**2. SCNA overview and incidence**

**Figure 1.** (Left) Possible changes in a single chromosome's architecture. One chromosome is shown; each copy of a chromosome can have different SCNA or SNV. (Right) A plot of the major cancers in the TCGA database, showing total genetic lesions (as percentage of genes) vs. the number of SCNA for each SNV. In each case SCNA are more abundant,

This categorization has now had clinical impact. Genes that are known as *drivers* are prioritized for diagnostic testing, and have become a focus for "molecular tumor boards" that review patient data in hospitals across the United States. These boards focus foremost on reviewing a molecular profiling of the tumor, rather than on histopathological features. Thus, tumors with similar genetic features may call for similar therapy regardless of whether they originate in the colon, breast or lung. The division of mutation into drivers and passengers fosters an environment where new mutations may be missed, because we are focused on the pre-established clinical screening protocols, because we both profile and act upon well characterized genetic problems. Even when they are reported, their impact may not be appreciated if they have not had a role as a driver assigned to them in prior peer-reviewed study. The *driver* assignment comes from a breadth of work that focuses on a type of mutation called a somatic single-nucleotide variant (SNV). Driver SNVs are noted for their critical roles in tumor formation, frequently occur at precise locations within *oncogenes*, and can now be rapidly identified. Notable examples include K-Ras, where mutation of the glycine residue at position 12 (G12) inhibits GTPase activity, leaving the protein in an active, GTP-bound effector state. A second example is phosphoinositide 3′kinase, where mutation of the histidine residue at 1047 (H1047) similarly alters the ability of the protein to regulate activity. The gold standard for such driver mutations is their capacity to facilitate neoplastic disease in murine genetic models, most frequently by providing a dysregulated positive stimulus that drives mitosis and cell survival. Transcription factor mutations, such as the FOXL2 C243W mutation found in all adult type granulosa cell tumors, provide a good example of a key genetic driver. A second class of drivers involve SNV-mediated inactivation of *tumor suppressor* genes, which act to ameliorate the effects of oncogenes, shunt tumors towards programmed cell death, and maintain the fidelity of DNA replication and repair. Tumor formation requires both oncogenic activation and the disruption of tumor suppressors. Mutation in TS genes do not require the same precision as those in oncogenes; SNV's occur across a swath of locations, any of which may be sufficient to disrupt tumor suppressor function. This chapter will focus on serous

112 Ovarian Cancer - From Pathogenesis to Treatment

with serous ovarian cancer (SOC, denoted as OVCA in the green box) bearing the greatest number.

A gene normally occurs in the human nucleus twice. This normal 2N "dosage" of copy number, which originates from zygote formation, consists of one paternal gene and one maternal gene. SCNAs, which alter this occur in two types: amplifications and deletions. An amplification occurs when a chromosomal region containing a gene is copied. That gene will no longer have the normal 2N copy number, but, depending upon the number of times it is copied, could be 3N, 4N, or in cases of massive amplification, up to 200 N and more. Contrasting with this expansive range, SCNAs that result from deletions most frequently reduce the copy number to 1N. Total gene loss (0N) can occur in rare cases, and is associated with a very small fraction of the overall number of deletions. Nonetheless, these rarer SCNA-derived genotypes will obviously impact function most, since the lack of any gene copy means that the encoded protein cannot be produced. SCNAs are the most common lesions in cancer, occurring much more commonly than SNVs (**Figure 1**, right panel).

SCNAs occur via a variety of mechanisms in cancer [3]. Entire chromosomes may be gained/ lost during cell division, generating 3N or 1N copy number status for all genes on the chromosome. This occurs due to failed cell-division checkpoints resulting in chromosome missegregation. In contrast to such gains at the total chromosome level, tiny "focal" SCNAs may alter a single gene (or even part of a gene). The most common example of this is *CDKN2A*, a checkpoint protein which is fully deleted (0N) in 3% of SOC tumors. These focal deletions typically occur during repair of double-stranded DNA (dsDNA) breaks. During the attempted repair, short regions of homology can result in accidental deletion of DNA in between [4]. Focal amplifications occur through unknown mechanisms [5] and can form "double minute" chromosomes containing hundreds of copies of a gene, such as *ERBB2* or *EGFR* [6]. Finally, between the focal alterations and the whole chromosome losses, SCNAs can also encompass large regions of DNA through similar defects in dsDNA break repair. These intermediate sized SCNAs can contain many genes. However, they rarely contribute to a 0N copy numbers (loss on both chromosomes) since the regions affected frequently contain essential genes [7].

Within the Cancer Genome Atlas (TCGA) data sets, the presence of 3N and 1N gene copies dominate the SCNA genomic landscape. This is true across all tumors, including those tumors where SCNAs are highly prevalent, such as SOC [8]. SCNAs are prevalent in SOC. In fact, only about one third of all genes in primary tumors have a normal 2N gene dosage. Roughly a quarter of the total genes in the tumors show an extra gene copy (to 3N) and just over a third lose a gene copy (to 1N). By contrast, only 0.7% loses both gene copies (0N), while 4.2% are amplified to 4N or greater. In practice, the focus on understanding tumor biology has been only on these last two cases (total deletion and gross amplification, respectively). This has a reasonable basis; the effects of total loss or gross amplification are easiest to study.

0.12% of the average SOC genome [12]. Less than 10% of SOC patients are mutated in a targe-

Genomic Copy Number Alterations in Serous Ovarian Cancer

http://dx.doi.org/10.5772/intechopen.72695

115

It seems self-evident that an understanding of "driver SCNAs" is absolutely essential to our capacity to target the biology of the disease. Genetic disorders such as Down's syndrome (trisomy 21) and Cri du Chat (5p monosomy) and DiGeorge Syndrome (loss of only 30–40 alleles on 22q11) clearly indicate the penetrative biology of multiple SCNA. More importantly, such lesions affect only ~2% of the genome, while SCNA in SOC affect 67% of genes. Other subtypes of ovarian cancer vary widely in their SCNA burden, but are typically much lower, and

As most SCNA are "monoallelic" changes resulting in a 1N or 3N genotype, is there any reason to expect a phenotype, given our understanding of recessive alleles? Recurrent patterns in serous ovarian cancer suggest that frequently affected regions may be selected for as the tumor evolves. In high grade SOC, the most prevalent SNVs could have been predicted from literature preceding the genomics era. For decades, the mutation of *TP53,* the "guardian of the genome" has been appreciated due to its master control of multiple DNA repair pathways, cell cycle control, and metabolism. Interestingly, there is selection for SCNA deletion of the chromosome with the wild-type copy of p53, suggesting further suppression or misdirection of p53 furthers SOC development [14]. Inheritance studies have associated the *BRCA1/2* mutation with an increased risk of ovarian cancer, and not surprisingly these mutants contain opposite-chromosome deletions just like p53. *BRCA* genes are necessary to maintain the genome. Like p53, they play a coordinating role in facilitating homology-directed repair of DNA. However, single nucleotide variant mutation is not the most common mechanism of BRCA gene disruption. Only ~6% of patients display non-germline SNVs, while copy number deletions (to 1N) occur in more than 70% of tumors. PTEN, a tumor suppressor commonly mutated in many tumor types but not ovarian, was found as early as 2001 to have reduced

Aside from very infrequent gene losses paired with mutations, there are also a few SCNAs which drive cancer through amplification of oncogenes. The stem-cell transcription factor *MYC* is the most amplified gene in the TCGA cohort (42% with at least a 4N copy number, and an additional 37% with 3N). Myc has been appreciated as a common SOC driver oncogene since 1990 [16]. Homozygous deletions in Rb were discovered around the same time [17, 18], and occur in 9% of tumors. *KRAS* amplifications and gene overexpression were discovered around the same time, but in a smaller minority (13%) of patients [19]. Her2, encoded by *ERBB2*, can be overexpressed but this appears to be a case unrelated to SCNA amplification, which occurs in only 3% of cases [20, 21]. Drug resistance can occur following increases in drug efflux genes, and one of the first identified was *MDR1* (*ABCB1*) [22]. Again, this is only found in a small minority of patients (4%). Comparative genomic hybridization in 2006 identified significantly amplified CCNE1 (cyclin E1) and MDM2 (a negative regulator of p53 from its E3 ubiquitin ligase activity) [23, 24]. The year these studies were published provides

table driver gene [12, 13].

are associated with SNVs.

**3. Ovarian cancer and copy number alterations**

expression due to shallow deletions across ~40% of samples [15].

The common gene changes (i.e., 1N and 3N) have not been the subject of focused study. Many scientists assume that the deletion, or addition, of a single gene copy has limited effect. Recessive genetic alleles are not uncommon in nature, supporting the idea that the loss of a single gene copy can be compensated for. However, the loss of a single gene may not reflect the situation in ovarian cancer, where massive genetic alteration occurs, and compensation may not be possible if the same cellular pathway is repeatedly targeted by SCNAs (**Figure 2**).

More than 80% of genes affected by SCNAs show concordant alteration of mRNA levels [9, 10]. For ~70% of genes, this correlates with steady-state protein levels [11]. Thus, SCNAs offer a predictable, but not absolute, indication of protein expression. This is relevant to ovarian cancer, as SCNAs modify on average 67% of the SOC genome, whereas SNVs modify only

**Figure 2.** Model showing how SCNA changes resulting in differences in protein expression might impact overall function within a single multiprotein complex. The relative function of the complex as a % is shown at left. The right side shows how a sequence of SCNA changes within a pathway could cumulatively impact its function.

0.12% of the average SOC genome [12]. Less than 10% of SOC patients are mutated in a targetable driver gene [12, 13].
