结果：导管内乳头状粘液性肿瘤（IPMN）表现出被激活的免疫微环境（TME，tumor immune microenvironment），包括免疫抑制细胞和调节细胞的浸润：Foxp3+，CD68+以及免疫检查点蛋白的表达：LAF3,PD-1,PD-L1,VISTA。虽然在IPMN和浸润性PDAC之间未发现CD45+浸润细胞的差异，免疫细胞亚群在癌前病变发展至浸润性癌的过程中有明显的调节作用。相比IPMN，在相关浸润性PDAC中发现以下细胞亚群密度明显增加：CD68+细胞， p<0.001; Foxp3+ 细胞, p=0.04; LAG3+ 细胞, p=0.017;PD-L1+ 细胞, p=0.03。而CD20+, CD4+, CD8+, PD-1+,VISTA+细胞密度无明显差异。
1.Progression of the Immune Response in Matched Intraductal Papillary Mucinous Neoplasms and Associated Invasive Ductal Adenocarcinomas
（Naziheh Assarzadegan, Braxton Alicia, Dwayne Thomas, et al.）
Results: IPMNs demonstrated an active TME with infiltration of immunosuppressive and regulatory cells: Foxp3+ and CD68+ and expression of immune checkpoint proteins: LAG3, PD-1, PD-L1 and VISTA. While no overall difference in CD45+ infiltrating cells was seen between IPMN and matched invasive PDAC on average, there was marked modulation of immune cell subsets with progression from precursor lesion to invasion. The density of the following cell subsets increased significantly in PDAC as compared to their matched IPMN: CD68+ cells, p<0.001; Foxp3+ cells, p=0.04; LAG3+ cells, p=0.017 and PD-L1+ cells, p=0.03. No significant differences were seen in density of CD20+, CD4+, CD8+, PD-1+ or VISTA+ cells.
Conclusions: The infiltration pattern of IPMNs suggests that groundwork for the immunosuppressive TME in PDAC may begin far prior to the presence of an invasive PDAC. Immune cell subsets evolved as tumors progressed from IPMNs to PDAC in matched patients, with increases seen in immunosuppressive cells and in regulatory immune checkpoint proteins. The presence of immunosuppression in precursor lesions, paired with modulation of the TME as PDAC develops, suggests multiple time points for potential intervention with immune-based therapies, both in a prophylactic and treatment paradigm. Additional studies are crucial to best understand when and how immune-based treatments can be used to alter the TME at different stages of tumor development.
2.High Tumor Mutational Burden Identifies Specific Subsets of Pancreatic Cancer Patients with Prolonged Survival and Improved Anti-tumor Immunity
（Eva Karamitopoulou-Diamantis, Andreas Andreou, Anna-Silvia Wenning, et al.）
Results: Median and mean TMB values were 3.36 and 5.64 respectively. Overall, 12 TMB-high cases (median:14.025, range:10.21-129) were detected. They comprised all five MSI-high cases (100%), five LTSs (20%) and two conventional MSS PDACs (1.66%). TMB-high PDACs exhibited high T cell density and significantly increased CD3+CD4+FOXP3-T cells (P=0.0398) and DC-LAMP+dendritic cells (DC; P=0.0093), compared with TMB-low cases. Five TMB-high cases (41.66%) were also PD- L1+ exhibiting various PD1/PD-L1 staining patterns. ARID1A- and ERBB3-alterations were more frequent in TMB-high tumors. The OS of TMB-high cases ranged between 5-71 months (median:27), whereas that of the TMB-low cases between 3-161 months (median:13), P<0.001.
Conclusions: High TMB mostly identifies patients from specific PDAC-subsets such as LTS (MSS) and MSI-high cases. Their microenvironment displays strong anti-tumor immune response, mediated by increased DC counts, which have the capacity to initiate and regulate T cell responses, as well as CD3+CD4+FOXP3-T cells, known to exhibit direct cytotoxicity against tumor cells as well as potentiate the DCs. TMB-high PDACs frequently harbor other actionable alterations, such as defective mismatch repair (MSI) and DNA Damage Response and Repair (ARID1A), as well as ERBB3-alterations. These results suggest that PDAC-patients with TMB-high tumors might be good candidates for combinatorial treatments including immunotherapy.
3.Loss of PTEN and Expression of GLUT1 Predict the Metastatic Progression of Pancreatic Neuroendocrine Tumors
（Azfar Neyaz, Jasmijn Westendorp, Abigail Wald, et al.）
Results: Aberrant expression of PTEN, GLUT1, or both proteins were seen in 46 (13%), 43 (12%), and 17 (5%) PanNETs. Both loss of PTEN and overexpression of GLUT1 correlated with large tumor size, high WHO grade, advanced T-stage, loss of ATRX/DAXX, and presence of synchronous and metachronous metastases (p<0.02). Among 271 patients without synchronous metastasis, the 5-year RFS rate for PTEN-negative and/or GLUT1-positive patients was 40% as compared to 89% for PTEN-positive/GLUT1-negative patients (p<0.01). By multivariate analysis, aberrant expression for PTEN and/or GLUT1 was a negative prognostic factor for RFS, and independent of tumor size, WHO grade, lymphovascular and perineural invasion, N-stage, and ATRX/DAXX loss (p<0.01). Moreover, aberrant expression of PTEN, GLUT1, and/or ATRX/DAXX was associated with 5-year RFS rate of 47% as compared to 94% for patients without aberrant expression for these proteins (p<0.01), and remained an independent, negative prognostic factor for RFS (p<0.01).
Conclusions: Loss of PTEN and expression of GLUT1 in PanNETs represent prognostic biomarkers of poor patient outcome; thus, genomic alterations in PTEN and VHL may play a significant role in PanNET pathogenesis.
结果：721例(7.6%)PDAC为KRAS野生型(KRASwt)和8723例（92.4%）PDAC为KRAS突变型（KRASm）。年龄相似，KRASwt在男性中更常见。基因组改变（GA，genomic alterations）x相似。对于目前无法靶向的基因组改变，KRASwt组中ARID1A和RB1的基因组改变频率更高，而KRASm组中以TP53、CDKN2A/B、MTAP、SAMD频率更高。除了在1.6%KRASm胰腺导管腺癌中已发现的KRAS G12C突变，目前一些潜在靶向基因在KRASwt中突变频率较高，包括ERBB2(扩增或短变异GA）,BRAF、PIK3CA、FGFR1/2、PTEN、ATM。虽然频率较低，KRASwtzu 胰腺导管腺癌具有更高频率的与免疫检查点抑制剂有效性（IPCI）有关的生物标志物，包括PBRM1基因组突变和TMB＞10个突变/Mb，而KRASm组胰腺导管腺癌患者有较高频率的PD-L1免疫组化低表达。
4.Genomic Landscape of Clinically Advanced KRAS Wild-Type Pancreatic Ductal Adenocarcinoma (PDA)
（Serenella Serinelli, Daniel Zaccarini, Vamsi Parimi, et al.）
Results: 721 (7.6%) PDAs were KRAS wild-type (KRASwt) and 8,723 (92.4%) PDAs were KRAS mutated (KRASm), Table 1. Ages were similar, KRASwt were more often male gender. GA/tumor were similar. For currently not targetable GA, KRASwt featured greater frequencies of ARID1A and RB1 GA and KRASm featured greater frequencies in TP53, CDKN2A/B, MTAP, and SMAD4. With the exception of the KRAS G12C GA identified in 1.6% of the KRASm PDA, greater frequencies in currently potentially targetable genes were seen selectively among KRASwt including GA in ERBB2 (amplifications and short variant GA), BRAF, PIK3CA, FGFR1/2, PTEN, and ATM. Although frequencies were low, the KRASwt PDA featured greater frequencies of biomarkers associated with immune checkpoint inhibitor efficacy (IPCI) including PBRM1 GA and TMB > 10 mutations/Mb although KRASm PDA had a higher frequency of PD-L1 low but not high IHC expression.
Conclusions: Therapeutic management of PDA remains an important challenge. Our results confirm those reported in the literature, according to which many targetable genes and a high TMB are significantly more common among KRAS wild-type PDAs. Moreover, classification of PDAs into KRAS mutated vs KRAS wild-type categories may be clinically beneficial in summarizing the genomic profile and the available targeted therapies. Our findings highlight the importance of comprehensive genomic analysis to find molecular targets including gene fusions for precision oncology.
5.BLM, MYH9, and PCK1 Mutations are Possible Markers of Metastatic Potential in Pancreatic Solid Pseudopapillary Tumors
（Benjamin VanTreeck, Hee Eun Lee, Colton McNinch, Lizhi Zhang）
Results: Clinicopathologic data for the 9 PSPT cases is summarized in Table 1. RNA sequencing revealed no significant gene fusions identified in all PSPT cases. All cases of PSPT demonstrated biallelic CTNNB1 missense mutations in exon 3. Variant analysis of all cases showed 101 gene mutations exclusively shared by metastatic PSPT that were not identified in nonmetastatic PSPT. Of the 101 genes, the following 3 genes may potentially act as tumor suppressors and participate in metastatic progression: BLM, MYH9, and PCK1. Each case of metastatic PSPT demonstrated one or more biallelic mutations in each of the previously mentioned genes leading to missense, frameshift, stop gained, splice donor, splice acceptor, or splice region protein alterations.
A single mutation in MYH9 (c.5797C>T) is the only confirmed pathogenic mutation based on a review in GeneReviews/OMIM databases. Two addition mutations, BLM (c.377C>T) and PCK1 (c.228G>T), each have an uncertain significance in INVITAE and Illumina databases, respectively. The remaining mutations have an unknown pathogenic potential.
Conclusions: We demonstrated that all non-metastatic and metastatic PSPT in our cohort contain a CTNNB1 missense mutation in exon 3. Furthermore, we identified 3 genes only mutated in metastatic PSPT, BLM, MYH9, and PCK1, that may potentially participate in PSPT metastatic progression. Additional investigation is required to determine if these 3 genes play a significant role in PSPT metastatic progression and are definitively associated with metastatic potential.
设计：61例病例（39PDC,12iCCA，10eCCA）使用自定义设计的213基因二代测序进行检测。为了检测基因融合，使用了市售的FusionPlex CTL panel (ArcherDx)。从显微切割、福尔马林固定和石蜡包埋组织中提取总核酸，用于NGS检测，并在Illumina NextSeq进行测序。数据分析使用BWA和Pisces V2 (Illumina)用于变式调用(variant calling)，ArcherDx软件用于融合，以及内部自定义的拷贝数变异(CNV)。
6.Genomic Profiling of Pancreatic Ductal Adenocarcinoma, Intrahepatic and Extrahepatic Cholangiocarcinoma
（Matthew Gosse, Ramakrishna Sompallae, Natalya Guseva, et al.）
Background: In the work up of a carcinoma of unknown primary, the distinction between pancreatic ductal adenocarcinoma (PDC) and cholangiocarcinoma (CCA) is a challenging endeavor due the overlap in morphology and lack of immunohistochemistry (IHC) markers. The distinction has important ramifications for treatment and prognosis. Genomic analyses of these tumor types have been described. Intra- and extra-hepatic cholangiocarcinoma (iCCA and eCCA) had distinct molecular profiles. iCCA are enriched in IDH1/2 and BAP1 mutations and FGFR2 fusions whereas eCCA and PDC had a higher rate of TP53 and KRAS mutations. In this study, we directly compared molecular profiles of our institutional cohort of iCCA, eCCA, and PDC to aid in the distinction of the
diagnosis of these neoplasms.
Design: Sixty-one cases (39 PDC, 12 iCCA, and 10 eCCA) were tested using a custom-designed 213-gene next generation sequencing panel. For detection of gene fusions, the commercially available FusionPlex CTL panel (ArcherDx) was used. Total nucleic acid extracted from microdissected, formalin-fixed, paraffin-embedded tissue was used to generate NGS libraries and sequencing was performed on Illumina NextSeq. Data were analyzed using the BWA and Pisces v2 (Illumina) for variant calling, ArcherDx software for fusion, and an in-house built custom pipeline for copy number variation (CNV).
Results: The demographics of the cohort are shown in Table 1. Somatic mutations detected in all tumors are summarized in Fig. 1. Mutations in IDH1 and BAP1, and FGFR2 fusions were most commonly detected in iCCA; eCCA and PDC shared a similar mutation profile of TP53 and KRAS variants present in 70-80% of cases. KRAS G12R (9) and Q61H (3) were only detected in PDC; G12C in 2 eCCA, and G13D in 1 iCCA (Fig. 2). Gene rearrangements were detected in 4/11 iCCAs (3 with FGFR/BICC1 and 1 with FGFR/ SORBS1). No fusions were detected in eCCA (10) or PDC (32). CNV analysis showed CDKN2A/B loss in 18% PDC (n=39), 1 eCCA (n=12) and none in iCCA (n=10). All 4 iCCAs with BAP1 mutations showed BAP1 lost by IHC and all 4 cases of intact BAP1 expression by IHC showed no BAP1 mutation.
Conclusions: Our data show that the presence of IDH1, BAP1 mutation and FGFR2 fusion supports the diagnosis of iCCA. Tumors with loss of CDKN2A/B and/or SMAD4 and ATM mutations were most likely PDC. Our results support the previous molecular findings in CCA and PDC. The difference in KRAS variants in these two tumor types observed in this small cohort also warrants further investigation.
设计：对37例GBC患者的石蜡包埋FFPE组织中提取DNA，进行高深度、均匀覆盖的测序，(ION, Personal Genome)，使用一个与大肠癌相关的22基因panel进行分析。胚系突变使用变体识别从血液和非肿瘤对照样本中进行筛选。
结果：共检测到178个突变，平均每个肿瘤有4.8个突变（范围1-15）。在SMAD4 (60.60%), NOTCH1 (45.45%), ERBB2 (45.45%), PIK3CA (33.33%) and MET (30.30%), PTEN (30.30%), EGFR (24.24%), KRAS (21.21%), BRAF (9.09%) and NRAS (6.06%) 检测到38个具有个性化治疗潜能的体突变。在66.7%（2/3）突变相关病例中淋巴结转移不明显。在2/3患者中BRAF基因突变与PIK3CA、TP53和EGFR基因突变互斥。在GBC中还观察到EGFR、KRAS和NRAS的热点突变，在结肠癌中也存在（见表1）。表1进一步详细说明了共存的突变。EGFR阳性的病例中有3/8例发生KRAS突变。TP53突变与组织病理分化相关（P=0.0001），ERBB4&ALK突变与坏死相关（P=0.012，0.027），EGFR突变和粘液性肿瘤相关（P=0.023），ERBB2基因突变和T分期相关（P=0.036）。脉管侵犯、神经浸润和淋巴结转移没有显著的遗传相关性。
结论：该研究为胆囊肿瘤的遗传改变和参与通路提供了一个概述。89.1%病例存在靶向突变，包括SMAD4, NOTCH1, ERBB2&4, PIK3CA, MET, PTEN, EGFR, KRAS, BRAF ,NRAS，与结肠癌基因突变存在重叠。这一研究支持了跟随结肠癌治疗运用基因图谱靶向治疗胆囊癌的可能性。
7.Targeted Genomic Profiling of Gallbladder Carcinoma
（Nuzhat Husain, Sridhar Mishra, Swati Kumari, et al.）
Background: Gallbladder cancer (GBC) often presents in late stage of disease with poor prognosis. Mutations for selection of targeted therapies are limited. Next-generation sequencing (NGS) using frequently mutated genes for GBC may provide a reference for clinical management. The current study identifies cancer-related genetic alterations by NGS in cases of GBC and their association with clinicopathological features.
Design: DNA from FFPE tissue of 37 cases of GBC was sequenced to high depth, uniform coverage (ION, Personal Genome Machine) and analysed for genomic alterations using a 22 gene panel related to colorectal cancer. The germline variants were filtered using variant calls from blood and non-tumor control samples.
Results: A total of 178 alterations were identified for an average of 4.8 alterations per tumor (range 1–15). A total of 38 different genomic alterations with the potential to personalize therapy in SMAD4 (60.60%), NOTCH1 (45.45%), ERBB2 (45.45%), PIK3CA (33.33%) and MET (30.30%), PTEN (30.30%), EGFR (24.24%), KRAS (21.21%), BRAF (9.09%) and NRAS (6.06%) gene. In 66.67% (2/3) co-mutated cases lymph node metastasis was not evident. BRAF gene mutation was mutually exclusive with PIK3CA, TP53 (n=3/3) and EGFR in 2/3 patients. Hot spot mutations of EGFR, KRAS and NRAS as seen in colorectal cancers were also observed in GBC (Table 1).Table 1 further details coexisting mutations. KRAS mutation was observed in 3/8 in EGFR positive cases. TP53 mutation was associated with histopathological differentiation (p=0.0001), ERBB4 & ALK mutation was associated with necrosis (p=0.012, 0.027), EGFR mutation was associated with mucinous tumors (p=0.023) and ERBB2 gene mutation was associated with T stage (p=0.036). No significant correlation of genetic profile with lympho-vascular invasion, perineural invasion, lymph-node metastasis was observed.
Conclusions: The current study provides an overview of genetic alterations and pathways involved in gallbladder tumorigenesis. Targetable mutations were identified in 89.91% cases and included SMAD4, NOTCH1, ERBB2&4, PIK3CA, MET, PTEN, EGFR, KRAS, BRAF and NRAS and were overlapping those of colorectal carcinoma. The study supports the possible use of genetic profiling and targeted therapy along lines of colorectal cancer.
结果：我们检测了10例GBC患者，通过SEMA4对其进行分子分析（男性5例，女性5例，中位年龄68岁）。肿瘤平均大小2.2cm，良性2例，中分化6例，差分化2例，其中pT1 2例，pT2 6例，pT3 2例。组织学亚型上有8例未见明显特异，1例有粘液性表现，1例有印戒细胞特征。总体来看，42个体细胞突变包含34个基因被检测到。在这些突变中，在22个基因中的28个变异属于Tier1或Tier2改变，在治疗、预后和诊断方面具有很强的临床意义，而14例是Tier3，在一般或特殊的数据库中观察到显著的等位基因频率。平均检测的突变数与患者比值为4.4（Tier1/2 3.0,Tier3 1.4,整体范围1-11）。
在7例病例中检测到TP53突变（6个不同变异），MDM2扩增2例，ARID1A SNVS 2例，与已公布的频率相似，但6例TP53变异中有4例未在GBC中报道过。其他每个改变可以1名患者中见到。我们发现了RHOA和CDK2基因改变，以前从未在GBC病例中报道过。在ATR, BAP1, NOTCH1, SETD2, CTNNB1, PIK3R1, PIK3CA,EGFR发现了新的Tier1/2变异，在ARID1A, ATXR, CHEK1, RAD51C, FANCA, NOTCH 3, SETD2, SMARCB1, PIK3CB, NF1, ESR1发现了Tier3变异。通过Cytoscape，一种可视化分子相互作用网络的生物信息平台，初步分析强调了ERBB,PI3K和RAS信号，非RTK信号的激活和DNA损伤反应。
8.Molecular Analysis Reveals Novel Somatic Alterations in Resected Gallbladder Carcinomas
（Zhen Zhao, Stephen Ward）
Background: Gallbladder cancer (GBC) often presents at an advanced stage and has a dismal prognosis. Molecular characterization of GBC has thus far been limited. Our institution utilizes the SEMA4 solid tumor panel, a next generation sequencing analysis of 161 of the most relevant cancer drivers. We report several novel molecular alterations in GBC from our institution identified by the SEMA4 panel.
Design: SEMA4 solid tumor panel was applied to 10 cases of surgically resected GBC. Demographics, tumor size, grade, stage, and histologic subtype were recorded. Molecular findings were compared with existing published data.
Results: We identified 10 patients with GBC that underwent molecular analysis by SEMA4 (5 male, 5 female, median age 68 years). Mean tumor size was 2.2 cm, 2 were well-, 6 were moderately-, and 2 were poorly-differentiated while 2 were pT1, 6 were pT2 and 2 were pT3. The histologic subtype was not otherwise specified in 8 and mucinous in 1, while 1 had signet ring cell features. Overall, 42 somatic genetic alterations involving 34 genes were identified. Of these, 28 variants in 22 genes were Tier 1 or Tier 2 changes with strong or potential clinical significance of therapeutic, prognostic and diagnostic actionability, while 14 were Tier 3, yet observed at a significant allele frequency in the general or specific databases. The average detected alteration number/patient was 4.4 (3.0 Tier 1/2, 1.4 Tier 3; overall range 1-11).
TP53 mutation was seen in 7 cases (6 different variants), MDM2 amplification in 2, and ARID1A SNVs in 2, similar to published frequencies, though 4 of the 6 TP53 variants have not been previously reported in GBC. All other alterations were seen in 1 patient each. We found alterations in RHOA and CDK2, previously not reported in GBC. Novel Tier 1/2 alterations were seen in ATR, BAP1, NOTCH1, SETD2, CTNNB1, PIK3R1, PIK3CA and EGFR, and novel Tier 3 variants of ARID1A, ATXR, CHEK1, RAD51C, FANCA, NOTCH 3, SETD2, SMARCB1, PIK3CB, NF1, and ESR1 were identified. Using Cytoscape, an open-source bioinformatics platform for visualizing molecular interaction networks, preliminary analysis highlights activation of ERBB, PI3K and Ras signaling, non-RTK signaling, and impaired DNA damage response.
Conclusions: Our work highlights several new genetic alterations that may be important in the pathogenesis of GBC. Validation of these findings may lead to potential targeted therapies and improved patient outcomes for these devastating tumors.
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