FOLFIRINOX and translational studies: Towards personalized therapy in pancreatic cancer

Pancreatic cancer is an extremely aggressive disease; although progress has been made in the last few years, the prognosis of these patients remains dismal. FOLFIRINOX is now considered a standard treatment in first-line setting, since it demonstrated an improved overall and progression-free survival vs gemcitabine alone. However, the enthusiasm over the benefit of this three-drug regimen is tempered by the associated increased toxicity profile, and many efforts have been made to improve the feasibility of this schedule. After a more recent phase Ⅲ trial showing an improved outcome over gemcitabine, the combination of gemcitabine/nab-paclitaxel emerged as another standard first-line treatment. However, this treatment is also associated with more side effects. In addition, despite initial promising data on the predictive role of SPARClevels, recent studies showed that these levels are not associated with nab-paclitaxel efficacy. The choice to use this treatment over FOLFIRINOX is therefore a topic of debate, also because no validated biomarkers to guide FOLFIRINOX treatment are available. In the era of actionable mutations and target agents it would be desirable to identify molecular factors or biomarkers to predict response to therapy in order to maximize the efficacy of treatment and avoid useless toxic effects for non-responding patients. However, until today the milestone of treatment for pancreatic cancer remains chemotherapy combinations, without predictive or monitoring tools existing to optimize therapy. This review analyzes the state-of-the-art treatments, promises and limitations of targeted therapies, ongoing trials and future perspectives, including potential role of microR NAs as predictive biomarkers.

Tissue microarray-chip featuring computerized immunophenotypical characterization more accurately subtypes ampullary adenocarcinoma than routine histology

BACKGROUND Ampullary adenocarcinomas(AACs)are heterogeneous tumors currently classified into three important sub-classes(SC):Intestinal(INT),Pancreato-Biliary(PB)and Mixed-Type(MT).The different subgroups have similar clinical presentation and are treated by pancreatoduodenectomy with curative intent.However,they respond differently to chemotherapy and have different prognostic outcomes.The SC are often difficult to identify with conventional histology alone.The clinical outcome of all three remains unclear,particularly for MT.AIM To identify two main subtypes of AACs,using an immunohistochemical(IHC)score based on CDX2,CK7 and CK20.METHODS Tissue samples from 21 patients who had undergone resection of AAC were classified by HE histology and IHC expression of CDX2,CK7 and CK 20.An IHC score was obtained for each marker by counting the number of positive cells(0=no stained cells;1<25%;2<50%and 3>50%)and their intensity(1=weak;2=moderate and 3=strong).A global score(GS)was then obtained by summation of the IHC scores of each marker.The MT tumors were grouped either with the INT or PB group based on the predominant immuno-molecular phenotype,obtaining only two AACs subtypes.The overall survival in INT and PB patients was obtained by Kaplan-Meier methods.RESULTS Histological parameters defined the AACs subtypes as follows:15%INT,45%PB and 40%MT.Using IHC expression and the GS,75%and 25%of MT samples were assigned to either the INT or the PB group.The mean value of the GS was 9.5(range 4-16).All INT samples had a GS above the average,distinct from the PB samples which had a GS score significantly below the average(P=0.0011).The INT samples were identified by high expression of CDX2 and CK20,whereas PB samples exhibited high expression of CK7 and no expression of CK20(P=0.0008).The INT group had a statistically significant higher overall survival than in the PB group(85.7 mo vs 20.3 mo,HR:8.39;95%CI:1.38 to 18.90;P=0.0152).CONCLUSION The combination of histopathological and molecular criteria enables the classification of AACs into two clinically relevant histo-molecular phenotypes,which appear to represent distinct disorders with potentially significant changes to the current therapeutic strategies.

Detailing the ultrastructure’s increase of prion protein in pancreatic adenocarcinoma

BACKGROUND Recent evidences have shown a relationship between prion protein(PrPc)expression and pancreatic ductal adenocarcinoma(PDAC).Indeed,PrPc could be one of the markers explaining the aggressiveness of this tumor.However,studies investigating the specific compartmentalization of increased PrPc expression within PDAC cells are lacking,as well as a correlation between ultrastructural evidence,ultrastructural morphometry of PrPc protein and clinical data.These data,as well as the quantitative stoichiometry of this protein detected by immuno-gold,provide a significant advancement in understanding the biology of disease and the outcome of surgical resection.AIM To analyze quantitative stoichiometry and compartmentalization of PrPc in PDAC cells and to correlate its presence with prognostic data METHODS Between June 2018 and December 2020,samples from pancreatic tissues of 45 patients treated with pancreatic resection for a preoperative suspicion of PDAC at our Institution were collected.When the frozen section excluded a PDAC diagnosis,or the nodules were too small for adequate sampling,patients were ruled out from the present study.Western blotting was used to detect,quantify and compare the expression of PrPc in PDAC and control tissues,such as those of non-affected neighboring pancreatic tissue of the same patient.To quantify the increase of PrPc and to detect the subcellular compartmentalization of PrPc within PDAC cells,immuno-gold stoichiometry within specific cell compartments was analyzed with electron microscopy.Finally,an analysis of quantitative PrPc expression according to prognostic data,such as cancer stage,recurrence of the disease at 12 mo after surgery and recurrence during adjuvant chemotherapy was made.RESULTS The amount of PrPc within specimen from 38 out of 45 patients was determined by semi-quantitative analysis by using Western blotting,which indicates that PrPc increases almost three-fold in tumor pancreatic tissue compared with healthy pancreatic regions[242.41±28.36 optical density(OD)vs 95±17.40 OD,P<0.0001].Quantitative morphometry carried out by using immuno-gold detection at transmission electron microscopy confirms an increased PrPc expression in PDAC ductal cells of all patients and allows to detect a specific compartmentalization of PrPc within tumor cells.In particular,the number of immuno-gold particles of PrPc was significantly higher in PDAC cells respect to controls,when considering the whole cell(19.8±0.79 particles vs 9.44±0.45,P<0.0001).Remarkably,considering PDAC cells,the increase of PrPc was higher in the nucleus than cytosol of tumor cells,which indicates a shift in PrPc compartmentalization within tumor cells.In fact,the increase of immuno-gold within nuclear compartment exceeds at large the augment of PrPc which was detected in the cytosol(nucleus:12.88±0.59 particles vs 5.12±0.32,P<0.0001;cytosol:7.74.±0.44 particles vs 4.3±0.24,P<0.0001).RESULTS In order to analyze the prognostic impact of PrPc,we found a correlation between PrPc expression and cancer stage according to pathology results,with a significantly higher expression of PrPc for advanced stages.Moreover,24 patients with a mean follow-up of 16.8 mo were considered.Immuno-blot analysis revealed a significantly higher expression of PrPc in patients with disease recurrence at 12 mo after radical surgery(360.71±69.01 OD vs 170.23±23.06 OD,P=0.023),also in the subgroup of patients treated with adjuvant CT(368.36±79.26 OD in the recurrence group vs 162.86±24.16 OD,P=0.028),which indicates a correlation with a higher chemo-resistance.CONCLUSION Expression of PrPc is significantly higher in PDAC cells compared with control,with the protein mainly placed in the nucleus.Preliminary clinical data confirm the correlation with a poorer prognosis.

Use of zebrafish embryos as avatar of patients with pancreatic cancer:A new xenotransplantation model towards personalized medicine

BACKGROUND The response to chemotherapy treatment of patients with pancreatic ductal adenocarcinoma(PDAC)is difficult to predict and the identification of patients who most likely will benefit from aggressive chemotherapy approaches is crucial.The concept of personalized medicine has emerged in the last years with the objective to tailor the medical treatment to the individual characteristics of each patient,and particularly to the tumor biology of each patient.The need for invivo xenotransplantation models for cancer patients has increased exponentially,and for this reason zebrafish avatars have gained popularity.Preliminary studies were conducted also with PDAC tissue.AIM To develop a simple,not expensive,diffusible zebrafish embryo model as avatar for patients affected by PDAC.METHODS Tumor tissue was taken from the surgical specimen by the histopathologist.After its fragmentation into small pieces,they are stained with CM-Dil.Small pieces of stained tissue were transplanted into the yolk of wt AB zebrafish embryos with a glass capillary needle.Embryos were incubated at 35°C in E3 medium supplemented with 1%Pen/Strep in the presence or absence of drugs for the following days in respect of the treatment plan(Gemcitabine;Gemcitabine and Oxaliplatin;Gemcitabine and nab-Paclitaxel;5-Fluorouracil and Folinic acid and Oxaliplatin and Irinotecan).The response of zebrafish xenografts to the chemotherapy options has been analyzed by monitoring the fluorescent stained area at 2 h post injection(hpi),1 d and 2 d post injection(dpi).In each time point,the mean size of the stained area was measured by ImageJ and it was normalized with respect to the 1 dpi time point mean relative tumor area(RTA).We evaluated the effect of the chemotherapy exposition comparing the mean RTA of each treated subgroup and the control group and evaluating the percentage reduction of the mean RTA by comparing each treated subgroup with the control group.RESULTS Between July 2018 and October 2019,a total of 15 patients with pancreatic cancer were prospectively enrolled.In all cases,it was possible to take a fragment of the tumor from the surgical specimen for the xenotransplantation in the zebrafish embryos.The histological examination confirmed the presence of a PDAC in all cases.In absence of chemotherapy(control group),over time the Dil-stained area showed a statistically significant increase in all cases.A statistically significant reduction of the mean RTA in the treated subgroups for at least one chemotherapy scheme was reported in 6/15(40%)cases.The analysis of the percentage reduction of the RTA in treated subgroups in comparison to the control group revealed the presence of a linear relationship in each subgroup between the percentage reduction of the RTA and the number of cases reporting each percentage threshold considered for the analysis.CONCLUSION Our model seems to be effective for the xenotransplantation of PDAC tissue and evaluation of the effect of each chemotherapy scheme on the xenotransplanted tumor tissue.