Molecular, morphological and survival analysis of 177 resected pancreatic ductal adenocarcinomas (PDACs): Identification of prognostic subtypes

Pancreatic ductal adenocarcinoma (PDAC) has generally a poor prognosis, but recent data suggest that there are molecular subtypes differing in clinical outcome. This study examines the association between histopathologic heterogeneity, genetic profile, and survival. Tumor histology from 177 resected PDAC patients with follow-up data was subclassified according to predominant growth pattern, and four key genes were analyzed. PDACs were classified as conventional (51%), combined with a predominant component (41%), variants and special carcinomas (8%). Patients with combined PDACs and a dominant cribriform component survived longer than patients with conventional or other combined PDACs. Genetic alterations in at least two out of four genes were found in 95% of the patients (KRAS 93%, TP53 79%, CDKN2A/p16 75%, SMAD4 37%). Patients with less than four mutations survived significantly longer (p = 0.04) than those with alterations in all four genes. Patients with either wildtype KRAS or CDKN2A/p16 lived significantly longer than those with alterations in these genes (p = 0.018 and p = 0.006, respectively). Our data suggest that the number of altered genes, the mutational status of KRAS and certain morphological subtypes correlate with the outcome of patients with PDAC. Future pathology reporting of PDAC should therefore include the KRAS status and a detailed morphological description.


Histological features. The results are summarized in
TP53 wildtype and TP53 mutations were associated with distinct p53 immunolabelling patterns depicted in Fig. 4. TP53 Mutations were found in 139/177 tumors and detected in the known hotspots (exon 5-8), but not in exon 9. Mutation type 1 (defined as nuclear expression in ≥ 25% of tumor cells and missense mutation) was more common (52%, 92/177) than mutation type 2 (26.6%, 47/177; defined as absence of expression and presence of an intragenic deletion, a nonsense, a frameshift or splice site mutation) (Fig. 4).
CDKN2A/p16 alterations, recorded in 75% of the tumors, were detected by loss of protein expression (130 tumors), which was confirmed by loss of heterozygosity (LOH) in 58 tumors. Three additional tumors showed intact expression despite presence of LOH. Loss of SMAD4 expression was recorded in one third of the cases ( Table 3, Fig. 5).
Interestingly, altered CDKN2A/p16 was significantly more common in patients with positive lymph nodes (p = 0.02). No significant differences between lymph node negative and positive patients were observed for number of mutations, KRAS, TP53, SMAD4 and tumor morphology.
Four investigated metastases (3 from the liver and one from the peritoneum) had the same KRAS mutations as the primary. In case # 5 a low-level mutation in the primary tumor (p.G12D, 3%) was associated with a high-level mutation (p.G12D) in the liver metastasis. In case # 9 intact TP53 in the primary tumor was associated with a strong (> 90%) nuclear labeling in the peritoneal metastasis.
No differences were observed in the mutational status of patients who received neoadjuvant therapy compared to the large group of patients without neoadjuvant therapy (data not shown).
Correlation of molecular with morphologic features. KRAS wildtype was significantly more commonly detected in variants than classical PDACs (conventional or combined) (p = 0.035) ( Table 4). In detail, the group of KRAS wildtype tumors (7.3%, 13/177) included conventional PDACs (7/13), combined PDACs (3/13), and one colloid, one medullary and one tubular carcinoma. The KRAS wildtype tubular carcinoma and one KRAS-mutated tubular carcinoma also lacked alterations in the three other genes (Table 5), a result that was confirmed by additional extended gene analysis. One of the two KRAS-mutated tubular carcinomas harbored CDKN2A/p16 mutations ( Table 5). All papillary carcinomas were associated with mutated KRAS and lacked mutated GNAS 18 , irrespective of the presence of an associated IPMN. CDKN2A/p16 alterations were more strongly associated (p = 0.016) with combined PDACs (60/71) than with conventional PDACs (29/91) ( Table 4).  Patients with colloid, medullary, tubular or papillary carcinoma survived significantly longer than patients with conventional and combined PDACs (p = 0.04) ( Table 2). Longest survival was seen in two of the patients with a tubular carcinoma. They were still alive at the completion of the study, with a survival of > 68.8 and > 55.3 months, respectively. On the contrary, patients with adenosquamous carcinomas had an extremely poor outcome (4.1 and 10.0 months) ( Table 2).
Detailed analysis of the large group of patients with conventional and combined PDACs revealed that patients with a conventional PDAC and those with a cribriform type combined PDAC showed the most favorable overall  Table 2). Combined PDACs with gyriform and complex components were associated with poor survival (12.5 and 10 months).
The median patient survival correlated significantly with the number of mutations (p = 0.04, Fig. 6A). Patients with no or low number of mutations (no mutation > 68.6; one mutation > 45; two mutations > 25.3 months) survived longer than patients with tumors that harbored three (17.6 months) or four mutations (14.5 months).
Correlation of patient outcome and molecular features revealed a significant survival benefit in patients with wildtype KRAS (7.3%, 11/146) compared with that of KRAS mutated patients (92.7%, 135/146) (p = 0.018, median survival > 45 vs. 19.7 months, Fig. 6B). Likewise, patients with intact CDKN2A/p16 (22.8%, 33/145) lived significantly longer than those with altered CDKN2A/p16 (77.2%, 112/145) (p = 0.006; 36.9 vs. 18.8 months, Fig. 6C). Significant prognostic results were further obtained by the combination of CDKN2A/p16 and KRAS. All patients from the cohort followed for survival with an intact status of both genes (n = 4) were still alive at the end of the study. A median overall survival could therefore not be calculated. No prognostic significance was observed for mutated TP53 (with no prognostic difference between TP53 type 1 and type 2 mutations) and SMAD4.

Discussion
Reliable prognostic markers for pancreatic ductal adenocarcinoma (PDAC) patients are so far rare. By analyzing the expression profile of primary PDACs, and human and mouse PDAC cell lines, Collisson recently identified molecular subtypes of PDAC that differed in clinical outcome and drug response 11 . Other studies revealed an association between expression as well as mutational status of key tumor suppressor and oncogenes and patient survival 16,19 . In all these studies, the molecular profile of PDACs is not or only vaguely correlated to the individual morphology of the tumors.
Here we present data of a correlative study on histopathology, molecular profile, and survival in PDACs and related carcinomas of 177 resected patients, with the aim to find prognostic relevant features. As expected, the overall outcome of our patient cohort was bad. Nearly two thirds of patients survived less than 24 months and the 5-year survival did not exceed 21%. However, within this cohort there were patients who survived for up to four years and longer, and whose tumors had a special histopathology and/or molecular status.
Histopathological heterogeneity in PDACs has long been recognized, but it has not been defined in detail, -with the exception of the definition of histological grade and histological variants. Here we classified the pancreatic carcinomas according to a defined growth pattern into three groups. The first group included the conventional type PDACs, which showed an equal mixture of various histological elements (for details see Material and Methods). The second group encompassed the combined PDACs, which were characterized by a dominant     histological component (defined as involving more than 30% of the tumor area). The third group contained PDAC variants (i.e. adenosquamous carcinoma, colloid carcinoma, papillary carcinoma) and special adenocarcinomas such as medullary carcinoma and tubular carcinoma. By analyzing the survival of patients who were ascribed to the various morphological types, we found and confirmed that the colloid carcinoma, the medullary carcinoma and the tubular carcinoma showed a better outcome than conventional PDACs and particularly adenosquamous carcinoma [20][21][22][23] . Patient with a conventional PDAC or a PDAC with dominant cribriform component survived longer than patients with combined PDAC and other histological components, such as the clear cell, the papillary, the gyriform and, in particular, the complex component. PDACs typically harbor KRAS mutations, followed by mutations of CDKN2A/p16, SMAD4 and TP53 10 . These genes are considered the driver genes of PDAC. In addition, there is a multitude of other, but much less frequent, gene alterations, as revealed by whole genome sequencing analysis 6,7 . In our patients, in whom the molecular results were obtained by Sanger methodology and completed in selected cases by NGS of a large PDAC gene panel including the 40 most commonly mutated genes, KRAS was found to be mutated in 92.7%, TP53 in 78.5%, CDKN2A/p16 in 72.9% and SMAD4 in 37.3%. Most of these alterations coexisted in individual tumors and two third of the PDAC patients (68%) harbored alterations in three or all four genes.
Correlation of the individual mutational status with the respective histopathology and survival data revealed a number of findings with prognostic significance. First, the number of mutations per tumor was of prognostic relevance. Patients with one, two or three mutations survived longer than those with alterations in all four genes (> 45 vs. 25.3 vs. 17.6 vs. 14.5 months). Although these results confirm previous reports 16 , so far the correlation between gene status and phenotype has not been analysed in detail. Among the tumors with low number of mutations and prolonged survival, there are particularly colloid carcinomas, medullary carcinomas and tubular type carcinomas, while adenosquamous carcinomas, papillary carcinomas, and the combined PDAC with a complex pattern belong to the carcinomas with a high mutational frequency and poor survival (Fig. 7). Medullary and colloid carcinomas are known for their low prevalence of somatic mutations and good prognosis [20][21][22]24 . Conversely, adenosquamous carcinoma is well known for its many somatic mutations and aggressive behavior 25,26 . The papillary carcinoma variant seems in many cases to be the invasive component of an IPMN 27 , since IPMNs either of the pancreato-biliary (2/6), intestinal (1/6) or gastric type (1/6) were found in four of our six cases. However, it may also occur without an associated IPMN, since in two cases an associated IPMN was not found, and this was also true in another recently reported series of 10 papillary cystic PDACs 28 .  Second, patients with either wild type KRAS or CDKN2A/p16 had a better outcome than those with mutations in these genes, while loss of SMAD4 and intact or altered TP53 was of no prognostic relevance. The statistical significance of wildtype KRAS proved to be so strong that multivariate analysis identified the mutational status of KRAS as an independent prognostic marker. This result confirmed the recently reported survival benefit for PDAC patients with intact KRAS [12][13][14][15] . The largest of these studies by Sinn, included 153 PDAC patients and reported a significantly decreased median survival of 12.7 months for patients with mutated KRAS versus 20.7 months for patients with wild type KRAS 15 . In our study, the corresponding data were 19.7 months for mutated KRAS versus > 45 months with wild type KRAS. The longer survival in our patients may be due to a better selection on the basis of a more extensive KRAS analysis including exon 2 to 4, in contrast to Sinn's study that only focused on KRAS exon 2. This methodological difference probably also explains, why in Sinn's study the KRAS mutation rate of 68% is much lower than in our study with a rate of 93%.
Among the tumors with a wildtype KRAS status were a medullary carcinoma, whose particular genetic and biologic features have been described [20][21][22][23] , and a tubular adenocarcinoma. This latter type of carcinoma, which corresponds by grade to a G1 tumor (WHO 2010), may be considered a conventional PDAC with a strictly well differentiated tubular differentiation. It shows great similarities regarding its morphology, low frequency, rare mutations and long survival to the tubular type carcinoma of the breast [29][30][31][32] . One of the three tubular adenocarcinomas that we identified, not only had an intact KRAS gene, but also harbored no alterations in the 40 most commonly altered PDAC genes, including CDKN2A/p16, TP53 and SMAD4. Moreover, the patient with this tumor is still alive (see Table 4, case # 62), with a follow-up for > 68.6 months. The other two patients with tubular carcinomas had KRAS and/or CDKN2A/p16 alterations, but no loss of SMAD4 or TP53 mutations (details see Table 4), and survived for > 55.3 and 19.3 months.
The relevance of mutated KRAS as a prognosticator in PDAC, a feature shared with bile duct cancer 33 , is biologically most likely related to its driver function. Evidence that KRAS, in interaction with TP53, CDKN2A/p16 and SMAD4, is a driver gene comes from genetically engineered KRAS mouse models of pancreatic cancer 10,34-41 . Moreover, recently developed mouse models in which mutated KRAS can be switched on and off, have impressively demonstrated that continuous oncogenic KRAS signaling is essential for both progression and maintenance of PDAC 42,43 and its metastases 44 .
Patients with wildtype CDKN2A/p16, like patients with intact KRAS, lived significantly longer than those with altered genes (p = 0.006; 36.9 months vs. 18.8 months). Though multivariate analysis failed to identify this gene constellation as an independent prognostic factor, CDKN2A/p16 seems to play a role in patient outcome. All our patients without alterations in CDKN2A/p16 and KRAS were still alive at the completion of the study. On the other hand, loss of CDKN2A/p16, as was recently reported, seems to be associated with lymphatic invasion and widespread metastasis 19 and was significantly associated with lymphatic spread in our study. Interestingly, subgroup analysis revealed a significant higher CDKN2A/p16 mutation rate in combined PDACs with a dominant histological component compared to conventional PDACs. This suggests that a driver gene might be linked to the presence of dominant histological components in PDACs.
The prognostic relevance of SMAD4 has been the subject of controversial discussions over the last years. While several studies associated the loss of SMAD4 with poor prognosis or early metastatic disease 14,19,45,46 , others were unable to confirm these results 16,47,48 . Likewise, no differences in the survival data in patients with intact and loss of SMAD4 were observed in our study (p = 0.15, median overall survival 22.2 vs. 17.6 months).
In summary, our findings specify the prognostic relationship between the histopathology and molecular profile, based on the morphologic stratification of PDACs in subtypes and variants, and the mutational status of the four driver genes, KRAS, CDKN2A/p16, SMAD4 and TP53. Because our data suggest that PDAC subgroups can be identified (low to intermediate aggressive PDACs versus highly aggressive PDACs, see Fig. 7), tailored therapy options may be discussed. Patients with altered CDKN2A/p16 might benefit from more aggressive preoperative therapies, whereas patients with wildtype KRAS might best be treated by upfront surgery followed by adjuvant therapies. In view of these potential therapies it should be considered to include the status of the four driver genes into the pathological reporting of PDACs in the future [12][13][14][15]49 . Given the potential clinical implications of our results, validation in independent PDAC cohorts is of utmost importance.

Material and Methods
The study was approved by the ethics committee of the TU München, Germany (documents no. 1926/2007 and 126/2016S). Written informed consent was obtained from all patients. All methods were performed in accordance with the relevant guidelines and regulations.   Table 2) and/or arterial resection (n = 4), perioperative death/death due to complications (n = 1) and recurrence surgery (n = 1) were excluded from survival analysis. Three patients were lost to follow-up.
Histologic analysis. All PDACs were histologically classified into conventional PDACs, combined PDACs in which, in addition to the classical tubular growth pattern, a special histologic component was present in more than 30% of the tumor area, and variants with a special pattern in at least 50% of the tumor area. PDACs with a conventional morphology were largely composed of well-to moderately developed tubular and duct-like structures and showed only few other structures, such as glands with clear cell morphology, cribriform architecture, papillary epithelial lining and individual pleomorphic cells 17 . Combined PDACs with dominant histological features showed either a clear-cell, cribriform, gyriform, papillary, micropapillary or complex component against a background of tubular architecture (see Fig. 1). The complex component was characterized by small irregular glands mixed with solid or cribriform cell sheets and individual pleomorphic cells. PDAC variants included colloid, adenosquamous, and papillary carcinoma (see Fig. 1A-F). Among the pancreatic carcinomas that have not yet been regarded as PDAC variants are medullary and tubular carcinoma. The tubular adenocarcinoma is separated from conventional PDAC by its entirely well differentiated tubular architecture that is characterized  by small tubular glands diffusely infiltrating the pancreatic parenchyma and difficult to distinguish from equally sized normal ducts (see Fig. 2).
Immunohistochemical analysis. All  Statistical analyses. The statistical analyses performed are described in Supplementary materials and methods.