Cytoplasmic Lipid Droplets Predict Worse Prognosis in Diffuse Large B-Cell Lymphoma: Next-Generation Sequencing Deciphering Lipogenic Genes.

Burkitt lymphoma is characterized by high cell turnover and numerous cytoplasmic vacuoles that are demonstrated to be lipid droplets (LDs) decorated by adipophilin. By contrast, cytoplasmic vacuoles are variably observed in diffuse large B-cell lymphoma (DLBCL) and less well characterized. In this study, we first validated in DLBCL that cytoplasmic vacuoles are indeed LDs by Oil-red-O stain, Bodipy fluorescent stain, and electron microscopy. Second, in a cohort of DLBCL patients (n=52) we showed that LDs in effusional lymphoma cells were associated with a poorer prognosis (P=0.029, log-rank test) and higher International Prognostic Index (IPI) score (94% vs. 66%, P=0.026) than those without. Moreover, using adipophilin as a surrogate marker for LDs, we found in another cohort of biopsy specimen (n=85) that expression of adipophilin by lymphoma cells predicted a poorer prognosis (P=0.007, log-rank test) and higher IPI score (63% vs. 30%, P=0.005). In addition, whole exome sequencing of effusional DLBCL cells showed LD-positive DLBCL shared genetic features with the MCD (MYD88 and CD79B mutations) subtype and highlighted OSBPL10 and CUBN as the most frequently mutated genes involved in lipogenesis. Whole transcriptome analysis by comparing effusional DLBCL cells with versus without LDs showed upregulation of EHHADH, SLC1A1, CD96, INPP4B, and RNF183 relevant for lymphoma lipogenesis and upregulation of epithelial-mesenchymal transition and KRAS signaling pathways. Higher expression of EHHADH and CD96 were validated in LD-positive clinical samples and LD-rich cell lines than LD-poor cells along with the known lipogenic gene, FASN. Our findings highlight the roles of LDs and adipophilin expression in DLBCL, suggest that these markers may predict prognosis and show that lipogenic genes may be potential therapeutic targets.

Frequent expression of PD-L1 in BLS-type diffuse large B-cell lymphoma: implications for aggressiveness and immunotherapy.

BLS-type diffuse large B-cell lymphoma (DLBCL) denotes an uncommon, aggressive variant of DLBCL presenting initially in bone marrow, liver and spleen without lymphadenopathy or mass lesion. Patients with BLS-type DLBCL present frequently with haemophagocytic syndrome which often leads to early patient demise. Programmed death ligand 1 (PD-L1) plays a negative regulatory role on effector T cells and is an important target of immunotherapy. Assessment of PD-L1 expression in BLS-type DLBCL may carry therapeutic implications and provide mechanistic insights. Standard immunohistochemical analysis for PD-L1 was performed in seven cohorts for this study: (1) DLBCL-not otherwise specified (NOS) (n=201); (2) Epstein-Barr virus (EBV)-positive DLBCL (n=26); (3) thymic (primary mediastinal) DLBCL (n=12); (4) intravascular LBCL (n=3); (5) high-grade B-cell lymphoma, NOS (n=12); (6) BLS-type DLBCL (n=37); and (7) systemic DLBCL involving bone marrow (n=28). We found that PD-L1 was positive in 12.9% of DLBCL-NOS cases, 46.2% of EBV-positive DLBCL, 91.7% of thymic LBCL, none of intravascular LBCL, 8.3% of high-grade B-cell lymphoma-NOS, and 56.8% of BLS-type DLBCL. By comparison, only 14.3% of bone marrow cases involved by systemic DLBCL were positive for PD-L1 (p<0.001). Interestingly, BLS-type DLBCL more frequently showed activated B-cell phenotype (86.5% vs 65.2%, p=0.010), a high Ki-67 proliferative index (97.1% vs 63.3%, p<0.001), MYC overexpression (90.9% vs 56.2%, p=0.023), presence of haemophagocytic syndrome (86.5% vs 4.0%, p<0.001), and poorer overall survival (p<0.001) than DLBCL-NOS. These data suggest that the poor prognosis of BLS-type DLBCL may be explained by both extrinsic tumour microenvironment factors and intrinsic genetic factors of tumour cells, such as PD-L1-associated inactivation of anti-tumour immunity for the former, and MYC pathway activation-related aggressiveness for the latter.

Clinical implications of PNA­sequencing as a complementary test for EGFR mutation analysis in human lung cancer.

Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) are the first-line regimen for the treatment of non-small cell lung cancer (NSCLC) patients with EGFR mutations. However, false-negative results are occasionally observed, even with FDA-approved molecular tests. Such examples in have been reported in our pilot study showing a slightly upward-shifted amplification curve using commercial reverse transcription-quantitative (RT-q)PCR. Verification using peptide nucleic acid (PNA) clamping-sequencing, which has a sensitivity of ~0.1%, may allow better prediction of which patients will benefit from EGFR-TKI therapy. To confirm this hypothesis, samples were prospectively collected from 1,783 lung cancer cases diagnosed in National Cheng Kung University Hospital between 2012-2018. An independent lung cancer cohort of 1,944 cases was also recruited from other hospitals. The clinical significance of mutant-enriched PCR with PNA-sequencing was analyzed and patient outcomes were followed. A total of 17 of 34 cases (50%) were found to harbor EGFR mutations by PNA-sequencing. A total of 22 cases were discovered in the independent lung cancer cohort, and 14 of these (63.6%) cases had EGFR mutations. TKIs were administered to 14 of the 17 mutation-positive patients, and a partial response was observed in 4 cases and stable disease in 10 cases. Patients with EGFR mutations receiving a TKI regimen had a longer overall survival (OS) (median: 40.0 vs. 10.0 months) compared with those without treatment. The difference in OS was not significant. Based on the results of the present study, combining RT-qPCR with PNA-sequencing may be a practical supplementary technology in a clinical molecular laboratory for a subset of lung cancer patients in selection of EGFR TKI therapy.

The experience of neoadjuvant chemotherapy versus upfront surgery in resectable pancreatic cancer: a cross sectional study.

BACKGROUND: Upfront resection (UR) followed by adjuvant chemotherapy remains the standard treatment for resectable pancreatic cancer. There is increasing evidence suggesting favourable outcomes toward neoadjuvant chemotherapy (NAC) followed by surgery. METHODS: All clinical staging with resectable pancreatic cancer patients treated at a tertiary medical centre from 2013 to 2020 were identified. The baseline characteristics, treatment course, surgery outcome and survival results of UR or NAC were compared. RESULTS: Finally, in 159 resectable patients, 46 patients (29%) underwent NAC and 113 patients (71%) received UR. In NAC, 11 patients (24%) did not receive resection, 4 (36.4%) for comorbidity, 2 (18.2%) for patient refusal and 2 (18.2%) for disease progression. In UR, 13 patients (12%) were unresectable intraoperatively; 6 (46.2%) for locally advanced and 5 (38.5%) for distant metastasis. Overall, 97% of patients in NAC and 58% of patients in UR completed adjuvant chemotherapy. As of data cut-off, 24 patients (69%) in NAC and 42 patients (29%) in UR were still tumour free. The median recurrence-free survival in NAC, UR with adjuvant chemotherapy and without adjuvant chemotherapy were 31.3 months (95% CI, 14.4-not estimable), 10.6 months (95% CI, 9.0-14.3) and 8.5 months (95% CI, 5.8-11.8), P =0.036; and the median overall survival in each group were not reached (95% CI, 29.7-not estimable), 25.9 months (95% CI, 21.1-40.5) and 21.7 months (12.0-32.8), P =0.0053. Based on initial clinical staging, the median overall survival of NAC was not significantly different from UR with a tumour less than or equal to 2 cm, P =0.29. NAC patients had a higher R0 resection rate (83% versus 53%), lower recurrence rate (31% versus 71%) and harvested median number lymph node (23 versus 15). CONCLUSION: This study demonstrates that NAC is superior to UR in resectable pancreatic cancer with better survival.

Investigation into the content of red material in EUS-guided pancreatic cancer biopsies.

BACKGROUND AND AIMS: The red material occupying the larger portion of the acquired sample in EUS fine-needle biopsy (FNB) is seldom investigated. We aimed to evaluate the composition of the red material. METHODS: Patients with a solid pancreatic mass who received EUS FNB from September 2020 to June 2021 were enrolled. The white or yellowish content with apparent bulk (white material) and the rest of pasta-like red content (red material) were separated immediately after puncture. Needle passes proceeded until 2 specimens with >4 mm of white material were obtained. An extra needle pass was conducted for DNA collection. The DNA amount, Kirsten rat sarcoma virus (K-ras) mutation type, and mutation allele frequency were compared between the white and red material. RESULTS: Forty patients were enrolled with 68 paired white and red materials. The diagnostic accuracy was slightly higher in the white material (92.5% vs 82.5%, P = .219). On the histology slides, the area of the tumor gland was comparable in both materials, but the total tissue area was larger in the red material (9.74 mm2 and 10.74 mm2 larger according to generalized linear model and generalized estimating equation, respectively; both, P < .001). The amount of DNA was significantly higher in the red material (2.99 [interquartile range, 1.59-7.29] µg vs .70 [interquartile range, .27-1.24] µg; P < .001). Common pancreatic adenocarcinoma K-ras mutation was identified at a rate of 85% for the white material and 95% for the red material. Regardless of whether red or white material was used, there was a high concordance of K-ras mutation types (34 of 40 [85%]) and a high correlation of mutation allele frequency (ρ = .66, P < .001). CONCLUSIONS: In EUS FNB, the red material contains a higher amount of tumor DNA and can be an alternative source for tumor DNA analysis.

Evolution of the Correa's cascade steps: A long-term endoscopic surveillance among non-ulcer dyspepsia and gastric ulcer after H. pylori eradication.

BACKGROUND: This study is aimed toward investigating the evolution of each Correa's step after Helicobacter pylori eradication in a long-term follow-up and exploring the factors correlated with a high-risk of gastric cancer. METHODS: A total of 1824 H. pylori-infected subjects were enrolled to receive screening endoscopy. Among them, 491 received surveillance endoscopy. The patients were divided into Correa's steps I to VI, from normal to gastric cancer. A group-based trajectory model was used to classify patients as persistent high-risk status or not. RESULTS: The prevalence rates of positive corpus-predominant gastritis index (CGI) were 20%-40% in all age groups and Correa's steps IV-V increased >35% after 50 years based on screening endoscopy. Successful eradication of H. pylori regressed CGI after the 1st year-and-thereafter (P < 0.05) and decreased Correa's step progression (Relative risk 0.66 [95% CI 0.49-0.89], P = 0.01); however, it did not regress OLGA and OLGIM. Not only in steps IV-V, but also in step III, the patients had a risk of developing gastric cancer (11.13-76.41 and 4.61 per 1000 person-years). Age (Hazard ratio 1.012 [1.003-1.020], P = 0.01), OLGA stages ≥ I (2.127 [1.558-2.903], P < 0.001), and OLGIM stages ≥ I (1.409 [1.119-1.774], P = 0.004) were correlated independently with a persistent high-risk status. CONCLUSION: The patients in Correa's steps III-V, but not I-II, were at risk of gastric cancer after H. pylori eradication. Age, OLGA stages ≥ I, and OLGIM stages ≥ I were independent factors correlated to a persistent high-risk of gastric cancer. The data may be useful when scheduling surveillance endoscopy for subjects in each Correa's step (NCT04527055).

Improved survival with induction chemotherapy and conversion surgery in locally advanced unresectable pancreatic cancer: a single institution experience.

Both efficacy and tolerability are critical issues in choosing neoadjuvant chemotherapy in patients with unresectable locally advanced pancreatic cancer (LAPC). The optimal regimen and the impact of conversion surgery on patient survival remains insufficiently reported in Asain population. Therefore, we conducted a retrospective study aiming to evaluate the resection rate after different induction chemotherapy regimen and its impact toward survival. All patients with pancreatic cancer treated in our institute from 2013 to 2020, a total of 730 patients, were reviewed and 131 patients with LAPC were identified. For cohort homogeneity, 14 patients receiving induction concurrent chemoradiotherapy initially were excluded and 117 patients receiving induction chemotherapy were included in the study. Most patients (90 of 117, 77%) received triplet induction chemotherapy, including the combination of S1, leucovorin, oxaliplatin and gemcitabine (SLOG) in 48, modified FOLFIRINOX in 21 and the combination of gemcitabine, oxaliplatin, fluorouracil and leucovorin (GOFL) in 21. The tumor response rate (19%-33%), the surgical exploration rate (38%-52%) and the mOS (15.4-23.0 months) were not significantly different among the three triplets. Both GOFL and SLOG regimen had comparable efficacy and less neutropenia as compared to mFOLFIRINOX. Conversion surgery was performed in 34 of 117 (29%) patients after induction chemotherapy. The median overall survival (mOS) in patients with and without conversion surgery were 29.1 and 14.1 months, respectively (P<0.0001). Radiological response alone was not a reliable indicator of successful conversion surgery. Patients who underwent conversion surgery had significantly better survival and thus highlighted the importance of surgical exploration in all patients who did not have progressive disease after induction chemotherapy.

Complex Hydrogels Composed of Chitosan with Ring-opened Polyvinyl Pyrrolidone as a Gastroretentive Drug Dosage Form to Enhance the Bioavailability of Bisphosphonates.

Complex hydrogels formed with chitosan (CS) and ring-opened polyvinyl pyrrolidone (roPVP) as a swellable mucoadhesive gastroretentive drug dosage form (smGRDDF) were prepared and characterized. CS/roPVP hydrogels were produced by blending CS with roPVP obtained by basic treatment of PVP. Effects of the heating time and NaOH concentration employed for preparing roPVP, and CS molecular weights (Mws), and roPVP/CS ratios on the swelling ability of the resultant hydrogels were characterized. Rheological characteristics were further examined. Results demonstrated that roPVP obtained in a 0.5 M NaOH solution heated to 50 °C for 4 h was suitable for producing complex hydrogels with CS. At a roPVP/CS ratio of 20:1, hydrogels composed of three different Mws of CS possessed optimal swelling and mucoadhesive abilities and rheological properties. In vitro dissolution revealed sustained drug release. A pharmacokinetic study exhibited that the plasma profile of alendronate followed a sustained manner with 3-fold enhancement of the oral bioavailability. In conclusion, the smGRDDF composed of CS/roPVP complex hydrogels was successfully developed and is potentially applicable to improve the clinical efficacy of bisphosphonates.

Quantitative analysis of alkaloidal constituents in imported fire ants by gas chromatography.

A method based on silica gel chromatography and GC-MS/GC-FID analyses was developed for the quantitation of alkaloidal compounds in imported fire ants, Solenopsis richteri, S. invicta, and their hybrid found in the southern United States. The cis and trans alkaloids from fire ant body extracts were successfully separated by silica gel chromatography, identified by GC-MS, and quantitated by GC-FID. Piperideine compounds were eluted together with the cis and trans piperidines, but were well-resolved on a nonpolar GC column. Eight pairs of piperidine isomers and 12 piperideines were quantitated. The ratios of trans alkaloids to corresponding cis isomers ranged from 87 to 378:1 in S. invicta and were significantly higher than in S. richteri and hybrid ants. The results were discussed in relation to the evolution of fire ant venom alkaloids and their role as host location cues for parasitic Pseudacteon phorid flies.