Tracking Subclonal Mutations in IGHV-Mutated CLL with Progressive Disease
Authors: Rose-Zerilli Matthew JJ , Jane Gibson , Wang Jun , Tapper William J , Parker Helen , Parker Anton , Davis Zadie , Gardiner Anne Catherine , Forster Jade , Steele Andrew J , Walewska Renata , McCarthy Helen , Collins Andrew , Oscier David G , Strefford Jonathan C 
Published: October 2019

Abstract
Most CLL is diagnosed with a low tumor burden with no indication for therapy. Biomarkers, such as unmutated IGHV genes, TP53 loss/mutation and raised β2M predict short time to first treatment and overall survival; however there remain patients with good risk biomarkers who nevertheless develop progressive disease. Advances in genomics and immunogenetics have lead to the discovery of new biomarkers and their integration with cytogenetic data refines outcome prediction. However these novel markers are predominantly found in IGHV unmutated cases (U-CLL).
To identify novel genetic mechanisms that may contribute to progression, we have studied 13 patients (pts) presenting with cMBL (n=3) or Stage Binet A/Rai 0 disease and good risk markers: IGHV-mutated, excluding poor risk stereotypes (n=13), no 17p or 11q deletion by FISH, (n=13), sole del13q14 (n=8), low CD38 expression (n=13) who all developed lymphocytosis (n=13), between two untreated timepoints (TP1 & TP2), 10 of whom subsequently required treatment.
Copy number analysis (SNP6), whole exome sequencing (WES; Agilent SureSelect & Illumina sequencing) of tumour-germline pairs and targeted deep sequencing (TDS; Haloplex, Agilent) of the the WES-identified variants and the 22 most frequently mutated genes in CLL, to a mean depth of 3681 fold, were performed at TP1 and TP2. TP1 was close to diagnosis (median of 1 yr, range 0.11-7.33) with a median time to TP2 of 4.5 yrs (0.2-8.9). In addition, TDS was undertaken at later time points in one patient described in point 4), who relapsed and ultimately transformed.
Our analysis shows the following potential mechanisms:
1. Our germline WES data revealed 5 heterozygous missense/frameshift variants in 5 genes in 5 pts, also known to be targeted by somatic mutation in CLL (eg: FBXW7, POT1, SAMHD1. Fig1).
2. We then established the somatically-acquired mutation profile of each patient. We validated 72% (224/312) of the mutations discovered by WES using TDS and identified clinically relevant mutations earlier on in disease, supporting the hypothesis that sub-clonal mutations in genes in addition to TP53 may drive a progressive clinical course. At diagnosis (TP1) by WES/TDS, 5/13 pts had mutations in CLL driver genes (ATM, NOTCH1, SF3B1, TP53) and 2/13 pts had mutations in genes of undetermined clinical significance (CHD2, NFKBIE, ZMYM3). One patient was MYD88 mutated at TP1 and remains untreated after follow up of 12 yrs. In total, the following 9 genes (ATM, CHD2, DDX3X, MYD88, NOTCH1, NFKBIE, SF3B1, TP53 & ZMYM3) were mutated in 62% (8/13) pts at TP1.
3. Of the remaining 5/13 pts lacking a detectable mutation in any of the established CLL genes, we observed on average 7 mutations/patient in genes involved in cancer and each patient harboured one or more mutated genes with a role in haematological malignancy (eg. ITGA6, KLHL6, LTF, TNFAIP3).
4. One patient exhibited a remarkable temporal shift in copy number changes and mutations. At TP2, SNP6 analysis could not detect the del13q observed at TP1, and a clonal trisomy 12 had emerged, along with several mutations associated with progressive disease (BIRC3, IRF4, NOTCH1), that predominate in U-CLL. As a consequence we re-analysed the IGHV mutational status at TP2, and showed that rather than the IGHV3-48 with 92% germline identity identified at diagnosis, our patient exhibited an additional and dominant IGHV5-10-1*01 (100% identity) clone at TP2, 8 yrs after TP1. Additional analysis of intermediate samples detected the unmutated clone as far back as 4 yrs post diagnosis, and TDS analysis showed the NOTCH1 mutation was a minor subclone at diagnosis (0.06% VAF). Ultimately, this patient developed Richters syndrome with expansion of the NOTCH1 mutation (27% VAF). Retrospective sequential immunogenetic analysis of the other 12 cases yielded no other example of this phenomenon.
In summary, IGHV-mutated cMBL/early stage CLL with a progressive outcome can be associated with, the presence of germline or subclonal gene mutations of known or putative importance in CLL, or the emergence of a IGHV-unmutated clone. Our data supports deep sequencing in the clinical setting for earlier detection of pathogenetic mutations and emerging immunogenetically distinct subclones in patients with early stage 'good risk' disease.
Figure 1: Heatmap representation of the cohorts clinical features and DNA mutation. Patient 287 haboured the IGHV-unmutated clone at TP2-5. Figure 1:. Heatmap representation of the cohorts clinical features and DNA mutation. Patient 287 haboured the IGHV-unmutated clone at TP2-5.

Disclosures
No relevant conflicts of interest to declare.