This study focuses on the complexity of the bladder cancer genome, and for the first time to our knowledge, on the possibility to compare genomic alterations of in vitro selected cancer stem-like cells and their original biopsy in order to identify different genomic signature already present in the early stages of tumorigenesis of LG and HG tumors. Despite the low number of cases enrolled and therefore, at the moment, the interpretation may be only hypothetical, the findings of this study may assume a very important significance to those with closely related research interest. Firstly, we performed a conventional karyotype on 20 biopsies (11 LG and 9 HG) in order to delineate the status of ploidy in bulk tumors. Despite this technique is far from being innovative, we observed that LG tumors generally have near diploid metaphases, while HG tumors have a tendency to triploidy with a greater dispersion in the number of chromosomes per cell. Furthermore, we revealed that only two samples out of nine, both non-invasive LG, show a strong tendency to Y chromosome loss. Conversely, the others maintain or, at most, show an opposite tendency to acquire multiple copies of this chromosome. Although the number of cases is very low, this observation is a little in contrast with a recent study showing that Y chromosome losses are equally frequent in urothelial bladder cancer of all grades and stages . Our data seem to agree with another study that showed a significant association of Y polysomies with HG invasive tumors . The overall observations obtained by conventional chromosomal analysis and FISH have confirmed a greater aggressiveness of HG tumors than LG ones; in addition, although a larger number of cases must be studied, we believe that the involvement of the Y chromosome has yet to be clarified and that it is no coincidence that men are more affected by this type of cancer .
In this work we present a comprehensive catalog of CNAs across 20 tumor fresh biopsies providing an overview of their common alterations. The overall data evidenced a general chromosomal instability, especially in HG tumors, with a general CNAs ratio of 4:1 respect to LG tumors, and a ratio of 10:1 considering only gains. A previous study by HR-CGH analysis had reached the same conclusion . In addition, in the present work we unveiled an opposite situation analyzing the genomic profiles of CSCs, as in HG tumors they were less affected than in LG tumors, with a general ratio of 1:3 (1:6 if we consider only the losses). To understand this anomalous behavior it would be useful to compare the genomic alterations of the original biopsy with their isolated cells because this approach may focus on the alterations most involved in tumorigenesis of TCCs. The overview of CNAs per chromosome (Figure 3) evidences a better conservation between cells and biopsies of HG tumors than LG tumors, even if isolated cells of HG group are less altered than their original biopsies while the situation is reversed in LG group. In two HG samples the complex pattern of CNAs involving chromosome 6 is consistent with “a chromothripsis like event”. Morrison et al. have recently reported the chromothripsis phenomenon in muscle-invasive TCCs . These authors postulated that chromothripsis is related to a defective replication-licensing complex and that it could lead to intratumoral mutational heterogeneity. However, a recent work suggests that more stringent criteria must be used to identify chromothripsis and that it cannot be distinguished from other complex genomic rearrangements . According to these authors, great caution should be exercised when labeling complex rearrangements as chromothripsis from genome hybridization and sequencing experiments. In our study we identified several shared gain/amplifications in chromosome 6 between the biopsy and the isolated cells of the same tumor, providing evidence in favor of a non-progressive mechanism.
The differences between HG and LG tumors also emerged by GO analysis, especially for two functional GO classes: transcription and apoptosis. Additional file 10: Table S7 shows a preponderance of GO terms for transcription class derived from gained genes of biopsies and isolated cells of HG tumors. Although some LG tumors exhibited a similar behavior (i.e. 34 sample), statistical significance for these tumors is determined by lost genes, especially in isolated cells where it has been shown many lost regions. Similarly, GO terms for apoptosis class were derived from lost regions of isolated cells of LG tumors, while the same GO terms were linked to gained regions of isolated cells of HG tumors. Thanks to the comparative analysis between biopsies and CSCs isolated from them, we can speculate that the driving forces of tumorigenesis are quite different in HG and LG tumors, even showing a complementary behavior. Specifically, we confirmed a good correlation between the total number of CNAs and genomic instability with increasing stage and grade of the biopsy . Moreover, we found that CSCs isolated from LG biopsies accumulate a disproportionate number of genomic losses, so the isolated cells would be much altered respect to their original biopsy. This phenomenon was not observed in HG tumors, then it would not seem the result of purely random alterations due to culture conditions, but to essential characteristics which diversify the two types of tumor. Furthermore, GO stat analysis and aCGH data evidenced a subgroup of LG tumors where this paradox seems to be more evident. It would be interesting to verify if this subgroup of LG tumors could have a more aggressive potential and a greater propensity to progress and invade.