Importance of Fluorescence in Situ Hybridization (FISH) Analysis on Bone Core Specimen for Detection of Relapse in Myeloid Malignancies with Fibrosis
Authors: Chu Lee Voon , Lim Tse Hui , Than Hein , Tien Sim Leng , Lim Soon Tiong Alvin , Lau Lai Ching , Ho Aloysius Yew Leng , Quek Jeffrey Kim Siang , Inng Lim Francesca Lorraine Wei , Chen Yunxin , Hwang William Ying Khee , Nagarajan Chandramouli , Ng Lawrence Cheng Kiat , Tan Melinda Si Yun , Tuy Tertius Tansloan , Ang Chieh Hwee , Linn Yeh Ching 
Published: December 2024

Background and Aim:Patients with haematological disorders often require bone marrow aspirate (BMA) and trephine biopsy as part of diagnostics and assessment of disease relapse. Conventionally, marrow is assessed by morphology, immunophenotyping, cytogenetics, fluorescence in situ hybridization (FISH), and mutation analysis. However, in myeloid malignancies such as myelofibrosis or myelodysplastic syndrome (MDS) with fibrosis, BMA may be dry or haemodiluted without representative marrow fragments, leading to failure in analysis or inaccurate findings. In such instances, cut sections of bone core from trephine biopsy, besides providing histopathology, can be analysed for informative markers by FISH on the formalin-fixed paraffin-embedded blocks (FFPE-FISH).
Methods:This is a retrospective study on patients followed up in Singapore General Hospital Department of Haematology who had informative results from FFPE-FISH performed on marrow bone core. FISH probes that detect chromosomal aberrations common in MDS, including chromosomes 5q, 7q, 20q, 17p, and trisomy 8, were used in the panel. In addition, FISH for X and Y chromosomes (X/Y-FISH) was done for patients with gender-mismatched haematopoietic stem cell transplant (HSCT). The result of FFPE-FISH was compared with FISH on BMA samples (asp-FISH), karyotype analysis, and a variable number of tandem repeat (VNTR) analysis.
Results and Discussion:Ten HSCT patients had a total of 40 marrow samples taken at different timepoints. There was a total of 65 (39 MDS and 26 X/Y chromosome) FFPE-FISH tests performed. Fourteen BMA samples had both MDS and X/Y-FISH performed, 14 had only MDS FISH, while 12 had only X/Y-FISH done.
Of 28 marrow samples with MDS FFPE-FISH done, 11 (39.3%) had results discordant from corresponding BMA tests, of which 90.9% (n=10) had disease detectable by FFPE-FISH which were missed on either asp-FISH, karyotyping or both. Conversely, FFPE-FISH failed to detect the expected complex karyotype only in 1 instance. Among these 11 discordant samples, 7 (63.6%) were dry or haemodiluted. On the other hand, for the 17 concordant samples, a significant number (n=8, 47.1%) did not have detectable MDS markers. For those with true disease presence, most (62.5%) had adequate BMA samples making asp-FISH and FFPE-FISH comparable.
In the 26 FFPE-X/Y-FISH done on 7 patients with gender-mismatched HSCT, 22 (84.6%) had incomplete donor chimerism detected by FFPE-X/Y-FISH where other tests failed to. Amongst them, 11 had parallel asp-FISH done where 8 (72.7%) failed to detect incomplete chimerism (62.5% haemodiluted); 14 cases had successful karyotyping done where all missed the presence of recipient-gender metaphases (50% haemodiluted). Similarly, 100% (n=17) available VNTR analyses done (82.4% from BMA) erroneously reported complete donor chimerism.
When analysed in totality, 6 of the 10 patients (60%), and 24 out of 40 unique bone marrow specimens (62.5%) would have had disease relapse or incomplete donor chimerism missed if FFPE-FISH had not been performed on the bone core.
Conclusion:We have shown the usefulness of FFPE-FISH in the detection of disease relapse and falling donor chimerism post-HSCT, in the context of fibrotic or inadequate BMA samples. Our observation provides compelling evidence that FFPE-FISH should be performed in such patients with seemingly normal asp-FISH or karyotyping reports.