Molecular features of adenoid cystic carcinoma with an emphasis on microRNA expression.

Abstract

Tumors of the salivary glands are rare and mainly affect young and middle-aged adults with an equal distribution between the genders 1. The majority of these tumors are of epithelial origin, but while benign tumors are the most common, the carcinomas pose a number of challenges and for some subtypes, namely adenoid cystic carcinoma (ACC), the prognosis may be grave 2. No other organ in the human organism gives rise to such a large spectrum of neoplasia, and the number of different entities has increased dramatically, from 7 carcinoma types in 1972 to 22 in 2017 3, 4. A quite unique feature for this group of tumors is the identification of a network of genes involved in the formation of a variety of fusion oncogenes, each being characteristic of particular tumor types 5. Although believed to be without prognostic significance, these fusion oncogenes have become valuable diagnostic markers and are instrumental in delineating the biology of salivary gland tumors 6. In 1974, Conley and Dingman recapitulate their experience of managing ACC with the following statement: ‘Of all tumors in the head and neck region, the adenoid cystic carcinoma is one of the most biologically deceptive and frustrating in management’ 7. ACC represents approximately 1% of head and neck malignancies and, in contrast to the rest of the world, it is the most frequent type of salivary gland carcinoma in Denmark 1. It is notorious for its unpredictable and often relentlessly progressive clinical course 2, 8. Although clinically slow growing, ACC is locally invasive and has a propensity for early invasion of peripheral nerves and blood vessels, resulting in a high incidence of local recurrence and distant metastases mainly to the lung, bone, and liver 2, 9. Interestingly, intraneural and not perineural invasion has recently been established to be associated with reduced overall and recurrence-free survival 10. Also, regional recurrence, mainly to the cervical lymph nodes, are highly dependent on the presence of high-grade transformation as well as on the T-site 11, 14, 13, 12. Further complicating the management of ACC patients is the continuing presentation of recurrences many years after primary treatment, causing ACC to constitute a disproportionate disease burden despite its low incidence 2. One reason for this is the shortage of prognostic factors beyond conventional cancer staging and classical histopathological parameters such as close or involved surgical margins and a special histological subset with a predominantly solid growth pattern 2. Metastatic disease is one of the main causes of ACC-related mortality as metastases are often surgically unresectable, are relatively resistant to radiotherapy, and lack effective chemotherapeutic regimens 15-17. Once metastatic disease is diagnosed, median survival is about 2 years but, especially for pulmonary metastases, patients can be asymptomatic for several years 2, 15. If inoperable, normal lung parenchyma is replaced by confluent metastatic masses and this evolution is inevitably fatal. Despite that metastatic disease is one of the main causes of ACC-related mortality, previous research on the biology of ACC has focused on exploring the biology of primary lesions 18-21. Adenoid cystic carcinoma is not a disease that is exclusive to the salivary gland, but is also found in the lacrimal gland and breast, as well as more rare locations including sweat gland, lung, and Bartholin's gland 22-26. Regardless of the site, ACC is characterized by recurrent fusion oncogenes and relatively few but diverse mutations 5, 18, 21, 24, 27-32. Lacrimal gland ACC shares the clinical characteristics of salivary gland ACC, with pronounced infiltrative growth and frequent recurrences in local as well as distant sites 29, 33. In contrast, ACC in the breast is a rare, indolent type of breast cancer, with recurrences and distant spread being vanishingly rare events 34. Also, breast ACC has phenotypic features in common with a subset of breast carcinomas with a very poor prognosis 28. This paradox in the clinical behavior of ACC depending on its origin in the salivary gland or breast has earned ACC the all but flattering title as the Dr. Jekyll and Mr. Hyde of exocrine gland carcinomas 35. But what makes this particular puzzling, besides the identical morphology of ACC in the breast and salivary glands, is that the genetic background, including mutations, copy-number alterations, and gene fusions, is identical 18-21, 32. In this series of studies, we compare the phenotypes, genetics, and microRNA (miRNA) expression profiles of ACC in the salivary gland, lacrimal gland, and breast in order to explore the differences between these seemingly identical tumors with markedly differing prognoses (I). Next, we sought to investigate the genetic and miRNA expressional evolution of salivary gland ACC by comparing paired samples of primary and metastatic lesions (II). Lastly, we performed global miRNA expressional profiling of a large cohort of salivary gland ACC with long-term follow-up to investigate the prognostic value of miRNA (III). In early fetal life, the major salivary glands arise from ectodermal proliferations invading the underlying mesenchyme (Fig. 1) 36. Through a carefully regulated process, these initially solid proliferations develop to form tubuloacinar units consisting of acinar cells, a segmental ductal tree, and myoepithelial cells (Fig. 1). Distinct tumor types arise from each of these compartments, with ACC arising from the intercalated duct segment 37. The salivary glands are divided into (i) major salivary glands, namely the paired parotid-, submandibular-, and sublingual glands, and (ii) minor salivary glands located throughout the mucosal membrane of the upper aerodigestive tract (Fig. 2). The acini form the secretory unit and are composed of inner luminal acinar cells and an outer myoepithelial layer, with the composition of the secretion depending on the site of the acinus producing it. The parotid gland contains almost exclusively serous acinar cells, whereas the palatal minor salivary glands are predominantly of mucous type. Saliva produced by the major and intraoral minor salivary glands facilitates mastication and swallowing and provides lubrication and protection of the mucous membranes and teeth. In addition, saliva contains amylase that initiates digestion of starch, but saliva also plays an essential role in preventing dental caries and infection by direct cleansing of foreign bodies and by an anti-bacterial activity mediated through multiple factors (e.g., IgA and histatins) 38. In contrast, the secretion of the minor salivary glands lining the sinonasal tract is devoid of amylase but mainly functions in lubrication and innate immune defense. Carcinomas of the salivary glands are rare, with an incidence of 1.1/100 000/year in Denmark which has been stable since at least 1990 1. Salivary gland malignancies constitute 0.3% of human cancers and 6% of head and neck cancers in the United States and are predominantly of epithelial origin, with the remaining tumors represented by lymphomas, sarcomas, and metastases mainly to parotid lymph nodes from cutaneous squamous cell carcinomas 39-43. Epithelial salivary gland tumors comprise one of the most diverse groups of tumors in the human organism, with 11 benign and 22 malignant entities listed in the current WHO classification 4. The distribution between benign and malignant tumors varies tremendously according to the gland of origin, with an inverse relationship between the proportion of malignant tumors and the size of the major salivary gland of origin. Accordingly, 17% of parotid gland tumors are malignant, increasing to 38% of submandibular gland tumors and 96% of sublingual gland tumors 44, 45. With the notable exception of the palate, minor salivary gland tumors are malignant in the majority of cases 46, 47. In all surveys on the distribution of different histological types of salivary gland carcinomas, mucoepidermoid carcinoma has been identified as the most frequent 48. However, Denmark is an exception to this, as a nationwide study on Danish salivary gland carcinomas convincingly demonstrated a relatively high incidence of 11 salivary gland carcinomas/1,000,000 Danes/year as well as ACC being the by far the most frequent, constituting more than 25% of all salivary gland carcinomas 1. The reason for this uniquely high proportion of ACC among salivary gland carcinomas in Danes is unknown. Although it is well-known that the Inuit in Greenland (part of the Danish Kingdom) contribute with a high incidence of EBV-related lymphoepithelial carcinoma in salivary glands, these relatively few cases cannot itself explain the higher incidence of salivary gland carcinomas in Danes 1, 49, 50). Epithelial salivary gland tumors are rare in children and adolescents and are, as in adults, predominantly benign 51. In early adulthood, the incidence of benign tumors increases, especially in females, but carcinomas are equally distributed between the genders in Denmark, whereas foreign studies have found a higher incidence of salivary gland carcinomas in males 1, 52, 53. With the exceptions of a markedly increased risk of extranodal marginal zone lymphomas in patients with Sjögren syndrome and salivary gland carcinoma in patients exposed to ionizing radiation, risk factors for and etiology of salivary gland carcinogenesis have not been identified, including in ACC 54, 55. As with all rare diseases, identifying disease-causing factors is complicated due to several factors: First, their rarity makes the accumulation of large patient populations difficult; second, there are no known stages of premalignancy; and third, there has only been identified very few cases of familial accumulation of salivary gland carcinoma that have been identified and no genetic disposition was found in the single case of ACC that underwent genetic testing 56-59. Notable exceptions to the lack of hereditability in salivary gland tumors are the rare hereditary syndromes Muir Torre syndrome (OMIM: 158320), with an increased risk of sebaceous carcinoma, Brooke-Spiegler syndrome (OMIM: 605041), with an increased risk of basal cell adenoma, and Birt–Hogg–Dubé syndrome (OMIM:135150), with an increased risk of oncocytomas 60-62. ACC is considered a sporadic disease. The symptoms caused by salivary gland tumors are very similar across the different histological subtypes and vary according to the anatomical site (Figs 3 and 4). Usually, they are slow growing and fixated to the surrounding tissues. More than 30% of ACCs arise in the parotid gland, and the presenting features for this location are an often painless lump with occasional affection of facial muscle function due to facial nerve invasion 1. The intraoral minor salivary glands constitute the second most frequent site, with more than 25% of ACCs, and this location, along with ACCs in the sinonasal tract and sublingual gland, more frequently cause pain as the presenting symptom due to the propensity of ACC for neural invasion (Fig. 3) 1, 45, 63. Other symptoms of sinonasal ACC include obstructive symptoms, epistaxis, and/or auditory symptoms 64. Ulceration of overlaying mucosa can be seen in oral sites 45, 63. Metastases to bones often cause pain, whereas metachronous pulmonary metastases are often an incidental finding due to chest imaging performed for unrelated reasons. As with other lesions located to the major salivary glands, the preoperative diagnosis is based on the clinical history, imaging, and fine-needle aspiration (FNA) (Fig. 3). FNA has an excellent specificity and good sensitivity in separating benign from malignant lesions in both the major and oral minor salivary glands 65. However, ACC shows occasional cytological overlap with pleomorphic adenoma, basal cell adenocarcinoma, and polymorphous adenocarcinoma 66. For sinonasal lesions, the diagnosis is made by incisional biopsy. The final diagnosis is made by histopathological evaluation of the resected lesion. For diagnostic purposes and for surgical planning, preoperative imaging includes contrast-enhanced computerized tomography (CT) for the identification of bone invasion and/or magnetic resonance imaging (MRI) in assessing soft-tissue extension and the extent of perineural invasion (Fig. 4) 67, 68. The use of fluorodeoxyglucose positron emission tomography (18F-FDG PET) in combination with CT can be employed for the initial staging, and although not all ACCs are PET-positive, the physiological high uptake of the major salivary glands can obscure assessment of the primary lesions (Fig. 4) 69. Of note, caution must be taken in using 18F-FDG PET-CT to exclude distant metastases, especially if the primary tumor does not show enhanced 18F-FDG uptake, which is not uncommon in ACC 69. In a seminal paper published by Robin and Laboulbene in 1853, a tumor of the salivary glands with ‘cylindrical’ histology and a pronounced tendency to grow along nerves was described for the first time 70. In 1859, Billroth designated this entity as ‘cylindroma’, a term since modified to adenoid cystic carcinoma by Spies in 1930 71, 72. Regardless of whether it originates in the salivary gland, lacrimal gland, or breast, ACC is a biphasic tumor consisting of relatively uniform ductal and modified myoepithelial cells (Fig. 5). The ductal component is the minor of the two, and the small true lumens formed by these are easily overlooked, as the majority of luminae in ACC are pseudoluminae formed by the dominant myoepithelial component (Fig. 5). The myoepithelial cells lining pseudoluminae produce excess amounts of basophilic, eosinophilic, and occasionally hyalinized extracellular material, often both types varying within a tumor (Fig. 5). The name cylindroma recapitulates the characteristic and most frequent cribriform (‘Swiss cheese’) growth pattern of ACC, one of the three architectural patterns seen in this malignancy. Tubular and solid patterns are usually seen intermingled with cribriform areas, but often in varying proportions within a single tumor. Especially tubular and cribriform areas usually occur together in the same specimen, and also, in the least frequent of the three, the solid type, minor foci of tubular or cribriform growth are usually present (Fig. 5). Solid ACC, defined as solid growth in >30% of the tumor, is known to have a particularly aggressive clinical course 73. The value of distinguishing tubular and cribriform types is debated, and the WHO defines only the solid type as prognostically relevant, thereby also recognizing the substantial subjectivity in assessing proportions of tubular and cribriform areas in a specimen 74, 75. Invasion of peripheral nerves is a frequent but not specific or universally found hallmark of ACC (Fig. 3). This particular feature enables ACC to invade a significantly larger area than the clinically apparent lesion, with only intraneural invasion being associated with overall and recurrence-free survival (Fig. 3) 10. The immunohistochemical profile of ACC is identical irrespective of anatomical location, with ductal cells being positive for CD117 and the myoepithelial component being positive for p63 and smooth-muscle actin. The proliferation marker ki-67 is positive in a variable proportion of tumor cells and is highest in ACC with solid histology 76. Steroid hormone receptors (estrogen receptor [ER], progesterone receptor [PR], androgen receptor [AR]) and human epidermal growth factor receptor 2 [HER2] are negative, whereas cytokeratin 5/6 (CK5/6) and/or epidermal growth factor receptor [EGFR] have been reported in varying proportions 77-80. Despite its indolent nature, breast ACC belongs to the otherwise aggressive basal-like (estrogen receptor and human epidermal growth factor receptor 2 [HER2] negative, CK5/6 and/or epidermal growth factor receptor [EGFR] positive) and triple-negative (negative for estrogen receptor, progesterone receptor, and HER2) subtypes of breast cancer 35. The phenotypic classification of breast carcinoma (i.e., luminal, basal-like, and HER2 phenotypes) carries significant prognostic and therapeutic implications and has been shown to apply to at least a subset of salivary and lacrimal gland carcinomas 35, 81-83. Interestingly, triple-negative breast carcinoma is generally regarded as a clinically aggressive group of malignancies, in sharp contrast to the case for breast ACC 85, 84. The large number of different types of salivary gland carcinomas are broadly separated into two types: high-risk and low-risk types 86. ACCs, solid as well as tubulocribriform types, are high-grade malignancies, and consequently, the treatment of choice is radical surgical resection and almost always followed by adjuvant radiotherapy (Fig. 4) 8, 87. The surgical approach depends on tumor location and stage at diagnosis, with the objective of tumor-free margins being a compromise between functional outcome and therapeutic benefit 2, 88. The highly infiltrative nature of ACC makes this a complex task to preserve vital structures, speech, and masticatory function, as well as an acceptable cosmetic outcome. There is universal agreement about performing therapeutic neck dissection in the case of either clinically or radiologically detectable involvement of cervical lymph nodes. However, the reported low incidence of occult lymph node metastases has made management of the neck in ACC patients without detectable nodal involvement a matter of controversy 8. While high-grade transformation makes neck dissection mandatory due to a high rate of nodal involvement, recent international collaborations have identified occult lymph node metastases in 17% of clinically node-negative patients, with the highest frequencies found in ACC of the oral cavity (22%) and the lowest in the major salivary glands (12%) 11, 89. Importantly, nodal stage was shown to be an independent prognostic factor for overall and disease-specific survival 89. In head and neck squamous cell carcinoma, the rationale for elective neck dissection in clinically node-negative patients is justified in case of a > 15% risk of occult lymph node metastases 90, 91. Hence, the recommendation of selective neck dissection of levels I–III in clinically node-negative patients, especially if the tumor is located to the oral cavity, now seems justified for therapeutic and staging purposes 2, 92, 89. The effect of radiotherapy in the management of ACC has been a matter of debate. No direct survival benefit is seen in patients receiving adjuvant radiotherapy, but it does improve locoregional control 2, 93. As no data from randomized clinical trials are available, Danish guidelines currently includes adjuvant radiotherapy to all patients with salivary gland ACC 87. Similarly, evidence for the use of chemotherapy, conventional cytotoxic agents, and targeted therapies is heavily lacking. One of the main constraints in obtaining these data is the rarity of the disease, making the conductance of well-designed clinical trials logistically challenging. Currently, no compelling evidence exists for any medical treatment, and one must conclude that none of the tested agents results in cure 94-96. A personalized genomically driven approach has shown promising findings in other types of salivary gland carcinomas, and recent reports of transient response to the Notch1-inhibitor brontictuzumab in patients with NOTCH1-mutant ACC lends support to this approach 5, 17, 97-99. Whether neo-adjuvant intra-arterial chemotherapy has a place in the management of salivary gland ACC remains to be clarified, but this approach has yielded promising results in lacrimal gland ACC 100, 101. Collectively, asymptomatic patients with disseminated disease should be spared the side effects of chemotherapy until symptoms arise, and this treatment is to be reserved for patients with symptoms or in the palliative setting 95, 15. At best, temporary partial response or stabilization of the disease may be achieved. When ACC was first discovered as an entity, its malignant potential was not recognized due to the protracted course but was instead regarded as a variant of the benign pleomorphic adenoma due to its ability to recur 102. This changed with the milestone publication by Foote and Frazell in 1953, which reported a 5-year cure rate of just 20% and distant metastases occurring in half of all ACC patients 103. The term ‘slow killer’ for ACC is earned by its relatively good short-term prognosis, but with late recurrences and tumor-related deaths continuously occurring at least 30 years following primary treatment 93. In a comprehensive characterization of histologically revised salivary gland ACCs in Denmark, the 5-, 10-, and 15-year recurrence-free survival was 72%, 60%, and 55%, respectively 2. Similarly, the 5-, 10-, and 15-year overall survival was 80%, 58%, and 46%, respectively 2. Prognostic factors for salivary gland carcinomas include advanced stage, close or involved surgical margins, vascular invasion, and high histological grade 1. Very little variance is found between these parameters and those shown to have prognostic value in ACC, which also include high stage, close or involved surgical margins, solid histology (i.e., high histological grade), and vascular invasion 2, 10. Importantly, while perineural invasion has been associated with involved surgical margins, it is not associated with prognosis 2, 10. The factors associated with unfavorable prognosis are not surprising, since these features are nearly universally adverse characteristics across human cancer types 104. But while these features are of prognostic significance across different types of salivary gland carcinoma as well as within individual entities, there are severe flaws in prognostication based on these features alone in ACC. Specifically, close or involved margins are found in approximately two-thirds of patients, vascular invasion is found only in only 16%, and a substantial proportion of patients with low-stage disease experience local- and/or distant recurrence (20–46%) 2, 105-107. Also, the majority of patients experiencing metastatic spread are initially diagnosed with low-stage disease, free surgical margins are only obtained in one-third of patients, and only 10% have solid-type ACC 2, 15. Therefore, more accurate prognosticators applicable to a larger proportion of patients are highly warranted. The concept of cancer as a result of acquired genetic changes in a single cell was first proposed by Boveri in 1914, a notion that has since been widely accepted 108. The ‘central dogma’, initially described by Crick in 1958, is a term used to describe the flow of genetic information in a biological system, principally including the transfer of genetic information from nucleic acid to nucleic acid or from nucleic acid to protein 109. Since the early 1970s, cancer research has identified a plethora of genes that are frequently mutated in cancer, resulting in (i) constitutively active or overexpressed proteins (i.e., proto-oncogenes) or (ii) inactive or absent proteins (i.e., tumor suppressor genes) 110, 111. Overactivity of proto-oncogenes confers a growth advantage which, when activated by mutations, result in cancer formation. Examples of these types of genes are numerous and include HER2, PIK3CA, and MYB, which are involved in several cancer forms 103, 112, 114. In contrast, tumor suppressor genes encode the normal counterbalancing constraints of proto-oncogene function, and inactivation of these types of genes consequently carries the same effect as activation of proto-oncogenes, that is, cancer. Well-described examples include the TP53 and PTEN genes, known to be involved in a variety of different cancer forms 115, 116. A third, more recently discovered class of genes is the ‘stability genes’, inactivation of which causes an increased rate of mutations throughout the genome, indirectly increasing the risk of mutations in oncogenes and tumor suppressors and thereby resulting in cancer formation 117, 118. In the context of salivary gland ACC, searching for mutations has identified only few and diverse mutations in primary tumors as well as the few metastatic lesions investigated 18-21, 119. Regardless of which type of gene is affected, the malignant phenotype is effectively the modeling of a variety of cellular functions, conceptualized by Hanahan and Weinberg in 2000 as the hallmarks of cancer 120, 121. Since being expanded to include additional characteristics, cancer is the result eight characteristic features, namely (i) sustaining proliferative signaling, (ii) evading growth suppressors, (iii) evading apoptosis, (iv) limitless replicative potential, (v) sustained angiogenesis, (vi) tissue invasion and metastasis, (vii) reprogramming of energy metabolism, and (viii) evading immune destruction. Besides mutations, there are two types of cytogenetic mechanisms that cause neoplasia: numerical and structural aberrations. Numerical changes include amplifications (primarily affecting oncogenes) and deletions (primarily affecting tumor suppressor genes). Structural changes include translocations, insertions, and inversions, and all three of these mechanisms can result in the generation of so-called fusion genes (Fig. 6) 122, 123. Fusion genes mediate their oncogenic properties as a result of the joining of two genes by one of two mechanisms 122. In one, gene breaks and subsequent fusion occur in the coding regions of one or both of the involved genes, forming a new chimeric fusion gene (Fig. 6). In the other, gene breaks occur outside the coding regions of both genes, resulting in exchange of the 5′ regulatory elements, which results in increased expression of a structurally normal gene, so-called promoter swapping (Fig. 6). The first fusion oncogene described is the BCR-ABL1, arising with formation of the Philadelphia chromosome in chronic myeloid leukemia 124, 125. This chimeric fusion gene is formed by the coding parts of both the BCR and ABL1 genes, which is the direct cause of the disease due to the formation of a constitutively active ABL1 kinase domain 126. This discovery sparked the identification of a specific inhibitor of the fusion protein, which has dramatically improved the prognosis for this patient group 127. Since then, numerous types of recurrent fusion genes have been identified across a wide spectrum of different tumor types, a large proportion of which are tumor-type specific, whereas others are more promiscuously involved in various malignancies 128-131. Initially, fusion genes were thought to be predominantly found in hematological malignancies, but they have since been shown to be frequent in sarcomas and carcinomas 123, 128. Hence, although gene fusions are not necessarily pathognomonic for specific entities, they have resulted in improved tumor classification, prognostication, and as promising venues for the development of targeted therapies 5. In salivary gland carcinomas, the most well-known numerical aberration with direct therapeutic implication is the amplification of HER2 in salivary duct carcinoma, in which anti-HER2 treatment has shown some effect in a subset of patients 132, 133. However, structural changes resulting in the formation of recurrent fusion genes have been shown to occur in a much larger number of different types of salivary gland carcinomas 5, 134. So far, the prognostic value has been debated for some of these, especially the CRTC1-MAML2 in mucoepidermoid carcinoma, but few, namely the ETV6-NTRK3 in secretory carcinoma and NCOA4-RET in a subset of salivary duct carcinoma, are directly targetable, with good responses reported 98, 135, 136. In ACC, the most frequent fusion gene consists of the 5′ part of the MYB proto-oncogene fused to the 3′ part of the NFIB transcription factor gene, which in most cases is the primary event in ACC formation (Figs 6 and 7) 27. Due to alternative splicing and different chromosomal breakpoints in both genes, multiple chimeric variants have been described, with most containing the first 8 exons of MYB and from exon 11 of NFIB 21, 137. Only very few non-NFIB fusions involving MYB have been described (Fig. 7) 20, 21. Interestingly, a substantial proportion of ACCs without MYB-NFIB fusions also overexpress the MYB protein, which is due to the translocation of super-enhancer sequences forcing MYB expression 138. Recently, an alternative fusion between the MYB proto-oncogene like-1 (MYBL1) and NFIB (MYBL1-NFIB) was shown to occur in a substantial proportion of ACCs without MYB-NFIB fusion 21. The MYB and MYBL1 genes share substantial homology in their functional domains, and the MYBL1-NFIB fusion includes the first eight exons of MYBL1, similar to the involvement of MYB in the MYB-NFIB fusion 21. However, while identification of these gene fusions are valuable ancillary diagnostic tools for ACC, only very few of these cases have been reported and the prognostic value is still unclear 21, 138-140, 137. Currently, no targeted therapies are available for any gene fusion found in ACC. Genetics undoubtedly plays a central role in the formation of human malignancy, and this field has managed to provide explanations to many fundamental questions in cancer. However, the inadequacy of genetics alone in explaining biology was recognized more than 70 years ago, and since then, several mechanisms regulating gene expression have been identified, including gene-silencing by methylation, modification of chromatin structure, and post-transcriptional gene regulation by RNA, especially by miRNA 141, 142. Lending strong support to the involvement of these genes in cancer is that genes involved in these mechanisms are frequently mutated in human cancer 143. In the context of fusion-driven tumors, it is interesting that miRNA genes are frequently located at fragile sites and genomic regions frequently involved in cancer, a