1. Case 291 Ana L. Ruano, Juraj Bodo, Megan O. Nakashima, Eric D. Hsi
Department of Clinical Pathology
Cleveland Clinic Foundation

2. Clinical History Previously healthy 53 year old man.
Three day history of a generalized petechial rash, hematuria and rectal bleeding following a flu-like illness.
WBC = 8.85 K/UL; Hgb=12.5 g/dL;
Plt= 40 K/UL.
Laboratory studies showed evidence of disseminated intravascular coagulation.

3. Peripheral blood, 1000x
Peripheral blood, 1000x
Bone marrow aspirate, 1000x
Bone marrow aspirate, 1000x

4. Cytochemical Stains
α-naphthyl butyrate esterase
Myeloperoxidase

5. Bone marrow biopsy, 100x
1000x

7. Interphase FISH for PML-RARA (dual fusion probes): Positive
Karyotype: 47,XY,+8,t(15;17)(q24;q21)[20]

8. Acute promyelocytic leukemia with (t15;17)(q22;q12)/PML-RARA.
Proposed Diagnosis
Acute promyelocytic leukemia with (t15;17)(q22;q12)/PML-RARA.
CONSENSUS DX: Acute promyelocytic leukemia
with t(15;17)(q22;q12)
PML-RARA

9. Clinical History (cont.)
Initially treated with systemic corticosteroids, vitamin K, platelets, fresh frozen plasma and cryoprecipitate.
Upon diagnosis of acute promyelocytic leukemia, ATRA was initiated.

10. Increasing shortness of breath, low SaO2, development of altered mental status and left hemiplegia. Head CT showed intraparenchymal hemorrhage.
Bilateral multifocal opacities on CXR; systemic corticosteroids initiated for apparent “differentiation syndrome”.
Clinical condition rapidly deteriorated. Brain death declared; patient expired shortly after removal from ventilator.
The next day the patient developed…

11. Acute Promyelocytic Leukemia
Time to diagnosis  prognosis
Expedient methods of diagnosis may significantly improve patient outcomes
This case exemplifies the seriousness of acute promyelocytic leukemia and the need for prompt diagnosis in order to start therapy as soon as possible. Although FISH is currently the mainstay of diagnostic confirmation, it is technically intensive and not available on a STAT basis in all intitutions. The need is clear for a simpler, faster method for diagnosis.

12. Patient sample
Negative control (HL60)
Proximity Ligation Assay: PML and RARA primary antibodies for detection of PML-RARA fusion protein
Positive control (NB4)
With this need in mind, we would like to share the application of a new, efficient protein-based method for the diagnosis of APL. It’s known as a proximity ligation assay. In this instance, primary antibodies separately bind to PML and RARA proteins. When in proximity, such as in the fusion protein, a chromogenic signal is produced. On the left, the brown chromagen indicates the abundance of PML-RARA protein in the neoplastic promyelocytes. On the right, AML cell lines serve as convenient negative and positive controls.

13. Proximity Ligation Assay
Method for co-localizing any two biologic features that can be targeted by antibodies
Formats
Homogenous mixtures
Solid phase
Localized (in situ)
The proximity ligation assay is not restricted to the diagnosis of APL. In its general principle, it is simply a method for co-localizing any two biologic features that can be targeted by an antibody. It is versatile in that it performs well in homogeneous sample mixtures, on a solid phase, and in situ. The latter is particularly useful for pathologic evaluation of cells and tissues, as we’ve seen, and may conceivably supplant FISH in some applications.

14. Applications Detect and quantify single protein expressionB
Detection of post-translational protein modifications (eg. phosphorylation, mucin glycoforms)
Detect and quantify protein interactions
Duolink Brightfield II User Manual Olink Bioscience Uppsala, Sweden

15. Procedure Primary antibodies PLA probes Ligation solution 1 2 3
Primary antibodies bind
PLA nucleic acid probes bind and are ligated into a circular template 3
Ligation solution
Duolink Brightfield II User Manual Olink Bioscience Uppsala, Sweden

16. Rolling circle amplification (RCA)4
Rolling circle amplification (RCA)
Rolliing circle amplification results in local amplification of the signal, and a chromogenic detection method is used.
Detection -HRP 5
Duolink Brightfield II User Manual Olink Bioscience Uppsala, Sweden

17. Patient sample
Negative control (HL60)
Proximity Ligation Assay: PML and RARA primary antibodies for detection of PML-RARA fusion protein
Positive control (NB4)
With this need in mind, we would like to share the application of a new, efficient protein-based method for the diagnosis of APL. It’s known as a proximity ligation assay. In this instance, primary antibodies separately bind to PML and RARA proteins. When in proximity, such as in the fusion protein, a chromogenic signal is produced. On the left, the brown chromagen indicates the abundance of PML-RARA protein in the neoplastic promyelocytes. On the right, AML cell lines serve as convenient negative and positive controls.

18. Advantages Amenable to bright field microscopy
Fast - Results in approximately 6 hours
Easy to quantify – use of image analysis
Single cell analysis possible
Independent of PML gene break point

19. Conclusion
The case illustrates the application of a an alternate technology to test for fusion proteins via an easily interpretable bright field method potentially adaptable to the clinical laboratory.

20. References
Bodo J, Lin JJ, Maciejewski JP, Schade AE, Hsi ED. Detection of PML/RARA Fusion Protein in APL Using Proximity Ligation Assay as an Alterative to FISH Testing Annual meeting of United States and Canadian Academy of Pathology, Baltimore, MD.
Duolink Brightfield II User Manual Olink Bioscience Uppsala, Sweden
Gustafsdottir SM et al. Proximity Ligation Assays for Sensitive and Specific Protein Analysis Anal Biochem. 345(1):2-9.
Zieba A et al. Bright-field microscopy visualization of proteins and protein complexes by in situ proximity ligation with peroxidase detection Clin Chem 56(1):99-110
Gullberg M et al. A sense of closeness: protein detection by proximity ligation Curr Opin Biotechnol 14(1):82-6.
Koos B et al. Analysis of Protein Interactions in situ by Proximity Ligation Assays Current Topics in Microbiology and Immunology Springer-Verlag Berlin Heidelberg 2013.
Figueiredo J et al. The importance of E-cadherin binding partners to evaluate the pathogenicity of E-cadherin missense mutations associated to HDGC Eur J Hum Genet;21(3):301-9.
Pinto R et al. Identification of new cancer biomarkers based on aberrant mucin glycoforms by in situ proximity ligation J Cell Mol Med Jul;16(7):
Leuchowius KJ et al. High content screening for inhibitors of protein interactions and post-translational modifications in primary cells by proximity ligation Mol Cell Proteomics Jan;9(1):
Chen TC et al. Protein phosphorylation profiling using an in situ proximity ligation assay: phosphorylation of AURKA-elicited EGFR-Thr654 and EGFR-Ser1046 in lung cancer cells PLoS One ;8(3):e55657.
Aubele M et al. In situ quantification of HER2-protein tyrosine kinase 6 (PTK6) protein-protein complexes in paraffin sections from breast cancer tissues Br J Cancer 103(5):663-7.