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Year : 2021  |  Volume : 64  |  Issue : 2  |  Page : 406-409
Cytogenetics and FISH negative cryptic acute promyelocytic leukemia with CD56 expression

1 Department of Oncopathology, Cancer Institute (WIA), Adyar, Chennai, Tamil Nadu, India
2 Department of Medical Oncology, Cancer Institute (WIA), Adyar, Chennai, Tamil Nadu, India

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Date of Submission21-Apr-2020
Date of Decision12-May-2020
Date of Acceptance27-Jul-2020
Date of Web Publication9-Apr-2021


Acute promyelocytic leukemia (APL) is characterized by reciprocal translocation t(15;17)(q22;q21) and has a favorable prognosis upon immediate recognition and treatment. However, rare cases of APL show a cryptic insertion of retinoic acid receptor alpha (RARA) gene into promyelocytic leukemia (PML) gene which is negative both by fluorescence in situ hybridization (FISH) and conventional cytogenetics (CC). Morphology, cytochemistry and flow cytometry play a key role in early identification of such cases. Polymerase chain reaction (PCR) remains the most efficient diagnostic modality for detection of cryptic APL and other variants. It is important to identify these cases as they show beneficial response to retinoids and favourable prognosis. We herein present a rare case of cryptic APL negative by FISH and conventional cytogenetics but positive for PML-RARA by PCR.

Keywords: Acute myeloid leukemia, acute promyelocytic leukemia, cryptic acute promyelocytic leukemia, promyelocytic leukemia-retinoic acid receptor alpha

How to cite this article:
Arumugam JR, Karthik Bommannan B K, Kalaiyarasi JP, Sundersingh S. Cytogenetics and FISH negative cryptic acute promyelocytic leukemia with CD56 expression. Indian J Pathol Microbiol 2021;64:406-9

How to cite this URL:
Arumugam JR, Karthik Bommannan B K, Kalaiyarasi JP, Sundersingh S. Cytogenetics and FISH negative cryptic acute promyelocytic leukemia with CD56 expression. Indian J Pathol Microbiol [serial online] 2021 [cited 2021 May 16];64:406-9. Available from: https://www.ijpmonline.org/text.asp?2021/64/2/406/313288

   Introduction Top

Acute promyelocytic leukemia (APL) is characterized by reciprocal translocation t(15;17)(q22;q21) leading to promyelocytic leukemia (PML) gene and retinoic acid receptor alpha (RARA) gene fusion in more than 90% of the cases.[1] A rapid response to all-trans retinoic acid (ATRA) that relieves the myeloid maturation arrest is a feature of the classical cases. Rapid diagnosis and immediate treatment is crucial in APL.[2] Unfortunately, rare cases of APL show a cryptic insertion of RARA gene into PML gene causing fluorescence in situ hybridization (FISH) and cytogenetically negative APL. Though the diagnosis in such cases can be established by polymerase chain reaction (PCR), morphology, cytochemistry and flow cytometry (FCM) plays a major role in making an early diagnosis, as the clinical challenge in such cases is in treating the patient with ATRA before the definitive diagnosis is established. We report a case of cryptic APL that was a diagnostic challenge.

   Case History Top

A 56-year-old female presented with generalized pruritis for 1 month, fever and headache for 1 week. On admission, patient had pancytopenia with haemoglobin 8 gm/dl, total count 700/mm3 and platelet count 96,000/mm3.

Bone marrow aspiration (BMA) showed 74% atypical cells which were large with abundant granular cytoplasm and some of them showed bi-lobed nuclei [Figure 1]a and [Figure 1]b. Myeloperoxidase (MPO) staining showed strong positivity [Figure 1]c. BMA was consistent with acute myeloid leukemia, suggesting APL. Bone marrow biopsy (BMB) showed sheets of promyelocytes [Figure 1d]. Flow cytometry showed 60% myeloid precursor cells expressing dim to moderate CD13, homogenous moderate CD33, moderate CD117, dim to moderate CD34, bright MPO, aberrant expression of CD56 and HLA DR was negative [Figure 2]. Based on morphology the patient was started on ATRA. PML RARA dual color dual fusion FISH and RARA break-apart FISH performed showed 100% nuclei negative for PML RARA fusion and RARA break-apart, respectively [Figure 3]. Conventional cytogenetics performed was normal, 46,XX[20/20] [Figure 4]. As cytogenetics and FISH were negative for PML RARA translocation, patient was started on AML induction therapy, a combination of Daunorubicin and Cytarabine (3 + 7). Meanwhile PCR showed positivity for PML-RARA fusion transcript, bcr1 isoform. A diagnosis of APL was made and the patient was restarted on ATRA from the 3rd day of induction. The patient developed respiratory distress, facial edema and arterial blood gas (ABG) analysis showed respiratory alkalosis. CT chest showed right lung lower lobe consolidation and was treated for probable differentiation syndrome. But due to the worsening respiratory distress, ATRA was witheld on sixth day. Soon the patient developed fever spikes with multi-organ dysfunction syndrome with worsening of liver and renal function and sepsis. Despite effective supportive care management, the patient developed refractory septic shock with encephalopathy and succumbed to the illness on the 12th day of induction.
Figure 1: (a and b): BMA shows promyelocytes (Leishman x 1000), (c) MPO Positivity (x 1000), (d) BMB shows sheets of promyelocytes

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Figure 2: Flow cytometry show atypical cell population (red) expressing CD34, CD117, CD56, CD13, CD33, cytoplasmic MPO and negative for HLAD

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Figure 3: (a) PML RARA DCDF FISH showing two green and two orange signals, negative for PML RARA fusion, (b) RARA break apart FISH showing two fusion signals, negative for RARA break apart

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Figure 4: Karyogram shows a normal karyotype

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   Discussion Top

About 10% of APL patients with successful cytogenetic analysis actually lack the classic t(15;17) which may not be detectable by routine FISH using standard probes.[3] Molecular analysis in approximately 92% patients of APL shows the balanced translocation t(15;17) (q24.1;q21.1) involving RARA gene on chromosome 17 and the PML gene on chromosome 15. However, 4% of patients do not have the classic t(15;17), but have the PML/RARA fusion gene due to a cryptic insertion of the RARA gene into the PML gene or vice versa, causing PMLRARA fusion without the reciprocal RARA/PML fusion. Such cases known as cryptic APL are not detected by conventional cytogenetics and FISH using standard probes but are diagnosed only by PCR. 2% of patients show simple variants/complex chromosomal rearrangements and 1% show no RARA rearrangement, detected only by PCR. In the remaining 1% cases, rearrangements of 17q21 lead to fusion of RARA to alternative partner genes, that is, PLZF (promyelocytic leukemia zinc finger), NPM (nucleophosmin), NuMA (nuclear mitotic apparatus), and STAT5b (signal transducer and activator of transcription) associated with t(11;17)(q23;q21), t(5;17) (q35;q12-21), t(11;17)(q13;q21), and der (17) respectively. Such cases can be identified by RARA break apart FISH or by PCR with specific primers for the variant translocation.[3],[4],[5],[6]

Treatment with ATRA and ATO (arsenic trioxide) in APL has completely changed its landscape with high remission rates and excellent long-term outcomes. Rapid diagnosis and immediate treatment is a requirement for favorable prognosis.[2] Hence morphology, cytochemistry and flow cytometry picture are crucial in making diagnosis and treatment decision before the arrival of PCR result. The importance of identifying the PML-RARA fusion protein by PCR is not only because the fusion protein causes differentiation block in APL, but in addition it is important for mediating the myeloid differentiation in response to ATRA. Hence, APL patients with cryptic PML-RARA fusion gene share the beneficial response to retinoids and the favorable prognosis associated with the group with documented t(15;17). This finding highlights the importance of establishing the presence of the PML/RARA rearrangement in patients with morphologic suspicion of APL, so that no APL patients miss the benefit of receiving differentiation agents.[7] In addition, patients with fusion genes involving NPM and NuMA appear to be sensitive to ATRA, whereas those associated with a PLZF/RAR rearrangement exhibit lack of differentiation response to retinoids.[4] Thus it is apparent that the nature of the fusion partner has a significant bearing upon disease characteristics, particularly the responsiveness to ATRA and ATO.

Cryptic insertion of RARA into PML cannot be demonstrated by FISH studies using a standard dual fusion probe but are readily identified by FISH using smaller probes. Probes of approximately 40 kb in size, 100× smaller than the standard probes helps in identification of cryptic fusion (we used standard FISH probe which is large, where the LSI RARA probe is approximately 827 kb in length, that spans either side of RARA locus by 410 kb and 417 kb and LSI PML probe that is approximately 517 kb in length which span either side of the PML locus by 239 kb and 278 kb). Very small RARA segment inserted into 15q24 allows the PML signal intensity to quench the RARA signal masking the fusion signal, whereas the less disparate intensities of the two signals using the smaller probes allows the fusion to be visualized. Hence, it is important to be aware of the false-negative FISH result using standard PML/RARA FISH probes to avoid missing a diagnosis of APL.[3],[8]

CD56 expression on promyelocytes in APL is rare and is also associated with poor prognosis and is an independent adverse prognostic factor for relapse. CD56 positive APL was significantly associated with high white blood cell counts; low albumin levels; bcr3 isoform and the co-expression of immaturity-associated and T-cell antigens like CD2, CD34, CD7, HLA-DR, CD15, and CD117 antigens.[9],[10] CD56 expression in the absence of standard FISH-detectable PML-RARA translocation, in the same patient is extremely rare.

   Conclusion Top

Cryptic APLs are negative by conventional cytogenetics and FISH using standard probes. It is essential to identify these cases as they show beneficial response to ATRA. PCR remains the most efficient diagnostic modality for detection of cryptic APL and other variants. All patients with suspected APL should have a morphological diagnosis followed by both FISH and PCR to make sure none of these rare variants are missed out causing delay in treatment.

Declaration of patient consent

The authors certify that they have obtained all appropriate patient consent forms. In the form the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.


Dr. Teena Koshy, Consultant Cytogeneticist, Department of Oncopathology, Cancer Institute (WIA).

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

   References Top

Amare PK, Baisane C, Nair R, Menon H, Banavali S, Kabre S, et al. Characterization of cryptic rearrangements, deletion, complex variants of PML, RARA in acute promyelocytic leukemia. Indian J Hum Genet 2011;17:54-8.  Back to cited text no. 1
[PUBMED]  [Full text]  
Rashidi A, Fisher SI. FISH-negative, cytogenetically cryptic acute promyelocytic leukemia. Blood Cancer J 2015;5:e320.  Back to cited text no. 2
Grimwade D, Coco FL. Acute promyelocytic leukemia: A model for the role of molecular diagnosis and residual disease monitoring in directing treatment approach in acute myeloid leukemia. Leukemia 2002;16:1959-73.  Back to cited text no. 3
Grimwade D, Biondi A, Mozziconacci MJ, Hagemeijer A, Berger R, Neat M, et al. Characterization of acute promyelocytic leukemia cases lacking the classic t(15;17): Results of the European Working Party. Blood 2000;96:1297-308.  Back to cited text no. 4
Kim MJ, Yang JJ, Meyer C, Marschalek R, Park TS. Molecular methods for genomic analyses of variant PML-RARA or other RARA-related chromosomal translocations in acute promyelocytic leukemia. Korean J Hematol 2012;47:307-8.  Back to cited text no. 5
Singh MK, Parihar M, Arora N, Mishra DK, Bhave SJ, Chandy M. Diagnosis of variant RARA translocation using standard dual-color dual-fusion PML/RARA FISH probes: An illustrative report. Hematol Oncol Stem Cell Ther 2019;12:50-3.  Back to cited text no. 6
Grimwade D, Gorman P, Duprez E, Howe K, Langabeer S, Oliver F, et al. Characterization of cryptic rearrangements and variant translocations in acute promyelocytic leukemia. Blood 1997;90:4876-85.  Back to cited text no. 7
Campbell LJ, Oei P, Brookwell R, Shortt J, Eaddy N, Ng A, et al. FISH detection of PML-RARA fusion in ins (15;17) acute promyelocytic leukaemia depends on probe size. Biomed Res Int 2013;2013:164501.  Back to cited text no. 8
Montesinos P, Rayón C, Vellenga E, Brunet S, González J, González M, et al. Clinical significance of CD56 expression in patients with acute promyelocytic leukemia treated with all-trans retinoic acid and anthracycline-based regimens. Blood 2011;117:1799-805.  Back to cited text no. 9
Murray CK, Estey E, Paietta E, Howard RS, Edenfield WJ, Pierce S, et al. CD56 expression in acute promyelocytic leukemia: A possible indicator of poor treatment outcome? J Clin Oncol 1999;17:293-7.  Back to cited text no. 10

Correspondence Address:
Jhansi Rani Arumugam
Department of Oncopathology, Cancer Institute (W.I.A.), Adyar, Chennai - 600 036, Tamil Nadu
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/IJPM.IJPM_409_20

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