time than standard cytogenetic analysis, so this would be the definitive test to conduct as soon as possible.
• In summary, the role of laboratory investigations is of crucial importance in diagnosing APL, since the disease is defined by a single, exclusive causative mutation. Even though APL often presents as an emergency where time is a critical factor, laboratory testing is still of major importance in providing a definitive diagnosis before initiating targeted treatment.

Question 6

-Genetics and pathophysiology of APML

Answer 6

• In order to explain targeted treatment, it is important to understand the pathogenesis, which is very specific as APL is exclusively caused by the presence of a PML-RARA fusion gene.
• Retinoic acid receptor alpha (RARA) is a protein that potentiates transcription that allows cells to differentiate from immature precursors.
• RARA is bound to and inhibited by a nuclear co-repressor and histone deacetylase complex (NCoR-HD). This complex deacetylates histones in the region, which leads to the inhibition of transcription.
• Under normal conditions, Retinoic acid (RA) (derived from vitamin A) is able to bind to RARA and produce a change in confirmation that releases the NCoR-HD complex and initiate transcription and differentiation of immature cells.
• However, in the case of APL with t(15;17)/PML-RARA, the variant form of RARA remains bound to the NCoR-HD complex and RA (In normal concentrations) is unable to exert its effect, leaving RARA to be constantly inhibited, leading to a decrease in cell differentiation.

Question 7

Treatment of APML

Answer 7

• APL has its own treatment protocol that is unique to any other leukaemia
• In the case of DIC as an emergency, there are a number of actions to take
• A combination of conventional and targeted chemotherapy
• Supportive care measures must also be taken, to correct the coagulopathy and to counteract complications of chemotherapy; these include fresh frozen plasma for factor replacement and cryoprecipitate for fibrinogen.
• In terms of specific targeted therapy, all trans retinoic acid (ATRA) is used with arsenic trioxide.
• ATRA binds to the RARA-PML and a change in conformation leads to release of NCoR-HD complex, acetylation of histones and resumption of transcription.
• In the presence of high dose ATRA, the RARA-PML protein is able to recruit factors that contribute to increased cell differentiation
• Arsenic trioxide works synergistically with ATRA, as it is able to bind to the PML portion of the PML-RARA protein, which initiates the degradation of PML-RARA and induces apoptosis.

Question 8

-Conclusion on APML

Answer 8

• In conclusion, APL often presents as a state of emergency. A swift, accurate diagnosis is of crucial importance and laboratory testing, namely FISH, plays a fundamental role in this.
• APL treatment involves a combination of general chemotherapy and targeted drugs. The main targeted treatment is ATRA combined with arsenic, where the ATRA is able to correct the function of the defective RARA-PML protein, to increase cell differentiation back to a physiological level.