[Short-term outcomes of the TRIANGLE operation after neoadjuvant chemotherapy in locally advanced pancreatic cancer].

Objective: To investigate the safety and efficacy of the TRIANGLE operation after neoadjuvant chemotherapy in locally advanced pancreatic cancer(LAPC). Methods: This study is a retrospective case series analysis. Between January 2020 and December 2022, a total of 103 patients were diagnosed as LAPC who underwent neoadjuvant chemotherapy at the Pancreas Center, the First Affiliated Hospital of Nanjing Medical University. Among them, 26 patients (25.2%) underwent the TRIANGLE operation. There were 15 males and 11 females,with a age of (59±7) years (range: 49 to 74 years). The pre-treatment serum CA19-9(M(IQR)) was 248.8(391.6)U/ml (range: 0 to 1 428 U/ml),and the serum carcinoembryonic antigen was 4.1(3.8)µg/L(range: 1.4 to 13.4 µg/L). The neoadjuvant chemotherapy regimens included: mFOLFIRINOX regimen in 6 cases(23.1%), GnP regimen in 14 cases(53.8%), and mFOLFIRINOX+GnP regimen in 6 cases(23.1%). The follow-up duration extended until June 2023 or until the occurrence of the patient's death or loss to follow-up. The Kaplan-Meier method was employed to estimate the 1-year and 3-year overall survival rates. Results: After neoadjuvant chemotherapy,CA19-9 levels decreased by 92.3(40.1)%(range:2.1% to 97.7%). Evaluation of the response to treatment revealed 13 cases(50.0%) of stable disease,11 cases(42.3%) of partial response,and 2 cases(7.7%) of complete response. The surgical operation consisted of 12 cases(46.2%) of pancreaticoduodenectomy,12 cases(46.2%) of distal pancreatectomy,and 2 cases(7.7%) of total pancreatectomy. Margin determination was based on the "standardised pathology protocol" and the "1 mm" principle. No R2 and R1(direct) resections were observed,while the R0 resection rate was 61.5%(16/26), and the R1(1 mm) resection rate was 38.5%(10/26).The R1(1 mm) resection rates for the anterior margin,posterior margin,transected margin,portal vein groove margin,and uncinate margin were 23.1%(6/26),19.2%(5/26),12.5%(3/24),2/14, and 1/12, respectively. The overall postoperative complication rate was 57.8%(15/26),with major complications including grade B/C pancreatic fistula 25.0%(6/24,excluding 2 cases of total pancreatectomy),delayed gastric emptying in 23.1%(6/26),wound complications 11.5%(3/26),postoperative hemorrhage 7.7%(2/26), chylous fistula 7.7%(2/26) and bile fistula 3.8%(1/26). No reoperation was performed during the perioperative period(<90 days). One patient died on the 32nd day postoperatively due to a ruptured pseudoaneurysm. A total of 25 patients were followed up,with a follow-up time of 21(24)months(range: 8 to 42 months). During the follow-up period,8 cases(32.0%) died due to tumor recurrence and metastasis,while 17 patients(68.0%) remained alive,including 11 cases of disease-free survival,5 cases of distant metastasis,and 1 case of local recurrence. The overall survival rates at 1- and 3-year after the initiation of neoadjuvant chemotherapy were 95.8% and 58.9%, respectively. The overall survival rates at 1- and 3-year after surgery were 77.7% and 57.8%, respectively. Conclusion: Performing pancreatoduodenectomy according to the Heidelberg triangle protocol in LAPC patients after neoadjuvant chemotherapy might increase the R0 resection rate without increasing perioperative mortality or the incidence of major postoperative complications.

[Clinical value of lymph node dissection of No. 14cd during pancreaticoduodenectomy in patients with pancreatic head carcinoma].

Objectives: To evaluate the positive rate of left posterior lymph nodes of the superior mesenteric artery (14cd-LN) in patients undergoing pancreaticoduodenectomy for pancreatic head carcinoma,to analyze the impact of 14cd-LN dissection on lymph node staging and tumor TNM staging. Methods: The clinical and pathological data of 103 consecutive patients with pancreatic cancer who underwent pancreaticoduodenectomy at Pancreatic Center,the First Affiliated Hospital of Nanjing Medical University from January to December 2022 were analyzed,retrospectively. There were 69 males and 34 females,with an age(M (IQR))of 63.0 (14.0) years (range:48.0 to 86.0 years). The χ2 test and Fisher's exact probability method was used for comparison of the count data between the groups,respectively. The rank sum test was used for comparison of the measurement data between groups. Univariate and multivariate Logistic regression analyzes were used for the analysis of risk factors. Results: All 103 patients underwent pancreaticoduodenectomy successfully using the left-sided uncinate process and the artery first approach. Pathological examination showed pancreatic ductal adenocarcinoma in all cases. The location of the tumors was the pancreatic head in 40 cases,pancreatic head-uncinate in 45 cases,and pancreatic head-neck in 18 cases. Of the 103 patients,38 cases had moderately differentiated tumor and 65 cases had poorly differentiated tumor. The diameter of the lesions was 3.2 (0.8) cm (range:1.7 to 6.5 cm),the number of lymph nodes harvested was 25 (10) (range:11 to 53),and the number of positive lymph nodes was 1 (3) (range:0 to 40). The lymph node stage was stage N0 in 35 cases (34.0%),stage N1 in 43 cases (41.7%),and stage N2 in 25 cases (24.3%). TNM staging was stage ⅠA in 5 cases (4.9%),stage ⅠB in 19 cases (18.4%),stage ⅡA in 2 cases (1.9%),stage ⅡB in 38 cases (36.9%),stage Ⅲ in 38 cases (36.9%),and stage Ⅳ in 1 case (1.0%). In 103 patients with pancreatic head cancer,the overall positivity rate for 14cd-LN was 31.1% (32/103),and the positive rates for 14c-LN and 14d-LN were 21.4% (22/103) and 18.4% (19/103),respectively. 14cd-LN dissection increased the number of lymph nodes (P<0.01) and positive lymph nodes (P<0.01). As a result of the 14cd-LN dissection,the lymph node stage was changed in 6 patients,including 5 patients changed from N0 to N1 and 1 patient changed from N1 to N2. Similarly,the TNM stage was changed in 5 patients,including 2 patients changed from stage ⅠB to ⅡB,2 patients changed from stage ⅡA to ⅡB,and 1 patient changed from stage ⅡB to Ⅲ. Tumors located in the pancreatic head-uncinate (OR=3.43,95%CI:1.08 to 10.93,P=0.037) and the positivity of 7,8,9,12 LN (OR=5.45,95%CI:1.45 to 20.44,P=0.012) were independent risk factors for 14c-LN metastasis; while tumors with diameter >3 cm (OR=3.93,95%CI:1.08 to 14.33,P=0.038) and the positivity of 7,8,9,12 LN (OR=11.09,95%CI:2.69 to 45.80,P=0.001) were independent risk factors for 14d-LN metastasis. Conclusion: Due to its high positive rate in pancreatic head cancer,dissection of 14cd-LN during pancreaticoduodenectomy should be recommended,which can increase the number of lymph nodes harvested,provide a more accurate lymph node staging and TNM staging.

[Distal pancreatectomy with celiac axis resection for pancreatic body cancer: a single center review of 89 consecutive cases].

Objective: To investigate the clinical efficacy of distal pancreatectomy with celiac axis resection(DP-CAR). Methods: A total of 89 consecutive patients (50 males and 39 females) who were diagnosed with pancreatic body cancer and underwent DP-CAR in Pancreas Center,First Affiliated Hospital of Nanjing Medical University between September 2013 and June 2022 were retrospectively reviewed. There were 50 males and 39 females,with age(M(IQR)) of 63(12) years(range:43 to 81 years). Perioperative parameters,pathology results and follow-up data of these patients were analyzed,χ2 or Fisher's test for categorical data while the Wilcoxon test for quantitative data. Survival results were estimated by the Kaplan-Meier survival method. Results: Among 89 cases,cases combined with portal vein-superior mesenteric vein or organ resection accounted for 22.5% (20/89) and 42.7% (38/89),respectively. The operative time,blood loss and postoperative hospital stay were 270 (110) minutes,300 (300) ml and 13 (10) days,respectively. The overall morbidity rate was 67.4% (60/89) while the major morbidity was 11.2% (10/89). The increase rate in transient liver enzymes was 42.7% (38/89),3.4% (3/89) for liver failure,53.9% (48/89) for clinically relevant postoperative pancreatic fistula,1.1% (1/89) for bile leak,3.4% (3/89) for chylous leak of grade B and C,11.2% (10/89) for abdominal infection,9.0% (8/89) for postoperative hemorrhage of grade B and C,4.5% (4/89) for delayed gastric emptying,6.7% (6/89) for deep vein thrombosis,3.4% (3/89) for reoperation,4.5% (4/89)for hospital mortality,7.9% (7/89) for 90-day mortality. The pathological type was pancreatic cancer for all 89 cases and pancreatic ductal adenocarcinoma made up 92.1% (82/89). The tumor size was 4.8(2.0) cm, ranging from 1.5 to 12.0 cm. The number of lymph nodes harvested was 14 (13)(range:2 to 33),with a positive lymph node rate of 13.0% (24.0%). The resection R0 rate was 30.0% (24/80) and the R1 (<1 mm) rate was 58.8% (47/80). The median overall survival time was 21.3 months (95%CI: 15.6 to 24.3) and the median disease-free survival time was 19.1 months (95%CI: 11.7 to 25.1). The overall survival at 1-year and 2-year were 69.60% and 39.52%. The median survival time of 58 patients with adjuvant chemotherapy was 24.3 months (95%CI: 17.8 to 32.3) while that of 13 patients without any kind of adjuvant therapy was 8.4 months (95%CI: 7.3 to 22.3). Seven patients accepted neoadjuvant chemotherapy and there was no significant morbidity among them,with a resection rate of R0 of 5/7. Conclusion: DP-CAR is safe and feasible for selective cases,which could be more valuable in improving long-term survival when combined with (neo) adjuvant therapy.

[Comparative clinical efficacy analysis of pancreatoduodenectomy for distal bile duct and pancreatic head cancer: a report of 1 005 cases].

Objective: To compare and analyze the clinical efficacy of pancreaticoduodenectomy for distal bile duct cancer and pancreatic head cancer. Methods: Clinical data of 1 005 patients who underwent pancreaticoduodenectomy and postoperative pathological examination confirmed the diagnosis of distal bile duct cancer and pancreatic head cancer at the Pancreas Center of the First Affiliated Hospital of Nanjing Medical University from January 2016 to December 2020 were analyzed retrospectively. There were 112 cases in the distal bile duct cancer group, 71 males and 41 females,with age (M(IQR)) of 65(15) years(range: 40 to 87 years); 893 cases in the pancreatic head cancer group, 534 males and 359 females,with age of 64(13)years(range: 16 to 91 years). The differences between clinicopathological characteristics and postoperative overall survival of the two groups were analyzed by χ2 test, Fisher's exact probability method, rank sum test or log-rank test, respectively. The difference in postoperative overall survival between the two groups was compared using Kaplan-Meier method after propensity score matching (1∶1). Results: Compared with the pancreatic head cancer group,the distal bile duct cancer group had shorter operative time (240.0(134.0) minutes vs. 261.0(97.0) minutes, Z=2.712, P=0.007),less proportion of combined venous resection (4.5% (5/112) vs. 19.4% (173/893), χ²=15.177,P<0.01),smaller tumor diameter (2.0(1.0) cm vs. 3.0(1.5) cm,Z=10.567,P<0.01),higher well/moderate differentiation ratio (51.4% (56/112) vs. 38.0% (337/893), χ²=7.328, P=0.007),fewer positive lymph nodes (0(1) vs. 1(3), Z=5.824, P<0.01),and higher R0 resection rate (77.7% (87/112) vs. 38.3%(342/893), χ²=64.399, P<0.01),but with a higher incidence of overall postoperative complications (50.0% (56/112) vs. 36.3% (324/892), χ²=7.913,P=0.005),postoperative pancreatic fistula (28.6% (32/112) vs. 13.9% (124/893), χ²=16.318,P<0.01),and postoperative abdominal infection (21.4% (24/112) vs. 8.6% (77/892), χ²=18.001,P<0.01). After propensity score matching, there was no statistical difference in postoperative overall survival time between patients in the distal bile duct cancer group and the pancreatic head cancer group (50.6 months vs. 35.1 months,Z=1.640,P=0.201),and multifactorial analysis showed that tumor site was not an independent risk factor affecting the prognosis of patients in both groups after matching (HR=0.73,95%CI:0.43 to 1.23,P=0.238). Conclusions: Patients with distal bile duct cancer are more likely to benefit from early diagnosis and surgical treatment than patients with pancreatic head cancer,but with a relative higher postoperative complication rates. The different tumor origin site is not an independent risk factor for prognosis of patients with distal bile duct cancer and pancreatic head cancer after propensity score matching.

[Application of left-sided uncinate process first approach in pancreaticoduodenectomy].

Objective: To evaluate the value of left-sided uncinate process first approach in pancreaticoduodenectomy. Methods: The clinical data of 152 patients who underwent the left-sided uncinate process first approach during pancreaticoduodenectomy at Pancreas Center, the First Affiliated Hospital of Nanjing Medical University from January 2020 to December 2020 were analyzed retrospectively. There were 64 females and 88 males,with age(M(QR)) of 62.0(14.7)years(range:16.0 to 84.0 years). The clinical date of 117 patients who underwent pancreaticoduodenectomy without using left-sided uncinate process first approach in the same period was selected as the control group,including 65 females and 52 males,with age of 64.0(13.0) years(range:13.0 to 84.0 years). Fisher exact probability method and t test were used to compare the data between the two groups,rank sum test was used for comparison of continuous variables between the two groups. Results: Pancreaticoduodenectomy was successfully performed in 152 patients in left-sided uncinate process first approach group. The operation time was 222.5(77.0) minutes(range:117.0 to 480.0 minutes),the time of uncinate process resection from left-side(the time from jejunum dissection to complete dissociation of the uncinate process) was 11.0(4.5) minutes(range:7.5 to 20.0 minutes),the time of pancreatic head resection (the time from jejunum dissection to pancreaticoduodenal specimen removal) was 26.0(8.5) minutes(range:20.0 to 41.0 minutes),the intraoperative blood loss was 200(150) ml(range:50 to 800 ml),and the intraoperative blood transfusion rate was 9.2% (14/152). Postoperative conditions:The postoperative hospital stay was 12 (9) d(range:6 to 55 d),the overall incidence of postoperative complications was 59.9%(91/152),and there was no perioperative death. Pathological results:The R0 resection rate of periampullary malignant tumor was 64.3%(77/112),with negative rate of uncinate process margin was 91.1%(102/112). The R0 resection rate of pancreatic ductal adenocarcinoma was 46.9%,with negative rate of uncinate process margin was 89.1%(57/64). Compared with the non-left-sided uncinate process first approach group(222.5(77.0) minutes, 9.2%(14/152)),the left-sided uncinate process first approach group had shorter operation time(246.0(94.0) minutes) (Z=3.964,P<0.01),less intraoperative blood loss (18.8%(22/117))(Z=4.843,P<0.01),and lower intraoperative blood transfusion rate(χ²=5.248,P=0.029). However,there were no significant differences between two groups in postoperative hospital stay(Z=1.682,P=0.093),postoperative overall complications(P=0.549),R0 resection rate of periampullary malignant tumor(χ²=2.012,P=0.156),and negative rate of uncinate process margin(χ²=2.108,P=0.147). Conclusions: The "left-sided uncinate process first approach" could completely resect uncinate process under a direct vision,especially when the uncinate process was behind the superior mesenteric artery or beyond the left lateral margin of the superior mesenteric artery. The "left-sided uncinate process first approach" might increase the negative rate of uncinate process margin and R0 resection rate for periampullary malignant tumor.

[Comparison of radical antegrade modular pancreatosplenectomy with conventional distal pancreatectomy for pancreatic adenocarcinoma of the body and tail].

Objective: To compare the short-term outcomes and long-term survivals of radical antegrade modular pancreatosplenectomy(RAMPS) and conventional distal pancreatectomy(CDP). Methods: A total of consecutive 304 patients including 176 male patients and 128 female patients who underwent RAMPS or CDP at Pancreas Center, the First Affiliated Hospital with Nanjing Medical University from May 2013 to June 2019 were retrospectively analyzed. The median age was 64.1 years old (range:39 to 85 years old). There were 101 patients underwent RAMPS and 203 patients underwent CDP. Measurement data with skewed distribution were presented as (M(Q(R))) and comparison between groups was evaluated with the Wilcoxon rank sum test. Count data were analyzed using the χ(2) test or Fisher exact probability. Survival analyses were performed by the Kaplan-Meier method after a one to one propensity score matching(PSM) conducted to balance several variables. Results: An eighty-one to eighty-one patients were enrolled after PSM. The overall morbidity was 32.1%(26/81)and there were no in-hospital mortalities in RAMPS. The median operative time was 225(95)minutes in RAMPS, not significantly longer as compared with CDP(210(130)minutes, P=0.916). The median greatest tumor diameter in RAMPS was 4.0(2.3)cm, not significantly larger as compared with CDP(4.5(2.2)cm, P=0.520).There were 34.6%(28/81)patients who presented with T4 tumors by 8(th) AJCC TNM staging system in RAMPS, which was not significantly different as compared with CDP(39.5%, χ(2)=0.574, P=0.902). The median number of examined lymph nodes was 9(9), not significantly greater in RAMPS as compared with CDP(10(11), P=0.992). The rate of negative posterior margins using 1 mm rule in RAMPS was 70.3%(52/74), significantly higher as compared with CDP(53.6%(30/56), χ(2)=3.817, P=0.044). The overall R0 resection rate was 44.6% (33/74) in RAMPS and 37.5% (21/56) in CDP, which was not significantly different(χ(2)=0.663, P=0.474). The median overall survival was 16.5 months for RAMPS, 25.2 months for CDP, and there was no statistical difference between two groups(P=0.981). The median overall survival was 16.0 months for patients with preoperative CA19-9≥300 U/ml who underwent RAMPS, 10.1 months for patients who underwent CDP, without significant difference(P=0.082). Conclusions: RAMPS can improve the rate of negative posterior margins by 1 mm rule and probably increase R0 resection rate and the harvest of lymph nodes. RAMPS may be beneficial to some patients with preoperative CA19-9≥300 U/ml.

[Analysis of risk factors and outcomes for delayed gastric emptying following pancreaticoduodenectomy: a single center experience of 492 cases].

Objective: To evaluate risk factors for delayed gastric emptying(DGE)following pancreaticoduodenectomy(PD). Methods: There were 492 consecutive patients who underwent PD in Pancreas Center, the First Affiliated Hospital with Nanjing Medical University between January 2012 and December 2014 were identified from a prospective database.There were 315 male and 177 female patients with a median age of 60.5 years.Univariate and multivariate analyses were performed to investigate the independent risk factors for clinically relevant DGE(CR-DGE). Results: The overall incidence of DGE was 29.5%, with Grade B and C occurring at 4.3% and 5.9%, respectively.In multivariate analysis, pancreatic duct diameter less than 3 mm(OR=1.888, P=0.042), pylorus-preserving pancreaticoduodenectomy(OR=2.627, P=0.005) and clinically relevant postoperative pancreatic fistula(OR=2.740, P=0.007) were independently associated with CR-DGE.Other main complications such as postoperative pancreatic fistula, pyoperitoneum, intraabdominal infection were also associated with the severity of DGE(χ(2)=21.360, 14.422, 14.378; P=0.011, 0.002, 0.002). DGE patients had a significantly prolonged postoperative length of stay(31(24-41)d vs. 13(11-17)d) and increased medical cost((122 367.5±66 068.3)yuan vs. (78 200.7±27 043.9)yuan)(both P<0.01). Conclusions: Small pancreatic duct, underwent pylorus-preserving pancreaticoduodenectomy and suffered postoperative pancreatic fistula might indicate a high risk of CR-DGE.

[Laparoscopic pancreaticoduodenectomy with a novel artery first and uncinate process first approach through Treitz ligament].

Objective: To explore the clinical effect of a novel artery first and uncinate process first approach for laparoscopic pancreaticoduodenectomy(LPD), emphasizing the left lateral and posterior dissection of uncinate process (UP) via Treitz ligament approach. Methods: From April to November 2016, 18 patients received LPD with a novel approach in Pancreas Center of the First Affiliated Hospital with Nanjing Medical University. All patients were diagnosed as pancreatic head or peri-ampulla tumor, without major vessel invasion nor distant metastasis. For resection, routine caudal view was used in the first step, to dissect the anterior medial border between uncinate process and superior mesenteric vein(SMV). Lymphatic tissues were completely dissected form anterior surface of hepatoduodenal ligament. In the second step, left lateral view with camera from left para-umbilical trocar was used, Treitz ligament was incised, SMA root was exposed. After anticlockwise rotation and retraction of mesentery, the anatomic relationship between SMA trunk, inferior pancreaticoduodenal artery(IPDA), jejunal branch of SMV, and distal part of UP, could be perfectly exposed from left lateral view. SMA was dissected from its root until the position above the uncinate process and duodenum, IPDA was transected, distal part of UP was freed from SMA. In the third step, right lateral view and caudal view were alternatively used; proximal UP mesentery was completely dissected out from SMA root, CA root and posterior surface of hepatoduodenal ligament. Pancreaticoduodenectomy was completed in the forth step after transection of pancreatic neck and common hepatic duct. Results: The SMA root and distal UP were successfully dissected out via Treitz ligament approach in all 18 patients, among them, distal UP was completely excised in 8 patients from left view. Postoperative pathology showed R0 resection rate in 69%. Postoperative complication included intra-abdominal hemorrhage in 1 patient, pancreatic fistula in 7 patients(6 cases with grade A and 1 case with grade B), delayed gastric emptying in 4 patients (2 cases with grade A, 2 cases with grade B). Average postoperative hospital stay was (15.5±6.8)days. Conclusion: The novel artery first and uncinate process first approach through Treitz ligament could help surgeons to completely dissect the full length of meso-pancreas along celiac axis-SMA axis in LPD.

Classification and diagnostic prediction of cancers using gene expression profiling and artificial neural networks.

The purpose of this study was to develop a method of classifying cancers to specific diagnostic categories based on their gene expression signatures using artificial neural networks (ANNs). We trained the ANNs using the small, round blue-cell tumors (SRBCTs) as a model. These cancers belong to four distinct diagnostic categories and often present diagnostic dilemmas in clinical practice. The ANNs correctly classified all samples and identified the genes most relevant to the classification. Expression of several of these genes has been reported in SRBCTs, but most have not been associated with these cancers. To test the ability of the trained ANN models to recognize SRBCTs, we analyzed additional blinded samples that were not previously used for the training procedure, and correctly classified them in all cases. This study demonstrates the potential applications of these methods for tumor diagnosis and the identification of candidate targets for therapy.

Revealing homogeneous plastic deformation in dendrite-reinforced Ti-based metallic glass composites under tension.

The tensile plastic deformation of dendrite-reinforced Ti-based metallic glass composites (MGCs) was investigated. It was found that there is a critical normalized strain-hardening rate (NSHR) that determines the plastic stability of MGCs: if the NSHR is larger than the critical value, the plastic deformation of the MGCs will be stable, i.e. the necking and strain localization can be effectively suppressed, resulting in homogeneous plastic elongation. In addition, dendrite-reinforce MGCs are verified as being intrinsically ductile, and can be used as good coatings for improving the surface properties of pure titanium or titanium alloys. These findings are helpful in designing, producing, and using MGCs with improved performance properties.

The primacy of NF1 loss as the driver of tumorigenesis in neurofibromatosis type 1-associated plexiform neurofibromas.

Neurofibromatosis type 1 (NF1) is a common tumor-predisposition disorder due to germline mutations in the tumor suppressor gene NF1. A virtually pathognomonic finding of NF1 is the plexiform neurofibroma (PN), a benign, likely congenital tumor that arises from bi-allelic inactivation of NF1. PN can undergo transformation to a malignant peripheral nerve sheath tumor, an aggressive soft-tissue sarcoma. To better understand the non-NF1 genetic contributions to PN pathogenesis, we performed whole-exome sequencing, RNASeq profiling and genome-wide copy-number determination for 23 low-passage Schwann cell cultures established from surgical PN material with matching germline DNA. All resected tumors were derived from routine debulking surgeries. None of the tumors were considered at risk for malignant transformation at the time; for example, there was no pain or rapid growth. Deep (~500X) NF1 exon sequencing was also conducted on tumor DNA. Non-NF1 somatic mutation verification was performed using the Ampliseq/IonTorrent platform. We identified 100% of the germline NF1 mutations and found somatic NF1 inactivation in 74% of the PN. One individual with three PNs had different NF1 somatic mutations in each tumor. The median number of somatic mutations per sample, including NF1, was one (range 0-8). NF1 was the only gene that was recurrently somatically inactivated in multiple tumors. Gene Set Enrichment Analysis of transcriptome-wide tumor RNA sequencing identified five significant (FDR<0.01) and seven trending (0.01⩽FDR<0.02) gene sets related to DNA replication, telomere maintenance and elongation, cell cycle progression, signal transduction and cell proliferation. We found no recurrent non-NF1 locus copy-number variation in PN. This is the first multi-sample whole-exome and whole-transcriptome sequencing study of NF1-associated PN. Taken together with concurrent copy-number data, our comprehensive genetic analysis reveals the primacy of NF1 loss as the driver of PN tumorigenesis.

Correlation of membrane lipid peroxidation with oxidation of hemoglobin variants: possibly related to the rates of hemin release.

Experiments were performed to delineate the biochemical mechanism of hemoglobin (Hb)-catalyzed lipid peroxidation in human red blood cells (RBCs). Using a modified Langmuir trough lipid monolayer technique, we found that oxidized Hb induced an increase in lipid monolayer surface pressure, suggesting that oxidized Hb readily releases its heme moiety into the lipid monolayer. To confirm our interpretation that oxidized Hb readily releases its heme moiety, we monitored the fluorescence of Hb tryptophan upon oxidation of Hb. We found an increase in Hb fluorescence in the aqueous phase of our monolayer system after the addition of H2O2. The increase in fluorescence should reflect the departure of heme from globin due to a decrease in fluorescent quenching effect by the heme moiety. The rate of increase in lipid monolayer surface pressure upon Hb oxidation differed from Hb to Hb with an order of Hb E > F > S > A. The ability of various Hbs to affect lipid peroxidation in the RBC membrane, as monitored by the parinaric acid oxidation technique, followed this same order. In addition, hemin was shown to be a more potent catalyst of lipid peroxidation in RBC membrane than nonheme irons.

Inhibition of squalene synthetase by farnesyl pyrophosphate analogues.

The pyrophosphates of the following farnesol analogues have been synthesized: 2-methylfarnesol; 7,11-dimethyl-3-ethyl-2,6,10-dodecatrien-1-ol; 3-demethylfarnesol; 4-methylthiofarnesol; 7,11-dimethyl-3-iodo-2,6,10-dodecatrien-1-ol; 7,11-dimethyl02-iodo-2,6,10-dodecatrien-1-ol; 7,11-dimethyldodeca-6,10-dien-2-yn-1-ol; phytol; 3,7,11-trimethyl-2-dodecen-1-ol; 3,7,11-trimethyldodecan-1-ol; and geraniol. The double bonds in all the above compounds were in the E configuration, except phytol, which was a 7:3 mixture of 2E and 2Z isomers. Each of the pyrophosphates inhibits the incorporation of labeled farnesyl pyrophosphate into squalene by a yeast enzyme preparation. Free alcohols and monophosphates are inactive. The analogues, listed in order of decreasing inhibitory strength, are, by kinetic analysis, competitive or mixed inhibitors. Irreversible inhibition is not observed. The results suggest that binding to the enzyme is primarily mediated by the pyrophosphate moiety assisted by relatively nonspecific lipophilic interactions. Decreasing the chain length and saturating double bonds severely reduces binding, while substitution at the 2,3, and 4 positions, and lengthening of the chain, is well tolerated.

Detection of a novel lactate dehydrogenase isozyme and an apparent differentiation-associated shift in isozyme profile in hepatoma cell lines.

A hitherto unreported lactate dehydrogenase (LD) isoenzyme, which migrates electrophoretically to the relative position between LD2 and LD3 has been identified in the electropheratogram in 7 of 7 (100%) cultured hepatoma cell lines with various degrees of differentiation and is thus given the name LD2-3. LD2-3 seems to be specific for hepatoma cells because this atypical isoenzyme can not be detected in other tumor cell lines. In addition, the hepatoma cell lines also show a distinct pattern of LD isoenzyme and the isoenzyme pattern varies with the degree of differentiation. Hence, the expression pattern of LD isoenzyme phenotypes may provide a good marker for the investigation of human hepatoma cell differentiation.

Hemin-induced membrane sulfhydryl oxidation: possible involvement of thiyl radicals.

Sublytic levels (microM) of hemin destabilized RBC membrane as indicated by ghost fragmentation pattern using a laser viscodiffractometer. Furthermore, electron microscopic study shows that 5 microM of hemin induced echinocytic transformation whereas higher hemin concentration (40 microM) induced spherocytic transformation. In addition, hemin oxidized sulfhydryl groups in a dose dependent fashion and Electron Spin Resonance study suggests that such oxidation may involve a thiyl radical. Moreover, sulfhydryl compounds enhanced hemin-induced lipid peroxidation. Desferroxamine could prevent hemin-induced sulfhydryl oxidation as well as hemin-induced decrease in membrane stability. In contrast, vitamin E could effectively prevent hemin-induced lipid peroxidation but could not prevent hemin-mediated membrane destabilization.

Alkaline phosphatase activity during sphinganine potentiation of retinoic acid-induced differentiation of human promyelocytic leukemia cell line, HL-60.

Sphinganine (SP) pre-treatment potentiated the retinoic acid (RA)-induced (4-96h exposures) differentiation and increase of alkaline phosphatase (ALP) activity. A higher percentage of SP pre-treated cells in RA exposures resembled mature myelocytes or granulocytes; greater increase in ALP activity was observed. In cells exposed to RA alone for only a period of 24h, the ALP activity could still increase and reach a similar maximum ALP activity (8.5-10.0 units/mg protein) at 48h as it was under continuous RA treatment. In all cells with longer exposures (24-96h) to RA, SP pre-treatment increased ALP activity to more or less the same higher maximum (14.0-15.5 units/mg protein). SP, added 24h before or concomitantly, but not 24 nor 48h after the addition of RA, could potentiate the RA-induced differentiation and increase of ALP activity.

Enhanced susceptibility of erythrocytes deficient in glucose-6-phosphate dehydrogenase to alloxan/glutathione-induced decrease in red cell deformability.

It has been hypothesized that enhanced oxidant sensitivity of glucose-6-phosphate dehydrogenase (G6PD) deficient red cells(RBCs) is the underlying mechanism for drug- or chemical-induced hemolytic crises in G6PD-deficiency. To further test this hypothesis, we used an alloxan-glutathione system to mimic oxidative stress and see how oxidative damage might affect RBC deformability. RBC deformability, a major determinant of RBC survival in vivo, was monitored by a laser viscodiffractometer. Under our experimental conditions, GSH alone had very little effect on the deformability of either normal or G6PD-deficient RBCs. In contrast, alloxan alone induced a small but significant decrease in the deformability of either normal or G6PD-deficient RBCs. Interestingly, alloxan and GSH together induced a further decrease in the deformability of either normal or G6PD-deficient RBCs. The decrease in deformability in G6PD-deficient RBCs was much more profound than in normal RBCs. In addition, an alloxan-vitamin C system produced a similar deleterious effect on RBC deformability as that produced by the alloxan-GSH system. Appreciable amount of hydroxyl radicals was generated by both alloxan-GSH and alloxan-vitamin C systems as evidenced by the production of hydroxylated products of salicylate which was used as a radical trap. Moreover, salicylate could ameliorate the deleterious effect of the alloxan system on the deformability of RBCs. Taken together, our results demonstrated that G6PD-deficient RBCs were particularly susceptible to oxidant-induced damage leading to a dramatic decrease in their deformability and thus provided strong support for the hypothesis that enhanced oxidant sensitivity of G6PD-deficient RBCs is the underlying mechanism for accelerated destruction of these RBCs in vivo.

Homocysteine thiolactone induces apoptotic DNA damage mediated by increased intracellular hydrogen peroxide and caspase 3 activation in HL-60 cells.

The cytotoxicity of homocysteine derivatives on chromosomal damage in somatic cells is not well established. The present study used reactive homocysteine derivative of homocysteine thiolactone (Hcy) to investigate its causal effect on apoptotic DNA injury in human promyeloid HL-60 cells. Our results demonstrated that Hcy induced cell death and features of apoptosis including increased phosphotidylserine exposure on the membrane surface, increased apoptotic cells with hypoploid DNA contents, and internucleosomal DNA fragmentation, all of which occurred in a time- and concentration-dependent manner. Hcy treatment also significantly increased intracellular reactive oxygen species H2O2, which coincided with the elimination of caspase 3 proenzyme levels and increased caspase 3 activity at the time of the appearance of apoptotic DNA fragmentation. Preincubation of Hcy-treated HL-60 cells with catalase completely scavenged intracellular H2O2, thus inhibiting caspase 3 activity and protecting cells from apoptotic DNA damage. In contrast, superoxide dismutase failed to inhibit Hcy-induced DNA damage. Taken together, these results demonstrate that Hcy exerted its genotoxic effects on HL-60 cells through an apoptotic pathway, which is mediated by the activation of caspase 3 activity induced by an increase in intracellular hydrogen peroxide.

Enhanced vesiculation exacerbates complement-dependent hemolysis in glucose-6-phosphate dehydrogenase deficient red blood cells.

Glucose-6-phosphate dehydrogenase (G6PD) deficient red blood cells (RBCs) are known to be more susceptible to oxidant-induced hemolysis. Erythrocytes from G6PD-deficient individuals are significantly more susceptible to Ca(2+)-induced vesiculation than normal control cells. The enhanced susceptibility of G6PD-deficient RBCs to Ca(2+)-induced vesiculation is not due to ATP depletion. The remnant G6PD-deficient RBCs following vesiculation are more sensitive to complement-mediated hemolysis than control normal RBCs. A strong positive correlation exists between the level of Ca(2+)-induced vesiculation and the extent of complement mediated hemolysis.

Purification and characterization of endogenous digoxin-like immunoreactive factors in chicken blood.

Studies have been performed to determine whether an endogenous material capable of binding to digoxin antibodies is present in the chicken plasma. In the blood of 12 chickens without feed control, endogenous digoxin-like immunoreactive factors (DLIF) binding of digoxin antibodies in enzyme immunoassays amounted to 866 / 302 pg digoxin equivalents/mL of plasma (mean +/- SEM). Immunoreactivity of DLIF increased to 1848***331 pg/mL with a double value of control after boiling and acid pretreating the plasma. The major purification steps employed in this report were gel filtration column chromatography, high performance liquid chromatography (HPLC) and isoelectric focusing (IEF). Using HPLC for the separation, at least 10 chicken DLIFs with different molecular weight (MW) have been found. The MW of the smallest is 300 daltons (Da) while the largest is 100 kDa. The value of the isoelectric point of the most abundant type of DLIF from untreated chicken plasma is 6.3 as determined by IEF. The partially purified DLIF inhibits Na+, K(+)-ATPase from a porcine cerebral cortex as well as three human red blood cell membrane preparations in a dose-response fashion.