Distal cholangiocarcinoma - from novel biomarkers to clinical management and outcome

Cholangiocarcinoma is an aggressive malignacy arising from the biliary tree. Anatomical subtypes ofcholangiocarcinoma differs in tumor biology and clinical management. Distal cholangiocarcinoma (dCCA)originates from the common bile duct. Radical resection is the only curative treatment, for dCCA it entails apancreatoduodenectomy (Whipple procedure). Other periampullary cancers treated with pancreatoduodenectomyinclude pancreatic cancer (PC), ampullary cancer (AC) and duodenal cancer (DC). There is a high rate ofrecurrence after resection for dCCA. This thesis aimed to evaluate the clinical management of dCCA but alsoimprove understanding of the tumor biology and identify novel biomarkers.In paper I, the outcome and prognostic factors of patients treated with pancreatoduodenectomy for dCCA from2008 through 2015 at Skane University Hospital were evaluated. We found the median survival to be 22 monthswhich was worse than most previous studies. The presence of lymph node metastasis was confirmed as animportant prognostic factor.In paper II, the expression of secreted protein acidic and rich in cysteine (SPARC) in resected dCCA speciemns,paired lymph node metastases and normal bile ducts were evaluated using immunohistochemistry (IHC). Wefound SPARC to be expressed in the stromal compartment of dCCA in 80% of samples. Stromal expression wasretained in 68% of lymph node metastases. There was no significant correlation between SPARC expression andsurvival.In paper III, bottom-up mass spectrometry (MS) followed by verification using parallel reaction monitoring (PRM)was used to identify differentially expressed proteins between dCCA samples and normal bile ducts. Bioinformaticanalysis highlighted stromal alterations in dCCA. Forty-six proteins were verified using PRM. Thrombospondin-2(THBS2) was further validated using IHC. We found THBS2 to be upregulated in dCCA epithelial and stromalcompartments. Stromal THBS2 expression was present in 72% of paired lymph node metastases. There was acorrelation between stromal THBS2 expression and poor disease-free survival.In paper IV, we studied the utility of serum THBS2 as a diagnostic biomarker for dCCA and PC. THBS2 levelswere similar in dCCA and PC. THBS2 + CA 19–9 had an area under the curve of 0.92 in differentiating dCCA +PC from healthy donors. THBS2 did not provide utility is discriminating benign disease however, it was diagnosisdependent.In paper V, we used Swedish National Registry for Pancreatic and Periampullary Cancer to study national trendsin frequency of tumor origin, survival, histopathological evaluation and diagnostic accuracy for patients withperiampullary cancers. We found PC diagnosis to be more common in unresected patients. Survival was better forAC and DC then dCCA or PC. Median survival was 33 months for dCCA. Regional differences in tumor originfrequency and histopathological outcomes were identified. Clinical rate of misdiagnosis was 15 % for PC and 23%for non-pancreatic periampullary cancers

Expression of fibroblast activation protein and the clinicopathological relevance in distal cholangiocarcinoma

Objectives: The current survival of patients with distal cholangiocarcinoma (dCCA) is poor. There is a need to develop new prognostic and predictive biomarkers to improve the survival of patients. Fibroblast activation protein (FAP) expression has been associated with survival in several solid malignancies. The goal of this study was to evaluate the expression pattern and prognostic significance of FAP in dCCA. Materials and methods: FAP expression was examined in 57 resected dCCA specimens and 28 paired lymph node metastasis specimens, as well as 10 benign bile ducts using immunohistochemistry. FAP expression was scored in the epithelial and stromal component of the dCCA specimens. The association between FAP expression and prognosis was evaluated using univariable and multivariable statistical modeling. Results: FAP expression was absent in the benign controls. FAP expression was evident in the epithelial 43 (75%) and stromal compartment 34 (60%) of dCCA. There was no association between epithelial or stromal FAP expression and clinicopathological factors. Epithelial FAP expression (HR 0.4 95% CI 0.20–0.78; p=.007) but not stromal FAP expression was significantly associated with better survival in univariable and multivariable analysis. Conclusions: FAP overexpression is evident in dCCA. There was a positive association between epithelial FAP expression and better survival which merits further evaluation

Expression of peritumoral SPARC during distal cholangiocarcinoma progression and correlation with outcome

Objectives: Distal cholangiocarcinoma (dCCA) is a malignancy with a dismal prognosis. One of the hallmarks is the presence of a rich desmoplastic stroma believed to contribute to tumor progression and treatment resistance. Secreted protein acidic and rich in cysteine (SPARC) is a matricellular glycoprotein implicated in tumor-stroma interaction with prognostic correlation across several malignancies. The aim of the present study was to evaluate the expression pattern and prognostic significance of SPARC in resected dCCA and paired lymph node metastasis. Materials and methods: SPARC expression was evaluated in 59 resected dCCA samples and 25 paired lymph node metastases as well as 10 benign bile duct samples using immunohistochemistry. Stromal SPARC expression was scored semi quantitatively. Survival was estimated using the Kaplan–Meier method with associated log-rank test. Results: SPARC expression was absent in normal bile ducts. In dCCA, peritumoral stromal SPARC was detectable in 47/59 (80%) of samples with 40/59 (68%) classified as high stromal SPARC expression. There was a significantly lower proportion of SPARC positive stroma in paired lymph node metastasis 17/25 (68%) than the corresponding primary tumors 24/25 (96%) (p =.016). Stromal SPARC expression was associated with the presence of lymph node metastasis; high SPARC expression 31/40 (78%) versus low SPARC expression 9/19 (47%) (p =.013). In the present material there was no significant association between stromal SPARC expression and survival. Conclusions: Stromal SPARC expression occurs frequently in dCCA. Although significantly lower than in primary tumors stromal SPARC is frequently retained in paired lymph node metastasis suggesting a possible role in the metastatic process of dCCA

Outcome and evaluation of prognostic factors after pancreaticoduodenectomy for distal cholangiocarcinoma

Background The aim of the present study was to examine the outcomes and prognostic factors after surgery with curative intent for distal cholangiocarcinoma during a modern timespan, in a Swedish tertiary referral center. Methods All patients who underwent pancreaticoduodenectomy for distal cholangiocarcinoma between April 2008 and December 2015 were identified. Survival was estimated using the Kaplan-Meier analysis. Demographic, clinical, laboratory and histopathological data were evaluated for prognostic factors relating to mortality, using univariable and multivariable statistical analysis. Results Fifty-four patients were included. The mean age was 68±8 years and 21 (39%) of the patients were female. Jaundice was present at diagnosis in 73% of the patients. There was no 90-day mortality. Complications graded as Clavien-Dindo ≥3 occurred in 10 (19%) of the patients. Twenty-eight (52%) received adjuvant therapy. Overall survival rates at 1, 3, and 5 years were 80%, 21%, and 9.2%, respectively. Median survival was 22.2 months. The presence of lymph node metastases was found to be the only independent predictor of survival (hazard ratio 2.88, 95% confidence interval 1.22-6.84; P=0.016). The total number of lymph node metastases, lymph node ratio or total number of resected nodes did not improve the prediction. Conclusions We found that the recurrence rate was higher and the survival poorer after surgery for distal cholangiocarcinoma than has previously been reported. Lymph node status at the time of resection was the most important prognostic factor for survival in the current material

Mass spectrometry-based analysis of formalin-fixed, paraffin-embedded distal cholangiocarcinoma identifies stromal thrombospondin-2 as a potential prognostic marker

Background: Distal cholangiocarcinoma is an aggressive malignancy with a dismal prognosis. Diagnostic and prognostic biomarkers for distal cholangiocarcinoma are lacking. The aim of the present study was to identify differentially expressed proteins between distal cholangiocarcinoma and normal bile duct samples. Methods: A workflow utilizing discovery mass spectrometry and verification by parallel reaction monitoring was used to analyze surgically resected formalin-fixed, paraffin-embedded samples from distal cholangiocarcinoma patients and normal bile duct samples. Bioinformatic analysis was used for functional annotation and pathway analysis. Immunohistochemistry was performed to validate the expression of thrombospondin-2 and investigate its association with survival. Results: In the discovery study, a total of 3057 proteins were identified. Eighty-seven proteins were found to be differentially expressed (q < 0.05 and fold change ≥ 2 or ≤ 0.5); 31 proteins were upregulated and 56 were downregulated in the distal cholangiocarcinoma samples compared to controls. Bioinformatic analysis revealed an abundance of differentially expressed proteins associated with the tumor reactive stroma. Parallel reaction monitoring verified 28 proteins as upregulated and 18 as downregulated in distal cholangiocarcinoma samples compared to controls. Immunohistochemical validation revealed thrombospondin-2 to be upregulated in distal cholangiocarcinoma epithelial and stromal compartments. In paired lymph node metastases samples, thrombospondin-2 expression was significantly lower; however, stromal thrombospondin-2 expression was still frequent (72%). Stromal thrombospondin-2 was an independent predictor of poor disease-free survival (HR 3.95, 95% CI 1.09-14.3; P = 0.037). Conclusion: Several proteins without prior association with distal cholangiocarcinoma biology were identified and verified as differentially expressed between distal cholangiocarcinoma and normal bile duct samples. These proteins can be further evaluated to elucidate their biomarker potential and role in distal cholangiocarcinoma carcinogenesis. Stromal thrombospondin-2 is a potential prognostic marker in distal cholangiocarcinoma