Neoadjuvant chemo- or chemo-radiation-therapy of pancreatic ductal adenocarcinoma differentially shift ECM composition, complement activation, energy metabolism and ribosomal proteins of the residual tumor mass.

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal cancer, often diagnosed at stages that dis-qualify for surgical resection. Neoadjuvant therapies offer potential tumor regression and improved resectability. Although features of the tumor biology (e.g., molecular markers) may guide adjuvant therapy, biological alterations after neoadjuvant therapy remain largely unexplored. We performed mass spectrometry to characterize the proteomes of 67 PDAC resection specimens of patients who received either neoadjuvant chemo (NCT) or chemo-radiation (NCRT) therapy. We employed data-independent acquisition (DIA), yielding a proteome coverage in excess of 3500 proteins. Moreover, we successfully integrated two publicly available proteome datasets of treatment-naïve PDAC to unravel proteome alterations in response to neoadjuvant therapy, highlighting the feasibility of this approach. We found highly distinguishable proteome profiles. Treatment-naïve PDAC was characterized by enrichment of immunoglobulins, complement and extracellular matrix (ECM) proteins. Post-NCT and post-NCRT PDAC presented high abundance of ribosomal and metabolic proteins as compared to treatment-naïve PDAC. Further analyses on patient survival and protein expression identified treatment-specific prognostic candidates. We present the first proteomic characterization of the residual PDAC mass after NCT and NCRT, and potential protein candidate markers associated with overall survival. We conclude that residual PDAC exhibits fundamentally different proteome profiles as compared to treatment-naïve PDAC, influenced by the type of neoadjuvant treatment. These findings may impact adjuvant or targeted therapy options.

Oncological recurrence following pathological complete response after neoadjuvant treatment in patients with esophageal cancer - a retrospective cohort study.

BACKGROUND: To evaluate recurrence in patients with post-neoadjuvant pathological complete response (pCR) and in patients with complete response of primary tumor but persisting lymphatic spread of disease (non-pCR, ypT0ypN +) of esophageal cancer. METHODS: Seventy-five patients (63 pCR, 12 non-pCR) were analyzed retrospectively. Pattern and incidence of local and distant recurrence as well as the impact on overall (OS) and disease-free survival (DFS) were evaluated. The efficacy of neoadjuvant chemotherapy according to FLOT protocol was compared to neoadjuvant chemoradiation according to CROSS protocol. RESULTS: In the pCR group, isolated local recurrence was diagnosed in 3%, while no isolated local recurrence was observed in the non-pCR group due to the high incidence of distant recurrence. Distant recurrence was most common in both cohorts (isolated distant recurrence: pCR group 10% to non-pCR group 55%; simultaneous distant and local recurrence: pCR group 3% to non-pCR group 18%). Median time to distant recurrence was 5.5 months, and median time to local recurrence was 8.0 months. Cumulative incidence of distant recurrence (with and without simultaneous local recurrence) was 16% (± 6%) in pCR patients and 79% (± 13%) in non-pCR patients (hazard ratio (HR) 0.123) estimated by Kaplan-Meier method. OS (HR 0.231) and DFS (HR 0.226) were significantly improved in patients with pCR compared to patients with non-pCR. Advantages for FLOT protocol compared to CROSS protocol, especially with regard to distant control of disease (HR 0.278), were observed (OS (HR 0.361), DFS (HR 0.226)). CONCLUSION: Distant recurrence is the predominant site of treatment failure in patients with pCR and non-pCR grade 1a regression, whereby recurrence rates are much higher in patients with non-pCR.

Circulating Epithelial Cells in Patients with Intraductal Papillary Mucinous Neoplasm of the Pancreas.

Intraductal papillary mucinous neoplasm (IPMN) is the most common pancreatic cyst and a precursor of pancreatic cancer (PDAC). Since PDAC has a devastatingly high mortality rate, the early diagnosis and treatment of any precursor lesion are rational. The safety of the existing guidelines on the clinical management of IPMN has been criticized due to unsatisfactory sensitivity and specificity, showing the need for further markers. Blood obtained from patients with IPMN was therefore subjected to size-based isolation of circulating epithelial cells (CECs). We isolated CECs and evaluated their cytological characteristics. Additionally, we compared Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations in CECs and the primary IPMN tissue, since KRAS mutations are very typical for PDAC. Samples from 27 IPMN patients were analyzed. In 10 (37%) patients, CECs were isolated and showed a hybrid pattern of surface markers involving both epithelial and mesenchymal markers, suggesting a possible EMT process of the cells. Especially, patients with high-grade dysplasia in the main specimen were all CEC-positive. KRAS mutations were also present in CECs but less common than in IPMN tissue. The existence of CEC in IPMN patients offers additional blood-based research possibilities for IMPN biology.

Cancer-associated Macrophage-like Cells in Patients with Non-metastatic Adenocarcinoma of the Esophagus - Cytomorphological Heterogeneity.

Introduction: Esophageal adenocarcinoma (EAC) often recurs systemically despite therapy with a curative aim. New diagnostic and therapeutic approaches are urgently needed. A promising field is liquid biopsy, meaning the investigation of tumor-associated cells in the peripheral blood, for example cancer-associated macrophage-like cells (CAML). The aim of this multicentric study was to investigate the presence and cytomorphological appearance of CAML in patients with non-metastatic and operable esophageal cancer. Methods: Blood samples from 252 patients with locally advanced EAC were obtained before starting curative treatment including surgery, and then processed using ScreenCell® filtration devices. Cytological analysis was performed via May-Grünwald-Giemsa staining. CAML were defined by their morphological characteristics. We also performed immunofluorescence staining with the mesenchymal marker vimentin on a subset of our study cohort. Results: We detected cytomorphologically heterogeneous CAML in 31.8% (n=80) patients. Their presence and cell count did not correlate significantly with pretherapeutic cTNM. Even in patients with small tumors and no lymph-node infiltration, cell counts were high. CAML showed heterogenous staining patterns for vimentin. Conclusion: This is one of the first studies demonstrating the presence and phenotype of CAML in a uniquely broad cohort of EAC patients. As they are believed to be representatives of the inflammatory tumor microenvironment shed into the bloodstream, their presence in non-metastatic EAC is a promising finding.

Pathological complete response in multimodal treatment of esophageal cancer: a retrospective cohort study.

To evaluate pathological complete response (pCR, ypT0ypN0) after neoadjuvant treatment compared with non-complete response (non-CR) in patients with esophageal cancer (EC), and 393 patients were retrospectively analyzed. Survival probability was analyzed in patients with: (i) pCR vs non-CR; (ii) complete response of the primary tumor but persisting lymphatic metastases (non-CR-T0N+) and (iii) pCR and tumor-free lymphnodes exhibiting signs of postneoadjuvant regression vs. no signs of regression. (i) Median overall survival (mOS) was favorable in patients with pCR (pCR: mOS not reached vs. non-CR: 41 months, P < 0.001). Multivariate analysis revealed that grade of regression was not an independent predictor for prolonged survival. Instead, the achieved postneoadjuvant TNM-stage (T-stage: Hazard ratio [HR] ypT3-T4 vs. ypT0-T2: 1.837; N-stage: HR ypN1-N3 vs. ypN0: 2.046; Postneoadjuvant M-stage: HR ypM1 vs. ycM0: 2.709), the residual tumor (R)-classification (HR R1 vs. R0: 4.195) and the histologic subtype of EC (HR ESCC vs. EAC: 1.688) were prognostic factors. Patients with non-CR-T0N+ have a devastating prognosis, similar to those with local non-CR and lymphatic metastases (non-CR-T + N+) (non-CR-T0N+: 22.0 months, non-CR-T + N-: mOS not reached, non-CR-T + N+: 23.0 months; P-values: non-CR-T0N+ vs. non-CR-T + N-: 0.016; non-CR-T0N+ vs. non-CR-T + N+: 0.956; non-CR-T + N- vs. non-CR-T + N+: <0.001). Regressive changes in lymphnodes after neoadjuvant treatment did not influence survival-probability in patients with pCR (mOS not reached in each group; EAC-patients: P = 0.0919; ESCC-patients: P = 0.828). Particularly, the achieved postneoadjuvant ypTNM-stage influences the survival probability of patients with EC. Patients with non-CR-T0N+ have a dismal prognosis, and only true pathological complete response with ypT0ypN0 offers superior survival probabilities.

Protocol of the HISTOTHERM study: assessing the response to hyperthermia and hypofractionated radiotherapy in recurrent breast cancer.

Introduction: Breast cancer is globally the leading cancer in women, and despite the high 5-year survival rate the most frequent cause of cancer related deaths. Surgery, systemic therapy and radiotherapy are the three pillars of curative breast cancer treatment. However, locoregional recurrences frequently occur after initial treatment and are often challenging to treat, amongst others due to high doses of previous radiotherapy treatments. Radiotherapy can be combined with local hyperthermia to sensitize tumor cells to radiation and thereby significantly reduce the required radiation dose. Therefore, the combination treatment of mild local hyperthermia, i.e. locally heating of the tissue to 39-43°C, and re-irradiation with a reduced total dose is a relevant treatment option for previously irradiated patients. The mechanisms of this effect in the course of the therapy are to date not well understood and will be investigated in the HISTOTHERM study. Methods and analyses: Patients with local or (loco)regional recurrent breast cancer with macroscopic tumors are included in the study. Local tumor control is evaluated clinically and histologically during the course of a combination treatment of 60 minutes mild superficial hyperthermia (39 - 43°C) using water-filtered infrared A (wIRA) irradiation, immediately followed by hypofractionated re-irradiation with a total dose of 20-24 Gy, administered in weekly doses of 4 Gy. Tumor and tumor stroma biopsies as well as blood samples will be collected prior to treatment, during therapy (at a dose of 12 Gy) and in the follow-up to monitor therapy response. The treatment represents the standard operating procedure for hyperthermia plus re-irradiation. Various tissue and blood-based markers are analyzed. We aim at pinpointing key mechanisms and markers for therapy response which may help guiding treatment decisions in future. In addition, quality of life in the course of treatment will be assessed and survival data will be evaluated. Registration: The study is registered at the German Clinical Trials Register, Deutsches Register Klinischer Studien (DRKS00029221).

Benchmarking of analysis strategies for data-independent acquisition proteomics using a large-scale dataset comprising inter-patient heterogeneity.

Numerous software tools exist for data-independent acquisition (DIA) analysis of clinical samples, necessitating their comprehensive benchmarking. We present a benchmark dataset comprising real-world inter-patient heterogeneity, which we use for in-depth benchmarking of DIA data analysis workflows for clinical settings. Combining spectral libraries, DIA software, sparsity reduction, normalization, and statistical tests results in 1428 distinct data analysis workflows, which we evaluate based on their ability to correctly identify differentially abundant proteins. From our dataset, we derive bootstrap datasets of varying sample sizes and use the whole range of bootstrap datasets to robustly evaluate each workflow. We find that all DIA software suites benefit from using a gas-phase fractionated spectral library, irrespective of the library refinement used. Gas-phase fractionation-based libraries perform best against two out of three reference protein lists. Among all investigated statistical tests non-parametric permutation-based statistical tests consistently perform best.

Survival Outcome and Prognostic Factors for Pancreatic Acinar Cell Carcinoma: Retrospective Analysis from the German Cancer Registry Group.

BACKGROUND: Pancreatic acinar cell carcinoma (PACC) is a distinct type of pancreatic cancer with low prevalence. We aimed to analyze prognostic factors and survival outcome for PACC in comparison to pancreatic ductal adenocarcinoma (PDAC), based on data from the German Cancer Registry Group. METHODS: Patients with PACC and PDAC were extracted from pooled data of the German clinical cancer registries (years 2000 to 2019). The distribution of demographic parameters, tumor stage and therapy modes were compared between PACC and PDAC. The Kaplan-Meier method and Cox regression analysis were used to delineate prognostic factors for PACC. Propensity score matching was used to compare survival between PACC and PDAC. RESULTS: There were 233 (0.44%) patients with PACC out of 52,518 patients with pancreatic malignancy. Compared to PDAC, patients with PACC were younger (median age 66 versus 70, respectively, p < 0.001) and the percentage of males was higher (66.1% versus 53.3%, respectively, p < 0.001). More patients were resected with PACC than with PDAC (56.2% versus 38.9%, respectively, p < 0.001). The estimated overall median survival in PACC was 22 months (95% confidence interval 15 to 27), compared to 12 months (95% confidence interval 10 to 13) in the matched PDAC cohort (p < 0.001). Surgical resection was the strongest positive prognostic factor for PACC after adjusting for sex, age, and distant metastases (hazard ratio 0.34, 95% confidence interval 0.22 to 0.51, p < 0.001). There was no survival benefit for adjuvant therapy in PACC. CONCLUSIONS: PACC has overall better prognosis than PDAC. Surgical resection is the best therapeutic strategy for PACC and should be advocated even in advanced tumor stages.

Tumor budding as a prognostic factor in pancreatic ductal adenocarcinoma.

In this retrospective study, we analyzed the association between tumor budding and perineural invasion as well as their prognostic role in pancreatic ductal adenocarcinoma. A total of N = 119 patients resected for pancreatic ductal carcinoma from 1996 to 2015 were included. Clinical and standard histopathological parameters were retrieved from the patient's records. One representative hematoxylin and eosin section from the tumor region was examined for perineural invasion and tumor budding using light microscopy. Tumor budding was assessed independently using two different methods: in the first approach, the number of buds was counted over three fields of 0.237 mm2 at 40-fold magnification; in the second approach, tumor budding was quantified according to the recommendation of the International Tumor Budding Consensus Conference (ITBCC) over a field of 0.785 mm2 at 20-fold magnification. Linear and logistic regression was applied to delineate association between perineural invasion, tumor budding, and other parameters; Kaplan-Meier and Cox regression were used in the survival analysis. Regardless of the quantification approach, high tumor budding was a significant negative prognostic factor in the univariable Cox regression (> 5 buds/0.237 mm2, hazard ratio (HR) 1.66, 95% confidence interval (CI) 1.06-2.61, p = 0.027; ≥ 10 buds/0.785 mm2, HR 1.68, 95% CI 1.07-2.64, p = 0.024). In the multivariable model adjusting for stage and standard histopathological parameters, lymph vessel invasion (HR = 2.43, 95% CI 1.47-4.03, p = 0.001) and tumor budding > 5 buds/0.237 mm2 (HR = 1.70, 95% CI 1.07-2.7, p = 0.026) were independent negative prognostic factors, while adjuvant therapy was a positive prognostic factor (HR = 0.54, 95% CI 0.33-0.86, p = 0.009). No significant prognostic value could be delineated for perineural invasion. In conclusion, tumor budding is an independent negative prognostic factor in pancreatic ductal adenocarcinoma associated with lymph node metastasis. The prognostic role of perineural invasion remains uncertain.

Circulating Tumor Cells in Pancreatic Cancer: Current Perspectives.

Pancreatic cancer is the fourth leading cause of cancer-related death in the USA and Europe; early symptoms and screenings are lacking, and it is usually diagnosed late with a poor prognosis. Circulating tumor cells (CTCs) have been promising new biomarkers in solid tumors. In the last twenty years (1999-2019), 140 articles have contained the key words "Circulating tumor cells, pancreatic cancer, prognosis and diagnosis." Articles were evaluated for the use of CTCs as prognostic markers and their correlation to survival in pancreatic ductal adenocarcinoma (PDAC). In the final selected 17 articles, the CTC detection rate varied greatly between different enrichment methodologies and ranged from 11% to 92%; the majority of studies used the antigen-dependent CellSearch© system for CTC detection. Fifteen of the reviewed studies showed a correlation between CTC presence and a worse overall survival. The heterogeneity of CTC-detection methods and the lack of uniform results hinder a comparison of the evaluated studies. However, CTCs can be detected in pancreatic cancer and harbor a hope to serve as an early detection tool. Larger studies are needed to corroborate CTCs as valid biomarkers in pancreatic cancer.

Putting the Hindgut Hypothesis to the Test in a Diabetic Zucker Rat Model.

BACKGROUND: The hindgut theory hypothesizes a key role of differential hindgut stimulation following metabolic procedures in ameliorating diabetes mellitus. We used two strategies to remove the hindgut from intestinal continuity in order to analyze its impact on diabetes mellitus. METHODS: Loop duodeno-jejunostomy (DJOS) with exclusion of one-third of total intestinal length was performed in 3 groups of 9-week-old Zucker diabetic fatty rats. In group 1, no further alteration of the intestinal tract was made. Group 2 received additional ileal exclusion (IE). Group 3 underwent additional resection of 50% of the ileum with side-to-side ileocecal anastomosis (IR). One, 2, and 4 months after surgery, fasting blood glucose levels, oral glucose tolerance tests (OGTT), and glucose-stimulated hormone analyses were conducted, and bile acid blood levels were compared. Body weight was documented weekly. RESULTS: In relation to DJOS, glucose control was not impaired in IR or IE. On the contrary, only IR could maintain preOP glucose values until 4 months. There were no significant weight differences between the groups. Confirming effective ileal diversion, bile acid blood levels were significantly higher in the DJOS group compared with both IR and IE (p = 0.0025 and p = 0.0047). Operative interventions had no impact on GLP-1 levels at any time point (ANOVA p > 0.05 for all). Insulin secretion was preserved in all groups. CONCLUSION: This data supports the hypothesis that the mechanisms driving amelioration of diabetes mellitus are complex and cannot be reduced to the ileum.

The Memorial Sloan Kettering Cancer Center Nomogram is More Accurate than the 2009 FIGO Staging System in the Prediction of Overall Survival in a German Endometrial Cancer Patient Cohort.

BACKGROUND: Despite the complexity of endometrial cancer (EC) tumor biology, treatment decisions are still mainly based on the post-surgical International Federation of Gynecology and Obstetrics (FIGO) stage. Prediction models considering more prognostic factors may represent a better risk assessment than FIGO stage alone. We tested the Memorial Sloan Kettering Cancer Center (MSKCC) nomogram for the prediction of overall survival (OS) in a German EC population. METHODS: Overall, 454 EC patients (322 type I and 132 type II) who received primary surgical treatment at our department between 1991 and 2011 were included in the analysis with a dataset of 68 covariates. Predicted OS was calculated using the online MSKCC nomogram and compared with the observed survival in our population. To estimate the discriminatory power, the concordance probabilities were calculated using the concordance probability estimate (CPE). Receiver operating characteristic curves were created and the area under the curve (AUC) values compared between predicted and actual OS. RESULTS: After a mean follow-up of 183 months, 211 patients were reported dead (47%). Mean OS for all stages was 101 months (standard deviation 66.7 months). The 2009 FIGO system showed an AUC value of 0.6 and a CPE of 0.63, while the 3-year OS prediction of the MSKCC nomogram showed an AUC value of 0.8 and a CPE of 0.77. CONCLUSION: This external validation of the MSKCC nomogram showed better discrimination and calibration values than the conventional FIGO classification system. The nomogram was externally validated and can serve as a tool for better risk-adapted treatment decisions and patient stratification, e.g. in clinical trials.

Clinical relevance of Cyr61 expression in patients with hormone-dependent breast cancer.

Tumor resistance to endocrine therapy triggers estrogen-independent cancer progression, which is a major obstacle to the successful treatment of hormone receptor positive breast cancer (BC). The underlying molecular mechanisms of endocrine resistance are not fully understood yet. The matricellular protein cysteine-rich angiogenic inducer 61 (Cyr61) is associated with tumor invasiveness and the induction of tumorigenesis in various malignancies in vivo and the induction of estrogen-independence and endocrine therapy resistance in BC. The present study evaluated the potential effects and clinical relevance of Cyr61 expression levels in 67 patients with primary non-metastatic BC. Immunohistochemical analysis of formalin-fixed paraffin-embedded tissue sections was performed, and the association between Cyr61 protein expression and clinicopathological factors and survival was analyzed. Cyr61 overexpression was revealed to be significantly associated with a positive estrogen receptor (ER)/progesterone receptor (PR) status (P=0.016) and to the molecular subtype of BC (P=0.039). Compared with patients without Cyr61 overexpression, patients with Cyr61 overexpression exhibited an increased recurrence rate (30.6 vs. 22.6%) and decreased long-term survival (10-year overall survival, 62.9 vs. 69.7%); however, these associations did not reach statistically significant levels in Cox regression model analysis. Similar results were identified in the subgroup analysis of patients with ER/PR positive BC. These results indicate that Cyr61 serves a role in the development of endocrine therapy resistance in BC and is thus a potential therapeutic target to overcome endocrine therapy resistance. However, additional long-term survival analyses with large patient populations are required.

Cyr61 Expression Pattern and Association with Clinicopathological Factors in Patients with Cervical Cancer.

BACKGROUND/AIM: The pro-angiogenic Cyr61 protein has been associated with tumorigenesis and cancer progression in different gynecological carcinomas. In this study, we evaluated the potential impact and clinical relevance of Cyr61 expression in patients with primary non-metastatic cervical cancer (CC). PATIENTS AND METHODS: Cyr61 expression was assessed in tissue specimen of 48 patients with primary CC by immunohistochemical analysis. Expression levels were scored and correlated to clinico-pathological factors and outcome data. RESULTS: High Cyr61 expression levels were present in 54.2% of CC tissues. Associations with histological grade (p=0.030), depth of tumor invasion (p=0.007) and GOG score (p=0.027) were observed. Patients who overexpressed Cyr61 displayed an increased death rate (30.8% vs. 18.2%) and a decreased 5-year-survival (76.9% vs. 86.4%). CONCLUSION: Our data indicate a potential functional impact of Cyr61 in development and the progression of CC. The definite tumor-relevant function (suppressive/promoting) of Cyr61 in CC and the prognostic relevance of Cyr61 overexpression has to be evaluated in larger cohorts.

BCG Induced Necrosis of the Entire Bladder Urothelium.

Instillation therapy with attenuated tuberculosis bacteria (BCG) can significantly reduce rates of recurrence of non-muscle invasive bladder cancer. Local and systemic side effects such as dysuria, irritative voiding symptoms or partial bladder contracture and systemic inflammation were reported. A 75 year-old male patient with recurrent non muscle invasive bladder cancer developed necrosis of the entire bladder urothelium more than six years after BCG instillation immunotherapy. The resulting irritative voiding symptoms and low bladder capacity required radical cystectomy. BCG instillation can cause severe side effects, which develop gradually and eventually need radical surgical therapy such as cystectomy without tumor recurrence.