Recurrent Loss of Heterozygosity in Pancreatic Neuroendocrine Tumors

Abstract

Chromosomal aneuploidies are prognostic markers across a wide variety of tumor types, and recent literature suggests that pancreatic neuroendocrine tumors are no different. In this study 214 patients with grade 1, 2, or 3 pancreatic neuroendocrine tumors had their tissue examined for chromosomal copy number alterations using next-generation sequencing. Univariate and multivariate statistical analyses were performed with all-cause mortality and disease-specific mortality as the end comparators. As such, the cohort stratified into 3 different clinically relevant chromosomal subgroups: an indolent subgroup characterized by loss of chromosome 11 in relative isolation, an aggressive subgroup characterized by losses of chromosomes 1, 2, 3, 6, 10, 11, 16, and 22 and with no loss of chromosomes 4, 5, 7, 12, 14, 17, 19, and 20, and finally a heterogeneous third group with a subset of cases that behave even more aggressively than the aforementioned.

Figure 1:. Visual Fingerprints of Recurrent Chromosome Loss and more objective 8+8 Signature Criteria

A) Copy number analysis is plotted per convention using the Log-ratio (LogR) of sample reads to expected reads [28]. The Y-axis for all graphs is LogR. The X axis for all graphs are the 23 chromosomes starting with autosomes in ascending order and terminating with X at right most position. Y is not plotted in males and X is normalized using sex-appropriate comparators. Mathematically, a region without gain or loss of genetic material will have a LogR of 0, which is indicated on the plot with a thick red line. Losses of chromosomes and sub-chromosomal regions will have a negative value, plotted below 0, and gains will have a positive value, plotted above 0. Although the axes are unlabeled, copy number analysis from eight different patients with the visual fingerprint of recurrent chromosome losses are pictured for a “low-power view” of the pattern of aneuploidies. A schematic representation of the visual fingerprint is presented on the bottom with all 22 autosomal chromosomes labeled and a red line at LogR=0. B) An example case with the visual fingerprint of chromosomal loss has been scored using 8+8 criteria. The LogR at 0 is indicated with a thick red line. Also, as above, the X-axis is the 23 chromosomes, autosomes first, in ascending order. Chromosome 23 is the X chromosome. And as above, the Y-axis is the log-transformed copy number measurements. For a case to meet criteria as having an “8+8 signature” it should have whole chromosome losses of chromosomes 1, 2, 3, 6, 10, 11, 16, and 22 while having no losses of chromosomes 4, 5, 7, 12, 14, 17, 19, and 20. To more objectively score, 1 point is assigned for each of the 16 listed chromosomes that matches expected criteria. All cases with a score of 14 or higher are considered positive for the signature. In this example case the patient had all 8 losses (diagramed below in red) and all 8 gains (diagramed in blue below) resulting in a score of 8+8=16, thus this case meets criteria for “8+8 signature.” If one of the chromosomes had not been lost, or had been lost where it should have been preserved, the patient would only receive a score of 7+8=15. Any chromosomes outside of the 16 listed, do not contribute to the assignment of an “8+8 signature”.

Figure 2:. Kaplan-Meier Survival Curve for All-Cause Mortality by PanNET Sub-groups.

The left Kaplan-Meier survival curve for all-cause mortality is stratified by pancreatic neuroendocrine tumor sub-group: mpNET1 (n=115), mpNET2 (n= 9), and mpNET3 (n=90). As in Lawrence et. al. mpNET2 is indolent with 100% survival in our small cohort. n.r. = not reached. mpNET1 tumors have an intermediate prognosis -- significantly better than the molecularly heterogeneous mpNET3 group (adjusted p=0.03), but trending toward significantly shorter survival than mpNET2 tumors (p=0.09). The right Kaplan-Meier survival curve for disease-specific mortality is again stratified by pancreatic neuroendocrine tumor sub-group. As discussed, mpNET2 has 100% survival. mpNET1 tumors again have an intermediate prognosis -- significantly better than the molecularly heterogeneous mpNET3 group (adjusted p=0.01), and significantly shorter survival than mpNET2 tumors (p=0.04).

Figure 3:. Genomic Features of mpNET1, mpNET2 and mpNET3 and Potential Non-NGS Workflow

This figure is adapted from Lawrence et. al. Figure 6. At left is a schematic depiction of cohorts. Chromosomal aneuploidy was used to separate the three PanNET subgroups. These aneuploidies separated very well with single gene mutations or focal losses in MEN1, DAXX, and ATRX and with overall survival in agreement with the known literature and index paper. mpNET1 is characterized by recurrent 10 chromosome monosomy, defined as an "8+8" signature in this paper. This chromosomal group had ATRX and DAXX mutations in 97% of samples and were often with MEN1 mutations, but not always. Their clinical outcome was a predictable slowly progressive course. mpNET2 is characterized by loss of chromosome 11 in relative isolation in conjunction with MEN1 mutations. Their clinical course is extremely indolent. mpNET3 is our 'wastebasket group' with variable aneuploidies and variable mutations. A not insignificant subset had MEN1 mutations (12%) but this group rarely had DAXX and ATRX mutations. Statistically our mpNET3 cases had worse overall and disease-specific survival compared to mpNET1. However, given the collection bias for our samples, we are not confident that this is generalizable to the broad population. Additionally, twice the number of mpNET3 patients are disease free compared to mpNET1. Given these limitations we have labeled this group as having "variable" outcome, specifically noting that a subset behave very poorly, more poorly than mpNET1. At right, a possible workflow, that does not require NGS, is illustrated. Low-grade pancreatic neuroendocrine tumors (notably grade 2 – which were present in all three mpNET groups) may benefit most from additional immunohistochemical stains and FISH analysis, as chromosome group does stratify with grade. Sensitivity and specificity of ATRX IHC in detecting ATRX mutations in panNET is still an area of required investigation. Additionally, the exact FISH probes and number of FISH probes required to confidently call isolated loss of chromosome 11 is an area of necessary future study.