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University of Pisa
Department of Surgical, Medical, Molecular Pathology and Critical Area
PhD Program in Clinical Physiopathology
Genomic signature in Intraductal Papillary Mucinous
Neoplasms (IPMNs) of the pancreas
Candidate Tutor
Dr. Andrea Cacciato Insilla Prof. Daniela Campani Academic year 2018-2019
1 Abstract ... 3 Riassunto ... 4 List of figures ... 6 List of tables ... 7 1 Introduction ... 8
Pancreatic pre-cancerous lesions ... 8
Intraductal papillary mucinous neoplasms (IPMNs) ... 9
1.2.1 Radiological management of IPMNs ... 11
1.2.2 Histology ... 12
1.2.3 IPMNs and invasive carcinoma ... 14
1.2.4 Molecular characteristics ... 16
1.2.4.1 KRAS and GNAS ... 17
1.2.4.2 SMAD4 and TP53 ... 18
1.2.4.3 RNF43 ... 18
Pancreatic ductal adenocarcinoma (PDAC). ... 19
2 Aim of the study ... 21
3 Materials and Methods ... 22
Intraductal papillary mucinous neoplasms (IPMNs) ... 22
3.1.1 Patients selection ... 22
3.1.2 Immunohistochemistry ... 22
IPMNs with associated invasive carcinoma ... 23
3.2.1 Patients selection ... 23
3.2.2 Immunohistochemistry ... 24
Pancreatic ductal adenocarcinomas (PDACs) ... 24
3.3.1 Patients selection ... 24
Molecular analyses ... 25
3.4.1 DNA purification ... 25
3.4.2 Custom Panel design and sequencing ... 25
Statistical analysis ... 26
4 Results ... 29
Intraductal papillary mucinous neoplasms (IPMNs) ... 29
4.1.1 Patients characteristics ... 29
4.1.2 Molecular characteristics ... 33
4.1.3 KRAS and GNAS ... 35
4.1.4 TP53 and SMAD4 ... 36
IPMNs with associated carcinoma (inv-IPMNs) ... 37
4.2.1 Patients characteristics ... 37
4.2.2 Molecular characteristics ... 41
4.2.3 KRAS and GNAS ... 41
4.2.4 TP53 and SMAD4 ... 43
Pancreatic ductal adenocarcinomas (PDACs) ... 45
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4.3.2 Molecular characteristics ... 48
4.3.3 KRAS, TP53 and SMAD4 ... 48
Comparison between IPMNs and inv-IPMNs ... 51
4.4.1 Pathological characteristics ... 51
4.4.2 Molecular characteristics ... 51
4.4.3 KRAS, GNAS, and TP53 ... 53
Comparison between inv-IPMNs and PDAC ... 54
4.5.1 Pathological characteristics ... 54
4.5.2 Molecular characteristics ... 56
4.5.3 KRAS, TP53, and SMAD4 ... 57
5 Discussion ... 58
6 Limits of the study ... 63
7 Conclusions ... 64
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Abstract
Different precursor lesions can give rise to invasive adenocarcinoma of the pancreas, like pancreatic intraepithelial neoplasms, intraductal mucinous papillary neoplasms (IPMNs), mucinous cystic neoplasms or more recently described intraductal tubulo-papillary neoplasms and atypical flat lesions. IPMNs are recognized as a morphologically and biologically heterogeneous group of neoplasms, but differently from other pancreatic precancerous lesions, little is still known about the molecular mechanisms that are involved in their development and progression to carcinoma. Previous reports on the behavior and prognosis of IPMNs with associated carcinomas (inv-IPMNs) have been inconsistent. Some have suggested that patients with inv-IPMNs exhibit a more favorable prognosis after surgery than those with conventional pancreatic cancer adenocarcinoma (PDAC), whereas others have reported many similarities between the two groups.
To partially answer to all these unsolved issues, this project wanted to study and compare some pathological, immunohistochemical and molecular aspects of IPMNs, inv-IPMNs and classic PDACs to highlight possible mechanisms involved in tumor genesis and development as well as differences or similarities among these groups of pancreatic neoplasms. An Ion AmpliSeq Custom Panel was designed for this study to perform multiplex PCR and sequencing of 26 genes, selected after literature review and known to be targeted in pancreatic neoplasms.
First, we evaluated the immunohistochemical mucine expression and the molecular profile of a selected group of IPMNs, defining their main clinic, pathological and biological characteristics. We performed same ancillary and molecular tests for a selected group of inv-IPMNs, and we compared the results obtained for the two groups. Finally, we defined the molecular profile of a selected control group of PDACs.
IPMNs showed a wider molecular heterogeneity than inv-IPMNs, presenting with higher rates of polyclonal mutations in driver genes, and supporting a recent theory of polyclonal origin of IPMNs, that anticipates the selection and expansion of single clones related to tumor progression. Inv-IPMNs, and in particular tubular carcinomas, were associated with some molecular aspects closer to IPMNs than PDACS, additionally to less aggressive pathological features, implying the existence of more differences than histology alone might suggest. However, no differences in overall survival and disease-free survival were observed between inv-IPMNs and PDACs.
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Riassunto
Diversi possibili precursori dell’adenocarcinoma del pancreas sono stati descritti, come ad esempio le neoplasie intraepiteliali, le neoplasie mucinose papillari intraduttali (IPMNs), le neoplasie cistiche mucinose, e più recentemente le neoplasie tubulo-papillari e le lesioni piatte atipiche. Le IPMNs sono riconosciute come neoplasie morfologicamente e biologicamente eterogenee, ma a differenza di altre lesioni precancerose del pancreas, ancora poco è noto riguardo ai meccanismi coinvolti nella loro genesi e progressione verso forme invasive. Gli studi fino ad oggi pubblicati, inerenti aspetti biologici e prognostici dei carcinomi pancreatici associati a IPMN, hanno fornito informazioni discordanti. Alcuni autori hanno evidenziato un comportamento meno aggressivo e una prognosi migliore per gli inv-IPMNs rispetto ai classici adenocarcinomi del pancreas non associati a IPMN (PDACs), mentre altri hanno sottolineato una sostanziale sovrapposizione delle loro caratteristiche patologiche e prognostiche.
Nel tentativo di dare alcune risposte a questi quesiti, questo progetto ha voluto studiare e comparare le principali caratteristiche patologiche, immunoistochimiche e molecolari di un gruppo selezionato di IPMN, inv-IPMNs e PDACs, in modo da evidenziare possibili meccanismi implicati nella genesi e nella progressione di queste lesioni oltre a chiarire possibili differenze o similitudini tra i diversi gruppi.
Per questo studio è stato ideato un proprio pannello per il sequenziamento e lo studio approfondito di 26 geni, selezionati dopo un’accurata revisione della letteratura.
Per prima cosa è stata valutata l’espressione immunoistochimica di alcune mucine e il profilo molecolare di un gruppo selezionato di IPMNs, in modo da definire le loro principali caratteristiche cliniche, patologiche e biologiche. Successivamente, le stesse analisi immunoistochimiche e molecolari sono state eseguite su un gruppo selezionato di inv-IPMNs, comparando i risultati ottenuti dai primi due gruppi. Infine, analisi molecolari sono state condotte su un gruppo di controllo di PDACs.
Le IPMNs hanno evidenziato un profilo molecolare eterogeneso, anche più degli inv-IPMNs, con la presenza di un maggior numero di mutazioni nei geni driver, a sostegno di recenti teorie sull’origine policlonale di queste lesioni, precedente la selezione di specifici cloni implicati nella progressione verso forme invasive. Gli inv-IPMNs, soprattutto i carcinomi tubulari, hanno evidenziato un profilo molecolare sotto certi aspetti più vicino alle IPMNs che non ai PDACs, oltre a presentare aspetti patologici meno aggressivi, suggerendo la presenza di differenze molecolari tra i due gruppi maggiori di quelle che la somiglianza
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istologica potrebbe far presupporre. Tuttavia, nessuna differenza significative è stata osservata nella sopravvivenza da chirurgica e nella sopravvivenza libera da malattia per i due tipi di tumore.
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List of figures
Figure 1 - Precursor lesions of pancreatic ductal adenocarcinoma. Top: low-grade PanIN (left); B: low-grade IPMN (right). Bottom: Atypical flat lesion with cellular atypia and
typical stromal reaction (left and right) [6] ... 9
Figure 2 - Mixed-type IPMN. Cystic dilatation of both main and peripheral pancreatic ducts is showed ... 10
Figure 3 - Algorithm for the management of suspect IPMNs [11] ... 11
Figure 4 - Different histotypes of IPMN. A: intestinal; B: gastric; C: pancreatobiliary. ... 14
Figure 5 - IPMN classification according to radiological and pathological evaluation ... 30
Figure 6 - Main and secondary histological patterns. Green: gastric; Blue: intestinal; Yellow: pancreatobiliary ... 30
Figure 7 - Low-grade and grade IPMNs. In 2 cases (*) of gastric-type IPMN, high-grade dysplasia ... 32
Figure 8 - Distribution of Clin and FAT-mutations among IPMNs ... 33
Figure 9 - Distribution of IPMNs and Carcinomas in the pancreas ... 38
Figure 10 - OSS by age ... 39
Figure 11 - Association between OSS and tumor grading (top) and between OSS and pN stage (bottom) ... 47
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List of tables
Table 1-Immunohistochemical profile of IPMNs ... 13 Table 2- Ion AmpliSeq Custom Panel reporting the 26 genes studied in this project ... 27 Table 3 - Baseline characteristics of 31 IPMNs ... 32 Table 4 - Molecular characteristics of the 31 IPMNs. Dark grey: Clin-mutations; light grey: FAT-mutations. P: multiple mutations ... 34 Table 5 - Clinico-pathological features of the 26 inv-IPMNs ... 40 Table 6 - Molecular characteristics of the 26 inv-IPMNs. Dark grey: Clin-mutations; light grey: FAT-mutations. P: multiple mutations ... 42 Table 7 - correlation between clinico-pathological features and KRAS, GNAS, and TP53 Clin-mutations in 26 inv-IPMNs ... 44 Table 8 - clinico-pathological features of the 16 PDACs ... 46 Table 9 - Molecular characteristics of 16 PDACs. Dark grey: Clin-mutations; light grey: FAT-mutations. P: multiple mutations ... 50 Table 10 - Comparison between clinico-pathological features of IPMNs and inv-IPMNs ... 52 Table 11 - Comparison between main clinico-pathological features of PDACs and inv-IPMNs ... 55 Table 12 - Distribution of gene mutations in inv-IPMNs and PDACs. Dark grey: genes with Clin-mutations ... 56
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1 Introduction
Pancreatic pre-cancerous lesions
Different precursor lesions can give rise to invasive adenocarcinoma of the pancreas. Pancreatic intraepithelial neoplasia, also known as PanINs, probably represent the most common ones. PanINs are small (< 5 mm), mucinous intraepithelial neoplasms, flat or micropapillary, confined to the pancreatic ducts and can be classified as low-grade or high-grade PanINs, according to the high-grade of dysplasia [1]. Two other well-known precursor lesions are represented by intraductal papillary mucinous neoplasms, or IPMNs, and mucinous cystic neoplasms, or MCNs. IPMNs can present as a dilatation of the main pancreatic duct or as peripheral mucinous cysts communicating with the main duct; coexistence of both aspects is also possible. MCNs usually present as large, well-defined cystic lesions without connection to the pancreatic ducts. Beyond the absence of communication with pancreatic ducts, the most characteristic finding in MCNs is the presence of a unique ovarian-type stroma, not found in other pancreatic neoplasms, that made many authors assume its possible origin from ectopic ovarian stroma incorporated in the pancreas [2]. Like PanINs, IPMNs and MCNs are also classified as low or high-grade according to the grade of dysplasia [1]. Intraductal tubulo-papillary neoplasia, ITPNs, described for the first time in 2009, represent another, uncommon, precancerous lesion and account for less than 1% of all pancreatic exocrine neoplasms [2], [3]. ITPNs are intraductal, grossly visible lesions, predominantly tubule-forming and with ductal differentiation, usually presenting with high-grade dysplasia and not characterized by mucin production [4]. Lastly, atypical flat lesions, AFLs, first described in genetically engineered mouse models, have been recently reported as a brand-new lesion characterized by the presence of cytological atypia and a peculiar stromal reaction, localized in the centro-acinar/acinar compartment; due to their immunophenotypical and molecular similarities with pancreatic carcinoma in mouse models, it has been hypothesized their possible precancerous nature (fig 1) [5], [6].
9 Figure 1 - Precursor lesions of pancreatic ductal adenocarcinoma. Top: low-grade PanIN (left); B: low-grade IPMN (right). Bottom: Atypical flat lesion with cellular atypia and typical stromal reaction (left and right) [6]
Intraductal papillary mucinous neoplasms (IPMNs)
Intraductal papillary mucinous neoplasms (IPMNs) of the pancreas were originally described in 1982, and have been recognized as a distinct entity by the World health Organization in 1996 [7]. IPMNs are grossly visible (typically > 5 mm) intraductal epithelial neoplasms of mucin-producing cells, and represent the most frequent resected cystic lesion of the pancreas [2], [6], [8]. Most IPMNs are diagnosed between 60 and 70 years of age, with a slightly higher prevalence in men than women [9]. IPMNs can occur anywhere in the pancreas, however, most are in the head. Multicentricity is reported in as many as 40% of cases [2], [4], [9]. They can be classified as main-duct (MD), branch-duct (BD) or mixed-type (MT) IPMNs, based on imaging studies and/or histology [10], [11]. MD-IPMNs are characterized by segmental or diffuse dilatation of main pancreatic duct (> 5 mm), without other causes of obstruction. MD-IPMNs are usually located in the head of the pancreas, but may occasionally involve the entire MD, including the ampulla of Vater. The MD is typically filled by mucin and usually lined by soft and friable papillary formations [2], [4].
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Instead, peripheral pancreatic cysts of more than 5 mm in diameter that communicate with the MD should be considered as BD-IPMNs [9]–[11]. BD-IPMNs mainly occur in the uncinate process as multilocular, mucinous cysts, and very often are incidentally discovered [10], [12]. MT-IPMNs appear as a combination of both two types (fig. 2).
One pitfall in this classification scheme is that correlation between imaging and histology is reported to be around 70% [11]. For example, in some cases the MD can be enlarged due to ductal hypertension related to protein plugs or mucin, and on the other hand many BD-IPMNs prove, by microscopic examination, to have some degree of involvement of the MD, even without significant duct dilatation [11]. However, since the classification is important for clinicians to plan the management, it should be based on preoperative images, with the pathological classification that can be specified later. This classification is important because MD and BD-IPMNs have significant differences in prevalence of cancer, ranging from 57 to 92% and 6 to 46% respectively [8], [10], [12].
IPMNs are often asymptomatic and they are frequently identified in patients undergoing CT or MRI for other reasons; when symptomatic, clinical presentation usually includes epigastric pain, abdominal discomfort, nausea; other symptoms, like weight loss, back pain, and jaundice have been often associated with malignant transformation of the IPMN [13]– [16]. The prognosis of patients with non-invasive IPMN is excellent, and the 5-year survival rate is reported to be 77-100% [17].
Figure 2 - Mixed-type IPMN. Cystic dilatation of both main and peripheral pancreatic ducts is showed
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1.2.1 Radiological management of IPMNs
Pancreatic cystic lesions are being recognized with increasing frequency by imaging studies, with a higher prevalence in MRI (up to 40%) compared to CT (< 5%) [11], [18]. Determining which patients are at higher risk of harboring or developing an invasive carcinoma, and therefore should undergo resection, and how to follow the remaining ones has been the matter of extensive studies and international meetings. The publication of the international consensus guidelines for management of IPMN in 2006 (the so called Sendai criteria), the Fukuoka guidelines published in 2012, and the following revision in 2017 contributed to define concepts helpful for physicians managing patients with pancreatic cystic lesions, taking to the definition of the so called “worrisome features” and “high-risk stigmata” [10], [11] (fig. 3). Criteria reported as worrisome features are: cyst of > 3 cm, enhancing mural nodule < 5 mm, thickened enhanced cyst walls, MD size of 5-9 mm, abrupt change in the MD caliber with distal pancreatic atrophy, lymphadenopathy, an elevated serum level of carbohydrate antigen (CA)19-9 and a rapid rate of cyst growth > 5 mm/2 years. For these patients, endoscopic ultrasonography is usually suggested. High-risk stigmata are described as obstructive jaundice in a patient with a cystic lesion of the pancreatic head, enhanced mural nodule > 5 mm, MD size of > 10 mm. Patients with so called “high-risk stigmata” have a 5-year risk of developing an adenocarcinoma of approximately 50%. For these patients, surgery without any further testing is usually indicated [11].
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Many studies reported how the development of worrisome features is frequently correlated with a significantly higher cyst growth rate [18]. In fact, malignant cysts grew at a greater rate than benign, and cysts that grew more than 2 mm/year or 2.5 mm/year, according to different studies, had a higher risk of malignancy. In this regard, cyst growth could be used as a useful predictor of malignancy [18], [19].
1.2.2 Histology
Microscopically, IPMNs are characterized by the intraductal proliferation of columnar mucin-producing cells, which can be flat or form papillae ranging from microscopic folds to grossly visible projections [2].
Based on the highest level of atypia, IPMNs can be graded as low or high grade, in a two-tiered grading system that recently replaced the former three-two-tiered one, where IPMNs were classified as “with low, intermediate or high grade of dysplasia”. The former “intermediate grade dysplasia” is now categorized as low grade IPMN, while the term high grade is reserved only for the uppermost end of the spectrum [1]. Low grade IPMNs are characterized by mild to moderate atypia and may or may not reveal papillary projections and mitoses; high grade IPMNs, instead are characterized by severe atypia, papillae with irregular branching and budding, nuclear stratification with loss of polarity, pleomorphism and prominent nucleoli; mitoses are numerous [2].
Based on the predominant cell differentiation, three different subtypes of IPMNs can be recognized: gastric, intestinal and pancreatobiliary. Differential diagnosis of these histopathological subtypes is accomplished by morphological aspects and immunohistochemical analysis of mucine (MUC) expression [2], [4], [12]. Mucines are high molecular weight glycoproteins produced by different types of epithelial cells. Some mucines are normally located in the cell membrane, like MUC1, while others are normally secretory products, including MUC2, MUC5AC and MUC6 [9]. The gastric-type IPMN is the most common one (60-70%) and usually occurs in BD-IPMNs. It is characterized by tall, columnar cells with basally oriented nuclei and pale mucinous cytoplasm resembling the gastric foveolar epithelium; scattered goblet-cells can be seen [2]. Immunoprofile of Gastric-type IPMN usually consist of diffuse expression of MUC5AC, without expression of MUC1 and MUC2 (scattered MUC2-positive goblet cells can be present); MUC6 can be focally expressed in basal, non-papillary areas with pyloric-like appearance [9], [20]. Gastric-type
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IPMNs usually present as low-grade lesions, although cases with high-grade dysplasia and associated invasive carcinoma have been described [21].
Intestinal-type IPMN is the second most common type (20%) and frequently occur in the MD [4]. It is characterized by intestinal-type epithelium that forms villous papillae composed of tall columnar cells with cigar-shaped enlarged nuclei and basophilic cytoplasm with variable amount of apical mucin. The immunoprofile of this type of IPMN is characterized by diffuse expression of MUC2 and MUC5AC, and usually negative expression of MUC1 and MUC6; nuclear positivity for CDX2 is commonly reported. Intestinal-type IPMNs usually show high-grade dysplasia [2], [9].
Pancreatobiliary type is the least common type of IPMN and is characterized by complex interconnecting papillae consisting of cuboidal cells with marked atypical nuclei and a basophilic cytoplasm, enlarge nuclei and prominent nucleoli. The neoplastic cells express MUC1 and MUC5AC, usually MUC6 but not MUC2. Pancreatobiliary type of IPMN frequently involve pancreatic MD and usually presents with high-grade dysplasia [2], [9]. Characteristic MUC immunoprofile of the three subtypes of IPMN is reported in table 1. The oncocytic subtype, previously considered as a fourth histotype of IPMN, has been recently classified as a separate entity, called intraductal oncocytic papillary neoplasm (IOPN), due to its peculiar molecular and clinical characteristics [4]. IOPNs are sometimes associated with different subtypes of IPMNs [22].
Table 1-Immunohistochemical profile of IPMNs
Subtype MUC1 MUC2 MUC5AC MUC6
Gastric - goblet cellsOccasional + -/+ Pyloric-like glands
Intestinal - + + -
Pancreatobiliary + - + +
Combinations of different histological patterns are often reported, most commonly intestinal or pancreatobiliary types mixed with gastric-type IPMNs, while association between
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intestinal and pancreatobiliary-type is uncommon; in this regard, it has been suggested that the low-grade gastric-type could be a common precursor to the other types of IPMNs [9], [14], [23], [24].
Presence of a singular histological pattern or combination of “polytypic” IPMNs could have a clinic and prognostic relevance. In one study, Shimizu et al described the main pathological aspects of monotypic and polytypic IPMNs, reporting that monotypic PB-IPMNs have more aggressive pathologic features, and are more often associated with invasive cancers, with general worse outcome. Radiological and molecular differences were also observed, with higher prevalence of GNAS mutations in polytypic IPMNs, and lack of clear cystic dilatation of pancreatic ducts by imaging in monotypic IPMNs [23].
Figure 4 - Different histotypes of IPMN. A: intestinal; B: gastric; C: pancreatobiliary. D: Intraductal Oncocytic Papillary Neoplasm
1.2.3 IPMNs and invasive carcinoma
Approximately one third of surgically resected IPMNs are found to have an associated invasive component, due to the extension of neoplastic cells through the basement membrane; the malignant potential depends on the type of IPMN and is usually higher for MD- and MT- than BD-IPMNs [7], [14], [25]. IPMNs can progress to two distinct types of invasive carcinoma, known as colloid or tubular carcinomas, and in this case, they are designated as “IPMNs with associated invasive carcinoma” (inv-IPMN). Mixed colloid-tubular carcinomas have been reported [9], [21], [26]. Colloid carcinoma is characterized by infiltrating epithelial elements separated by abundant stromal mucin, and typically arises in
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association with intestinal-type IPMNs; some authors actually argue that it virtually never exists without an associated IPMN, whose detection only depends on the extension of sampling [9]. Tubular carcinoma is morphologically similar to classic pancreatic ductal adenocarcinoma (PDAC) and arises more frequently in association with pancreatobiliary or gastric-type IPMNs, in both MD and BD-IPMNs [17]. Interestingly, although gastric-type IPMNs usually exhibit only low-grade dysplasia and the association with invasive carcinoma is uncommon, it has been observed that, when developing, invasive carcinomas have more aggressive aspects than carcinomas arising from intestinal or pancreatobiliary-types [9], [21]. It has been questioned, however, whether these gastric-type IPMNs are always related to the carcinoma or simply coexist in its background. In fact, IPMNs and invasive carcinoma can occur in the same pancreas, without necessarily connections: these cases are designated as “IPMNs with a concomitant invasive carcinoma” and it is noteworthy that they are often reported as BD-, gastric-type IPMNs [27], [28]. One explanation for this unrelated coexistence is that patients with IPMN can also have some concurrent precancerous lesions, like PanINs, accountable for the development of the carcinoma. Alternatively, the multifocality of IPMNs, and their genetic heterogeneity can support the hypothesis of a carcinoma arising from distant lesions, and resulting in anatomically separated neoplasms [14], [28]. However, it is not always possible to assess with certainty whether a carcinoma is concomitant or associated with an IPMN; in some cases, the histological transition might not be evident because serial section examination is not performed, or because the transition might have disappeared due to the extensive growth of the PDAC. In these cases, the only possibility is to refer to the IPMN and the carcinoma as to two different neoplasms [26].
The 5-year survival rate reported for inv-IPMNs ranges from 34% to 62%, and colloid carcinomas are usually associated with a more indolent behavior, and a more favorable outcome than tubular carcinomas [7], [14], [17]. The survival rate for inv-IPMNs appears significantly different from data usually reported for PDAC, for which the estimated 5-year survival rate is between 3% and 34% [29]. The reasons for this advantage remain controversial [17]. Many authors suggested that the improved survival for inv-IPMNs is not unconditional, but only observed in specific situations, such as in earlier stages and in the absence of nodal metastasis; moreover, the advantage of inv-IPMNs would be mainly affected by the different survivals between colloid carcinomas and PDACs, while tubular carcinomas and PDACs would show similar outcomes [9], [17], [27], [30]–[34]. Other studies, instead, reported a difference in the outcome of inv-IPMNs and PDACs in different
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groups matched by stage, suggesting that these carcinomas may have some biological differences that could explain this prognostic mismatch [26], [28], [35].
1.2.4 Molecular characteristics
Molecular studies suggest that progression from low to high-grade IPMN, and eventually to invasive carcinoma, is associated with an accumulation of genetic alterations in different oncogenes and tumor suppressor genes [36]. KRAS and GNAS mutations are universally recognized as the earliest driver gene alterations in IPMNs, usually followed by some other mutations mainly involved in the progression of the lesion, like TP53, RNF43, CDKN2A or
SMAD4. With the exception of GNAS, these mutations are also observed in other pancreatic
precancerous lesions or in invasive carcinomas, although with different frequencies [14], [36].
IPMNs are often described as neoplasms with genetic heterogeneity. Felsenstein et al compared the molecular aspects of two distinct regions of the same IPMN with a co-occurring carcinoma, observing that these areas were genetically related one to the other only in 39% of paired samples, with higher concordance in IPMNs with collateral colloid carcinomas (86%). In addition, a great proportion of mutations and a high molecular heterogeneity are typically reported in IPMNs, more often in low-grade than in high-grade IPMNs, and especially for driver genes. These data show that the identification of a mutation in one region of the neoplasm does not necessarily imply its occurrence throughout the entire lesion, questioning the model of progressive accumulation of genomic alterations, and highlighting the chance of a polyclonal origin for most IPMNs [28], [36].
IPMN heterogeneity is also stressed by the common presence of multiple mutations in single initiating driver genes, more than usually observed in PDACs, perhaps suggesting a more diverse mixture of clones in lower grade lesions [28], [36].
DNA-based analyses of cystic fluids have recently emerged as an adjunct to assess the nature of pancreatic cysts: although cellular content and fluid volume are often suboptimal for routine ancillary studies, DNA from lysed or exfoliated cyst epithelial lining can be analyzed for genetic abnormalities, giving relevant information for the clinical management of the patient. Sequencing studies, in fact, have identified distinct mutational profiles of most of the pancreatic cystic lesions, useful to discriminate between mucinous and non-mucinous lesions [15], [37]. Analyses of pancreatic juice collected from the duodenum has been described as another possible approach to investigate molecular aspects of pancreatic
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lesions, with good sensitivity in detecting mutations other than KRAS/GNAS, like TP53 and
SMAD4, useful to predict the presence of advanced precancerous lesions, although with
limitations for the adequate localization of the disease [38], [39].
1.2.4.1 KRAS and GNAS
Although the prevalence of KRAS and GNAS mutations varies among histologic subtypes, they are currently recognized as two of the most common driver genes in IPMNs [40], [41]. More than 90% of IPMNs have either a KRAS or GNAS mutation, and more than 50% have both [40], [42], [43]. KRAS mutations are not specific for IPMNs, and are often detected in other pancreatic neoplasms like PanINs or MCNs, as well as PDACs [8], [44]. Among IPMNs, they occur more frequently in gastric and pancreatobiliary subtypes and are also reported in about 80% of inv-IPMNs [17], [41]. KRAS mutations are frequently observed in codon 12 (G12V, G12D or G12R), as usually reported for other pancreatic neoplasms [8].
GNAS mutations, instead, are almost exclusively observed in IPMNs (48-78%) and are more
commonly detected in gastric (46%) and intestinal (59%) subtypes, than in pancreatobiliary types (27%). The vast majority of GNAS mutations occur at hotspot codon 201, with R201C and R201H as the most frequently reported [8], [23], [41], [45]; mutations in codon 227 have been occasionally described in IPMNs, but more frequently in colloid carcinomas [7]. Multiple KRAS and GNAS mutations have been reported within a single IPMN, and more frequently in IPMNs and inv-IPMNs than in PDACs, but also in low-grade than in high-grade IPMNs, suggesting a notably complex pattern of clonal precursor lesions [8], [28], [36], [45]. KRAS mutations are predominant in tubular carcinomas, while GNAS mutations are usually associated with colloid carcinomas: this different distribution of KRAS and
GNAS mutations in histological subtypes of IPMN and inv-IPMN probably reflects the
existence of different pathways of cancer progression [6], [7].
Detection of KRAS and/or GNAS mutations in pancreatic cystic fluids is reported to have up to 90% sensitivity and about 50% specificity for IPMNs, with a positive and negative predictive value, respectively of 71% and 81%. More importantly, KRAS and GNAS mutations have not been reported in cyst fluid from benign pancreatic cystic lesion [43]. Hence, these mutations might be useful markers to characterize pancreatic lesions, although, without enough specificity to distinguish low-grade from high-grade lesions. In fact, these mutations are reported to occur early in the neoplastic transformation of IPMNs, and no
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significant differences have been found across various grades of dysplasia in different studies [7], [8], [15], [40], [43], [45]–[47].
1.2.4.2 SMAD4 and TP53
SMAD4 and TP53 mutations are more frequently described in high-grade,
pancreatobiliary-type IPMNs as well as in inv-IPMNs, especially of tubular-pancreatobiliary-type; they are generally considered late-occurring gene mutations involved in malignant progression, although their presence in few low-grade IPMNs has been sporadically reported [28], [36], [48]–[51]. Differently from KRAS and GNAS mutations, high-grade IPMNs usually show higher heterogeneity for TP53 and SMAD4 mutations than low-grade IPMNs [36].
Detection of these mutations in cyst fluid is described by many authors as a promising tool to distinguish low-grade from high-grade premalignant lesions, separating patients who may benefit from immediate surgery from those who require follow up; in addition loss of
SMAD4 correlated with recurrence after surgery in some studies, highlighting its possible
role in assessing the clinical follow up [14], [28], [41], [44].
In this regard, TP53 and SMAD4 mutations have been reported in the pancreatic juice of patients with IPMN over 1 year prior to their pancreatic cancer diagnosis, at a time when no suspicious lesion was evident by imaging. However, pathogenic mutations of TP53 and
SMAD4, alone or in association with KRAS and GNAS mutations have also been reported in
low-grade IPMNs without concerning features of advanced neoplasia and without progression to malignancy on follow-up [15], [52].
1.2.4.3 RNF43
RFN43 mutations have been described from 10% to more than 70% of IPMNs and although these mutations were initially observed only in high-grade lesions, it is now clear that they are also present in low-grade neoplasms [36], [53], [54]. RNF43 mutations have been observed also in other pancreatic precancerous lesions and carcinomas, although frequency in IPMNs is reported to be higher [42], [52], [53], [55]. RNF43 mutations in IPMNs are often described in different regions, and presence of multiple mutations within the same patient have been reported, typically in high-grade lesions or inv-IPMNs; common association with KRAS and GNAS mutations has been observed [36]. In one study, RNF43 mutations were associated with radiological detection of mural nodules, suggesting their role
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in promoting proliferation processes [53]. However, the real correlation between RNF43 mutations and malignancy has still to be cleared [56].
Pancreatic ductal adenocarcinoma (PDAC).
PDAC is among the most aggressive cancer types, and is expected to be the second leading cause of cancer-related deaths in the USA by 2030 [57]. At present, PDAC usually shows a modest response to current therapies and surgery still offers the only possibility of cure, although less than 20% of patients are eligible for surgery at diagnosis, due to local spread or metastasis [14], [58]. Most PDACs arise in the head of the pancreas, and usually present as a solitary lesion, although multifocal disease can occur. Most PDACs are well to moderately differentiated carcinomas, with duct-like glandular structures characterized by angular contours and branching, lined by cuboidal or columnar epithelium with different degrees of nuclear pleomorphism, marked nucleoli and mitoses; common is the presence of desmoplastic reaction that encompasses the neoplastic glands, characterized by stroma composed of collagen fibers interspersed with fibroblasts, myofibroblasts, scattered lymphocytes and macrophages [2]. Different morphological variants of PDAC has been described including adeno-squamous, colloid, hepatoid, medullary, micropapillary, signet-ring cell, and undifferentiated carcinoma with or without osteoclast-like giant cells [2], [59]. In recent years, extensive next-generation sequencing studies have characterized the genomic landscape of PDAC, underling the fundamental role of four driver genes in the development of these cancers: KRAS, TP53, SMAD4 and CDKN2A [6], [28].
KRAS is by far the most frequently mutated gene in PDACs with a reported frequency
ranging from 20% to 100% [58]. Approximately 80-90% of PDACs have major hot spots mutations in codon 12 (G12D, G12V, and G12R) or other less frequent variants at codons 13, 61, 117 and 146 [14], [60], [61]. Presence of multiple mutations in KRAS have been reported, although not frequently as observed in some precancerous lesions [28], [36]. It has been suggested that different KRAS mutations are correlated with different survivals, with worse and better prognosis associated, respectively with mutations in codon 12 and codon 61 [58], [60], [62]; some authors also reported worse response to first-line gemcitabine-based chemotherapy in patients with KRAS mutations [61]. However, this is a controversial issue, since many studies have also discussed the association between KRAS mutations and survival in patients with PDACs and inv-IPMNs, demonstrating that these mutations were not associated with clinical outcome [46].
20
TP53 is one of the most frequently mutated genes in PDACs, and it is reported to be
inactivated in 50% to 75% of PDAC [28], [44]. Some clinical evidence suggests that TP53 could be used as biomarker for prognosis and therapy prediction, reporting that patients with mutations usually have a worse outcome than wild-type patients [61].
SMAD4 inactivating mutations are reported in about 55% of PDACs and are usually
described as markers of poor prognosis, often associated with early metastatic disease, although not all studies were able to confirm these results [28], [63]–[65]. SMAD4 mutations can be usually identified by loss of SMAD4 nuclear labelling by immunohistochemistry and, for some authors, this can be used in the differential diagnosis of carcinomas of unknown origin, due to the fact that loss of the protein expression is uncommon in extra-pancreatic tumors [44], [64], [66]. SMAD4 loss of function has been frequently reported in association with TP53 mutations, but not the opposite, suggesting that SMAD4 inactivation probably occurs later than TP53 mutation in the tumor progression [67].
CDKN2A is reported to be inactivated in up to 95% of PDACs [44]. CDKN2A seems to play
a role in patient outcome, and studies have shown that patients with wildtype CDKN2A live significantly longer than those with altered genes, supporting the concept of using CDKN2A mutations as a possible prognostic and predictive biomarker [61], [65]. In addition, loss of
CDKN2A has been reported to be associated with lymphatic invasion and widespread
metastasis [68].
Aside from these four genes, a large number of other genomic alterations have been described in PDACs, but usually at lower prevalence (< 5%).
Presence of driver genes that are not clinically targetable with current therapeutic regimens, and the high genomic heterogeneity of PDACs partly explain the relatively slow progress in the development of effective therapies. [69].
21
2 Aim of the study
IPMNs are recognized as a morphologically and biologically heterogeneous group of neoplasms, but differently from other pancreatic precancerous lesions, little is still known about the molecular mechanisms that are involved in their development and progression.
The objectives of this study are:
- to define some molecular aspects of different subtypes of IPMNs, identifying genes that may be involved in tumor genesis and progression, their association with main histological features and possible application as diagnostic markers
- to determine whether there is an association between histological subtypes of IPMNs and inv-IPMNs and to identify differences in their molecular characteristics that may be related to cancer progression or biological behavior
- to compare pathologic features and molecular profiles of inv-IPMNs and PDACs to assess differences in carcinomas that are classified separately, regardless of many histological similarities.
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3 Materials and Methods
Intraductal papillary mucinous neoplasms (IPMNs)
3.1.1 Patients selection
A total of 73 patients with a histological diagnosis of IPMN were retrospectively evaluated for this project. Most patients, 60 (82,2%) out of 73, underwent surgery at the General Surgery and Transplantation Unit of the University Hospital of Pisa (Italy) between 2009 and 2018, with a pre-operative radiological diagnosis of IPMN. Thirteen (17,8%) patients were organ donors and IPMN was incidentally detected during transplantation screening procedures. A histological review of all cases was performed to confirm the diagnosis, and all cases were re-evaluated according to the “2016 revised classification system from the Baltimore consensus meeting for pancreatic precancerous lesions” (5th ed. of WHO classification of digestive system tumors was published in 2019, after the beginning of this project) [1]. IPMNs were classified as MD, BD or MT-IPMNs, based on radiological and histological findings, and a database reporting clinical and pathological characteristic of all patients was created.
3.1.2 Immunohistochemistry
MUC1, MUC2, MUC5AC and MUC6 expression was evaluated by immunohistochemistry in the selected cases. Representative samples without factors that may invalidate immunohistochemistry (necrosis, calcifications, fibrosis) were selected for each case. All immunohistochemical analyses were performed automatically using the Ventana Bench-mark immunostaining system (Ventana Medical Systems, Inc.). Paraffin-embedded tissue sections (thickness 3 ) were deparaffinized in xylene, rehydrated through a graded series of ethanol and processed using a diaminobenzidine detection system, following the manufacturer’s instructions. MUC1 immunostaining was performed by using anti-MUC1 (H23) mouse monoclonal primary antibody; clear and diffuse cytoplasmic stain was considered as positive, while absence or occasional stain in the apical border of epithelial cells were considered as negative. MUC2 immunostaining was performed by using anti-MUC2 (MRQ-18) mouse monoclonal primary antibody. MUC5AC immunostaining was performed by using anti-MUC5A (MRQ-19) mouse monoclonal primary antibody. Finally,
23
MUC6 immunostaining was performed by using anti-MUC6 (MRQ-20) mouse monoclonal primary antibody. As for MUC1, also for MUC2, MUC5AC, and MUC6 only diffuse cytoplasmic stain was considered as positive. Based on histological features and mucines expression, IPMNs were classified as gastric, intestinal or pancreatobiliary, according to the predominant type. Presence of a secondary pattern was reported, when present.
IPMNs with associated invasive carcinoma
3.2.1 Patients selection
A total amount of 60 patients with a histological diagnosis of adenocarcinoma associated with an IPMN were retrospectively selected for this group. All patients underwent upfront surgery at the General Surgery and Transplantation Unit of the University Hospital of Pisa (Italy) between 2014 and 2018. A histological review of all cases was performed to confirm the diagnosis, and all cases were re-evaluated according to the “2016 revised classification system from the Baltimore consensus meeting for pancreatic precancerous lesions”, the AJCC cancer staging manual 8th edition, and the “College of American Pathologist (CAP) protocol for the examination of specimens from patients with carcinoma of the pancreas” [1], [70], [71]; special attention was paid to exclude invasive carcinomas coexisting with, but apparently separated from IPMNs, as well as cases with degenerative cystic changes of classic PDACs and secondary cystic dilatations of pancreatic ducts. Only pancreatic adenocarcinomas reasonably derived from an IPMN were selected, basing on radiological findings, macroscopic association between solid and cystic lesions, and histological evidence of transition from IPMN to inv-IPMN. In addition, cases with marked necrosis, abundant inflammatory infiltrate, calcifications as well as cases apparently not suitable for manual microdissection were excluded, to reduce any interference with molecular analyses. All cases were classified according to their radiological and histological features in MD, BT or MT-IPMNs with an associated colloid or tubular carcinoma; clinical data were collected from information obtained during routine surveillance visits. Finally, a database with the clinical and pathological aspects of the selected cases was created.
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3.2.2 Immunohistochemistry
Expression of MUC1, MUC2, MUC5AC, and MUC6 was evaluated to assess the histological subtype of IPMN associated with carcinoma. Immunohistochemistry was performed according to the same procedure described for IPMNs. MUC expression was evaluated in only one selected sample, where transition from IPMN to the invasive component was evident.
Pancreatic ductal adenocarcinomas (PDACs)
3.3.1 Patients selection
Forty-four consecutive cases of classic PDACs were retrospectively selected for this study, based on their histological diagnosis. All selected patients underwent upfront surgery at the General Surgery and Transplantation Unit of the University Hospital of Pisa (Italy) between 2016 and 2018. All cases with radiological and/or pathological findings consistent with the presence of collateral IPMN were not considered for this group. Only clear classic PDACs were selected, while other histological subtypes were not recruited, like adenosquamous, signet-ring cells, undifferentiated, and mixed carcinomas. Patients with collateral neoplasms as neuroendocrine tumors were also excluded. For all selected patients, tumor bed, collateral parenchyma and peripancreatic tissues were completely sampled and embedded during macroscopic examination of surgical specimens. Histological review of all cases was performed to confirm the diagnosis. All histological evaluations were reviewed according to AJCC cancer staging manual 8th edition, and to the College of American Pathologist (CAP) protocol for the examination of specimens from patients with carcinoma of the pancreas [70], [71]. To reduce possible interfering with molecular tests, tumors with marked necrosis, inflammatory infiltrate, degenerative changes or not suitable for manual microdissection were also removed. Post-operative follow-up data were collected from information obtained during routine surveillance clinic visits. Finally, a database reporting all clinical and pathological features of the selected PDACs was created.
25
Molecular analyses
3.4.1 DNA purification
For DNA isolation from formalin-fixed and paraffin-embedded (FFPE) tumor samples, hematoxylin and eosin–stained slides were reviewed to define tumor regions and percentage of tumor cells. Representative samples suitable for manual microdissection and without factors that may invalidate molecular analyses (necrosis, calcifications, fibrosis) were selected for each case. For High-grade IPMNs, samples representative of the histological grade were selected; because IPMNs are classified based on the highest grade of dysplasia present in the lesion, high-grade IPMNs were often associated with low-grade areas, also within the selected sample. For inv-IPMNs, sample showing transition from IPMN to the invasive component were selected. From 5 to 10 unstained slides per samples were deparaffinized using xylene, followed by protein digestion through proteinase K, and DNA purified using QIAamp DNA Mini Kit (Qiagen, Hilden, Germany). After purification, DNA quality and quantity were assessed by Qubit fluorometer using Qubit 1X dsDNA HS Assay Kit (Thermo Fisher Scientific, Massachusetts, United States).
3.4.2 Custom Panel design and sequencing
An Ion AmpliSeq Custom Panel was designed to perform multiplex PCR and sequencing of 26 genes selected after a literature review and known to be targeted in pancreatic neoplasms. (table 2). These chosen gene targets were entered into the Ion AmpliSeq Designer™ online tool to generate BED files and the resulting amplicons were divided by the online designer into two primer pools to maximize target specificity. The final target region of our panel was 92.85 kb with the amplicon range 125-175 bp in length and a predicted coverage of 88.17%. A total of 100 ng of DNA was amplified by polymerase chain reaction (PCR) using the 2 primer pools and the AmpliSeq HiFi Master Mix (Ion AmpliSeq Library Kit; Thermo Fisher Scientific, Massachusetts, United States). The multiplexed amplicons were treated with FuPa Reagent (Thermo Fisher Scientific) for partial digestion, and then the amplicons were ligated to adapters from the Ion Xpress Barcode Adapters 1–16 Kit (Thermo Fisher Scientific, Massachusetts, United States), in accordance with the manufacturer's instructions. After a purification step, the size and concentration of the produced amplicon libraries were checked using Qubit 1X dsDNA HS Assay Kit (Thermo Fisher Scientific, Massachusetts,
26
United States). Multiplexed barcoded libraries were diluted at a final concentration of 100 pM, then amplicon libraries were pooled for emulsion PCR using Ion One Touch 2 System (Thermo Fisher Scientific, Massachusetts, United States). The prepared libraries were then sequenced on an Ion S5 Sequencer using an Ion 530 Chip (all Thermo Fisher Scientific, Massachusetts, United States). In order to examine run metrics, including chip-loading efficiency and total read count and quality, all sequencing data generated in the present study were pre-processed using Torrent Suite Software V5.2.2 (Thermo Fisher Scientific, Massachusetts, United States) with the human genome hg19 as the alignment reference. Then the binary alignment map (BAM) files were analyzed using Ion Reporter™ Software v5.2 for variants class filtering and annotations. A workflow with a set of analysis components including location and type of mutation, filtered coverage and p-value variant caller was applied.
To identify confident somatic variants, annotated variants were filtered in accordance to the following criteria:
1) Synonymous single-nucleotide variants (SNV) were excluded
2) Allele coverage ≥ 500× and variant allele frequency (VAF) ≥ 5%, or coverage ≥ 1000× and VAF ≥3%
3) Minor allele frequency <1%
4) Annotated as “Pathogenic”, “Likely pathogenic”, or “Drug response” in the ClinVar database or predicted as deleterious/pathogenic by Functional Analysis Through Hidden Markov Models tools (FATHMM). Lastly, the resulting list of variants was manually reviewed.
Statistical analysis
Categorical variables were analyzed by the Pearson’s chi-square test. To identify cells within a contingency table in which observed values were significantly different from expected ones, the standardized residuals were analyzed. In detail, a Bonferroni correction was used adjust the significance value; the α value was then divided by the number of tests, and it was used to find the new critical absolute value of the standardized residuals [88], [89]. Whenever an expected value within a contingency table was lower than five, the Fisher’s exact test was run. For continuous variables, the normality distribution was tested by the Shapiro-Wilk’s test. Since these variables did not follow a normal distribution, a Wilcoxon’s test was used for two-group comparisons. For more than two groups, the
27 Table 2- Ion AmpliSeq Custom Panel reporting the 26 genes studied in this project
Number Gene References
1 KRAS [8], [42], [58], [72]–[76] 2 GNAS [7], [8], [42], [45], [60], [75], [76] 3 SMAD4 [53], [63], [66], [75], [76] 4 TP53 [3], [15], [42], [53], [75], [76] 5 CDKN2A [58], [64], [65], [75], [76] 6 PIK3CA [77]–[79] 7 STK11 [76] 8 GNAQ [41] 9 SERPINB1 [75], [76] 10 HIF1A [80] 11 SULF1 [75], [76] 12 RPL11 [75], [76] 13 TGFB [75], [76], [81] 14 TNF [75], [76] 15 RNF43 [53], [75], [76] 16 PDCD1 [75], [76] 17 PDL-1 [75], [76] 18 HER2 [75], [76] 19 EZH2 [82] 20 SETD2 [75], [76] 21 NOTCH1 [81], [83]–[85] 22 NOTCH3 [81], [83]–[85] 23 NOTCH4 [81], [83]–[85] 24 JAG1 [86] 25 AGTR1 [75], [76] 26 NFE2L2 [87]
Kruskal-Wallis’ test was used followed by the Dunn’s test for multiple comparisons with the Benjamini-Hochberg correction. Correlations within a single histological class were tested by Spearman’s correlation test. All tests were two-tier. Due to the non-normal distribution, continuous variables are presented as median and interquartile range. Main clinic-pathological features of inv-IPMNs and PDACs were correlated with Overall Survival from surgery (OSS), and Disease-Free Survival (DFS). OSS was defined as the time from surgery until the day of death, while DFS was defined as the time from surgery until the day of disease progression or death from any cause. Patients alive at the time of analyses were censored at the date of their last follow-up visit, whereas those without disease progression were censored at the time of the last radiologic assessment. Continuous variables were
28
dichotomized using the median value. Survival curves were estimated by the Kaplan-Meier method and compared by the log-rank test. P values below 0.05 were considered significant.
29
4 Results
Intraductal papillary mucinous neoplasms (IPMNs)
4.1.1 Patients characteristics
A total number of 73 patients with a histological diagnosis of IPMN were retrospectively evaluated for this project. A preliminary selection was performed, and 29 cases (39.7%) were removed due to an uncertain diagnosis of IPMN or because not suitable for manual microdissection, mainly for the paucity of material, the presence of marked collateral pancreatitis or degenerative changes. After immunohistochemical analyses, 3 more cases were excluded, due to morphological and immunohistochemical features consistent with a diagnosis of IOPN. In addition, ten more cases were excluded due to failing molecular tests. Finally, 31 (42.5%) cases out of the 73 initially selected were enrolled for the IPMN group, 15 (48.4%) females and 16 (51.6%) males. Age of patients ranged from 39 to 88 years (mean age: 66.2 years). Sixteen (51.6%) IPMNs were in the head of the pancreas, 11 (35.5%) in the body and/or tail, while 4 (12.9%) involved the entire pancreas. Sixteen (51.6%) cases were symptomatic at diagnosis, with abdominal discomfort and/or mobility disorders as onset manifestation; in addition, jaundice was also reported in 2 (12.5%) patients and weight-loss in 1 (6.25%). Remaining 15 (48.4%) cases were detected accidentally in the absence of clear symptoms. Three (18.7%) patients, all symptomatic at diagnosis, reported a history of chronic pancreatitis. Based on imaging studies, all cases were pre-operatively classified as MD, BD or MT-IPMNs, and 5 (16.1%) cases were reported as MD-IPMNs, 18 (58.1%) as BD-IPMNs, and 8 (25.8%) as MT-IPMNs. A pathological classification was also assessed after surgery. According to this latter classification, 4 (12.9%) cases resulted as MD-IPMNs, 14 (45.2%) as BD-IPMNs, and 13 (41.9%) as MT-IPMNs (fig 5). In particular, 1 case radiologically evaluated as MD-IPMN was pathologically classified as MT-IPMN, due to the presence of two small peripheral cysts of more than 5 mm in diameter; 4 radiological BD-IPMNs were later reported as MT-IPMNs, due to the small enlargement (6 mm) of the MD, associated with occasional foci of low-grade dysplasia; no discrepancies were noted for the already radiologically-classified MT-IPMNs. In general, concordance between radiology and pathology was observed in 83.9% of cases (p: 0.01).
30 Figure 5 - IPMN classification according to radiological and pathological evaluation
IPMNs involving the entire pancreas were observed more frequently in females (60% vs 25%), while IPMNs limited to the head or body/tail were more frequent in males (respectively 18.6% vs 6.7%, and 56.3% vs 33.3%; p: 0.049).
All 31 IPMNs were divided in three subtypes, based on immunohistochemical (MUC) expression: 19 (61.3%) gastric, 7 (22.6%) intestinal, and 5 (16.1%) pancreatobiliary; 8 (25.8%) cases presented with secondary histological aspects: 6 (75%) pancreatobiliary and 2 (25%) gastric (fig 6).
Fourteen (73.7%) out of 19 gastric-type IPMNs were BD, 2 (10.5%) were MD, and 3 (15.8%) MT-IPMNs; among the intestinal-types, 3 (42.9%) out of 7 were MD, while the other 4 (57.1%) were MT-IPMNs; finally, 4 (80%) out of 5 pancreatobiliary IPMNs were BD, and 1 (20%) was a MT-IPMN (fig. 7).
0 2 4 6 8 10 12 14 16 18 MD BD Mixed 5 18 8 4 14 13 N u mb er IPMN Radiology Pathology 1 6 1
19 Gastric 7 Intestinal 5 Pancreatobiliary
Figure 6 - Main and secondary histological patterns. Green: gastric; Blue: intestinal; Yellow: pancreatobiliary
31 Figure 7 - Association between radiological classification of IPMNs and histological evaluation
Lesions were assigned to only one category consisting of low- or high-grade dysplasia, based on the highest grade of dysplasia encountered in the specimen: 23 (74.2%) cases presented as low-grade IPMNs (low or intermediate grade of dysplasia), and 8 (25.8%) cases as high-grade IPMNs (meaning at least one area of high-grade dysplasia). Among the 23 low-grade IPMNs, 15 (65.2%) belonged to the gastric-type, 4 (17.4%) to the intestinal-type, and 4 (17.4%) to the pancreatobiliary type; regarding the 8 high-grade IPMNs, 4 (50%) were predominantly gastric-type, 3 (37.5%) intestinal-type, and 1 (12.5%) pancreatobiliary-type (fig. 4); however, in 2 (50%) out of 4 cases of gastric-type IPMN, the area with high-grade dysplasia was observed in the pancreatobiliary secondary pattern. Considering both primary and secondary histological patterns, high-grade dysplasia was more frequently related to the intestinal and pancreatobiliary-type IPMN (p: 0.06). Main clinico-pathological features of all IPMNs are reported in table 2.
2 3 0 14 0 4 3 4 1 0 2 4 6 8 10 12 14 16 Gastric Intestinal Pancreatobiliary Number H isto lo gy
32 Figure 7 - Low-grade and high-grade IPMNs. In 2 cases (*) of gastric-type IPMN, high-grade dysplasia
was observed in the pancreatobiliary secondary pattern .
Table 3 - Baseline characteristics of 31 IPMNs
Characteristic Number % Gender Female Male 15 16 48,4 51,6 Age Mean Range 66,2 39-88 Tumor location Head Body-tail Whole pancreas 16 11 4 51,6 35,5 12,9 Symptoms at diagnosis Yes No 16 15 51,6 48,4 Type of IPMN Main duct Branch duct Mixed 5 18 8 16,1 58,1 25,8 Histology Gastric Intestinal Pancreatobiliary 19 7 5 61,3 22,6 16,1 Dysplasia Low-grade High-grade 23 8 74,2 25,8 0 2 4 6 8 10 12 14 16 18 20
Gastric Intestinal Pancreatobiliary 15 4 4 4 * 3 1 Num ber Histology
33
4.1.2 Molecular characteristics
Presence of gene mutations was observed in all 31 IPMNs. Gene mutations were reported as “pathogenic/likely pathogenic/drug response-related” according to the ClinVar database (Clin-mutations), or as “predicted as deleterious/pathogenic” according to Functional Analysis Through Hidden Markov Models tools (FATHMM) (FAT-mutations), whenever the score was > 0.7 [90]. Eight cases (25.8%) out of 31 IPMNs presented with both Clin- and FAT-mutations, 15 cases (48.4%) had only Clin-mutations, and 8 cases (25.8%) had only FAT-mutations (fig 8).
Figure 8 - Distribution of Clin and FAT-mutations among IPMNs
Clin-mutations were observed in 6 different genes, with KRAS, GNAS, and TP53 as more frequently reported, while FAT-mutations were observed in 15 different genes, with JAG1,
TP53, and PIK3CA as the most frequent. Among the 26 genes evaluated in this study, 15
(57.7%) presented with at least one mutation, while no mutations were detected in 11 genes (42.3%), including RNF43, and CDKN2A. Thirteen (41.9%) cases out of 31 only presented with 1 Clin-mutation, and in 11 (84.6%) out of 13 cases this was the only mutation described. Molecular characteristics of the 31 IPMNs are reported in table 4.
Total number of mutated genes was statistically associated with the pathological classification of the IPMNs, and in particular, IPMNs limited to the head or to the body/tail had a higher number of mutated genes than IPMNs involving the whole pancreas (p: 0.02); same correlation was also observed when Clin+FAT mutations were examined (p: 0.03).
8; 25.8% 15; 48.4% 8; 25.8% Clin+FAT Clin only FAT only *
34 Table 4 - Molecular characteristics of the 31 IPMNs. Dark grey: Clin-mutations; light grey: FAT-mutations. P: multiple mutations
ID
Case
KRAS GNAS RNF43 TP53 SMAD4 PIK3CA CDKN2A NOTCH1 NOTCH3 NOTCH4
JAG1 STK11 EZH2 NFE2L2 SETD2 SULF1 AGTR1 GNAQ SE
R PIN B 1 HIF1A RPL11 TGFB TNF PDCD1 PDL -1 HER2 1 2 3 P 5 6 7 8 9 10 P 11 P 13 14 15 16 P 17 18 19 P 20 P P 21 22 23 25 P P 27 29 30 31 32 36 38 40 41 P
35
These correlations were not observed with the radiological classification of IPMNs. IPMNs with secondary histological patterns occurred with a slightly higher number of mutations, and cases with a pancreatobiliary secondary pattern presented with more Clin-mutations than cases with a gastric secondary pattern (p: 0.03). Clin+FAT Clin-mutations, and Clin-mutations were slightly higher in asymptomatic (p: 0.02) than in symptomatic IPMNs (p < 0.01). Gene mutations did not correlate with any other histological features of IPMN.
4.1.3 KRAS and GNAS
KRAS mutations were observed in 22 (71%) cases out of 31: 19 (86.4%) cases out of 22
presented with Clin-mutations of KRAS, and 3 (13.6%) cases with FAT-mutations. Coexistence of both Clin- and FAT-mutations for KRAS was not observed; 1 (4.6%) case out of 22 (IPMN 25) showed more than one FAT-mutation for KRAS, one in codon 143 and one in codon 52. Most KRAS Clin-mutations were in codon 12, exactly 17 (89.4%) out of 19, presenting the following characteristics: 2 cases showed the c.34G>C substitution resulting in p.Gly12Arg mutation, 4 cases showed the c.35G>A (p.Gly12Asp) and 11 cases harbored the c.35G>T (p.Gly12Val). Beyond mutations in codon 12, 1 sample (5.3%) presented a substitution in codon 13 (c.38G>A, p.Gly13Asp), and 1 (5.3%) in codon 61 (c.183A>T, p.Gln61His). Fourteen (73.7%) cases with Clin-mutation of KRAS were gastric-type IPMNs, 3 (15.8%) cases were intestinal-type, and 2 (10.5%) cases were pancreatobiliary-type IPMNs. Regarding the 3 cases with KRAS FAT-mutations, 1 case was a gastric-type IPMN, 1 was a pancreatobiliary-type, and 1 was an intestinal-type IPMN; in particular, the intestinal-type IPMN was the one to present with a double KRAS FAT-mutation. KRAS Clin-mutations were observed in 4 (50%) cases out of 8 with high-grade dysplasia, and in all 4 cases mutation was in codon 12. In 10 (32.3%) IPMNs out of 31, KRAS Clin-mutation was the only mutation reported. KRAS mutations were not significantly associated with any main clinico-pathological feature of IPMNs.
GNAS mutations were observed in 8 (25.8%) cases out of 31: 7 (87.5%) cases with a
Clin-mutation of GNAS, and 1 (12.5%) with a FAT-Clin-mutation. Coexistence of both Clin- and FAT-mutations was not observed; 2 (25%) cases out of 8 presented with more than one
GNAS mutation: 1 case with 2 Clin-mutations, in codon 165 and 294, and 1 case with 3
FAT-mutations, respectively in codon 61, 344 and 389. Five (62.5%) GNAS Clin-mutations out of 8 were observed in codon 201 (5 cases showed the c.602G>A, p.Arg201His mutation and 1 cases showed the c.601C>T, p.Arg201Cys mutation), while single mutation in codon
36
165 (p.Arg165Cys), 294 (p.Gln294Ter), and 258 (p.Arg258Trp) was reported in 3 different cases. Six (85.7%) Clin-mutations were observed in gastric-type IPMNs, and 1 (14.3%) in an intestinal-type IPMN; FAT-mutations in codon 61, 344, and 349 were reported in the same pancreatobiliary-type IPMN. High-grade dysplasia was reported in 2 cases with GNAS Clin-mutation, and in both cases, mutation was in codon 201. GNAS was the only mutation observed in 1 case (3.2%) out of 31, otherwise association with KRAS mutations was the most frequently observed, exactly in 5 (16.12%) out of 31 cases. Mutations of KRAS and/or
GNAS were present in 24 (77.4%) out of 31 cases, or in 21 (67.7%) cases considering
Clin-mutations only. GNAS Clin-mutations were not significantly correlated with the main clinico-pathological features of IPMNs.
4.1.4 TP53 and SMAD4
TP53 mutations were observed in 7 (22.6%) IPMNs out of 31: TP53 Clin+FAT mutations
were reported in 3 (42.9%) cases out of 7, while 3 (42.9%) cases presented with TP53 FAT-mutations only, and 1 (14.2%) case presented with TP53 Clin-FAT-mutations only. Two (28.6%) cases out of 7 presented with more than one Clin-mutation within the same sample. Two out of four IPMNs with TP53 Clin-mutations were MT-IPMNs, one gastric-type, low-grade IPMN (IPMN2) and one intestinal-type, high-grade IPMN; the others were BD-IPMNs, both pancreatobiliary-type, one with high-grade and one with low-grade dysplasia. TP53 Clin-mutations were associated with KRAS Clin-mutations in 50% of cases.
SMAD4 mutations were observed in 6 (19.4%) cases out of 31: 2 (33.3%) cases presented
with SMAD4 Clin-mutations and 4 (66.7%) with SMAD4 FAT-mutations; association between Clin- and FAT-mutations was not observed. One IPMN with SMAD4 Clin-mutation was a BD, gastric-type IPMN with low grade dysplasia; the other one was the already described IPMN2. SMAD4 Clin-mutations were associated with KRAS mutations in both cases.
