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Abstract Hepatocellular carcinoma (HCC) is a molecularly heterogeneous solid malignancy, and its fitness may be shaped by how its tumor cells evolve. However, ability to monitor tumor cell evolution is hampered by the presence of numerous passenger mutations that do not provide any biological consequences. Here we develop a strategy to determine the tumor clonality of three independent HCC cohorts of 524 patients with diverse etiologies and race/ethnicity by utilizing somatic mutations in cancer driver genes. We identify two main types of tumor evolution, i.e., linear, and non-linear models where non-linear type could be further divided into classes, which we call shallow branching and deep branching. We find that linear evolving HCC is less aggressive than other types. GTF2IRD2B mutations are enriched in HCC with linear evolution, while TP53 mutations are the most frequent genetic alterations in HCC with non-linear models. Furthermore, we observe significant B cell enrichment in linear trees compared to non-linear trees suggesting the need for further research to uncover potential variations in immune cell types within genomically determined phylogeny types. These results hint at the possibility that tumor cells and their microenvironment may collectively influence the tumor evolution process.

In Southeast Asia, the rhizome of Etlingera pavieana is commonly consumed and parts of the rhizomes have been used as a medicine for the treatment of several disorders. Its pharmacological effects have previously been reported. However, its potential toxicity has not been described. This study aimed to evaluate in vivo toxicity of E. pavieana rhizome extract (EPE) in Sprague Dawley rats. Acute toxicity testing of EPE at a single dose of 2,000 mg/kg produced no toxic effects in female rats after 14 days of treatment. Subchronic toxicity testing showed that all doses of EPE (500, 1,000, and 2,000 mg/kg/day) produced no sign of toxicity during 90 days of treatment. All biochemical and hematological values were within normal ranges. There were no significant histopathological differences in the internal organs among the tested groups. Therefore, the no-observed-adverse-effect level of EPE was 2,000 mg/kg/day in both male and female rats, thereby confirming the safety of EPE for use in traditional medicines.

Objectives: This study aimed to investigate the expression of programmed cell death-ligand 1 (PD-L1) and its associations with human papillomavirus (HPV) 16/18 DNA status, p16 expression, demographic, clinicopathologic and risk parameters in patients with oral squamous cell carcinoma (OSCC). Study design: A total of 85 formalin-fixed, paraffin-embedded OSCC specimens were collected. HPV16/18 DNA was detected by polymerase chain reaction. PD-L1 and p16 expressions were assessed using immunohistochemical technique. The immunostaining scores were calculated by combined positive score (CPS), previously described. The positive scoring value was determined at CPS ≥1, recommended by FDA. The associations between PD-L1 expression and HPV16/18 DNA status, p16 expression, demographic, clinicopathologic, and risk parameters were analyzed by Chi-square, Fisher's exact tests, and multivariate logistic regression. Results: PD-L1 expression was detected in 22 out of 85 cases of OSCC (25.9%). 16.5% of all cases were HPV 16/18-positive and 62.4% were p16-positive. Statistically, there were no significant associations between PD-L1 expression in OSCC and HPV16/18 DNA status, p16 expression, demographic and, clinicopathologic parameters or risk behaviors. Conclusion: Approximately one-fourth of OSCC cases were PD-L1-positive, suggesting candidacy for anti-PD-L1 immunotherapy. Furthermore, HPV infection and p16 expression were not involved with PD-L1 expression. Further clinical trials warrant the benefits of immunotherapy in patients with PD-L1-positive OSCC.

Summary: Background: An appropriate treatment of older lung cancer patients has become an important issue. The aim of this study is to evaluate the short and long-term surgical outcomes in lung cancer patients using 70 years as a cut-point, and to identify prognostic factors of cancer-specific mortality in patients older than 70 years. Methods: Medical records of non-small cell lung cancer (NSCLC) patients who underwent pulmonary resection at Chiang Mai University Hospital from January 2002 through December 2016 were retrospectively reviewed. Patients were divided into age less than 70 years (control group) and 70 years or more (study group). Primary outcomes were major post-operative complications and in-hospital death (POM); secondary outcome was long-term survival. Multivariable regression analysis was used. Results: This study included 583 patients, 167 for study group, and 416 for control group. There were no differences in POM, both at univariable and multivariable analyses, however, for long-term cancer-specific mortality, the study group was more likely to die (HRadj = 1.40, 95%CI = 1.03–1.89). Adverse prognostic factors for long-term mortality in study group were having universal coverage scheme (HRadj = 1.70, 95%CI = 1.03–2.79), the presence of intratumoral lymphatic invasion (HRadj = 2.83, 95%CI = 1.28–6.29), perineural invasion (HRadj = 2.80, 95%CI = 1.13–6.94), underwent lymph node sampling (HRadj = 2.23, 95%CI = 1.16–4.30) and higher stage of disease (HRadj = 2.02, 95%CI = 1.06–3.85 for stage III, HRadj = 3.40, 95%CI = 1.29–8.94 for stage IV). Conclusions: In-hospital mortality and composite post-operative complications are acceptable in pulmonary resection for NSCLC patients older than 70 years. However, these patients had shorter long-term survival, especially who have some adverse prognostic factors. Further studies with larger sample size are warranted. Keywords: Old age, Resectable NSCLC, Lobectomy, Prognostic factor, Propensity score

The incidence of lung cancer has increased while the mortality rate has continued to remain high. Effective treatment of this disease is the key to survival. Therefore, this study is a necessity in continuing research into new effective treatments. In this study we determined the effects of three different Thai herbs on lung cancer. Bridelia ovata, Croton oblongifolius, and Erythrophleum succirubrum were extracted by ethyl acetate and 50% ethanol. The cytotoxicity was tested with A549 lung cancer cell line. We found four effective extracts that exhibited toxic effects on A549 cells. These extracts included ethyl acetate extracts of B. ovata (BEA), C. oblongifolius (CEA), and E. succirubrum (EEA), and an ethanolic extract of E. succirubrum (EE). Moreover, these effective extracts were tested in combination with chemotherapeutic drugs. An effective synergism of these treatments was found specifically through a combination of BEA with methotrexate, EE with methotrexate, and EE with etoposide. Apoptotic cell death was induced in A549 cells by these effective extracts via the mitochondria-mediated pathway. Additionally, we established primary lung cancer and normal epithelial cells from lung tissue of lung cancer patients. The cytotoxicity results showed that EE had significant potential to be used for lung cancer treatment. In conclusion, the four effective extracts possessed anticancer effects on lung cancer. The most effective extract was found to be E. succirubrum (EE).

Abstract Purpose To evaluate clinical characteristics, treatments, and outcomes in patients with ocular surface squamous neoplasia (OSSN) at a tertiary center in Northern Thailand. Methods Patients diagnosed with either corneal-conjunctival intraepithelial neoplasia (CIN) or squamous cell carcinoma (SCC) from May 2000 to December 2015, were recruited. The patients’ demographics, symptoms, clinical characteristics, cytopathology, treatments, and outcomes were reviewed. Results Overall 171 eyes from 168 patients, 92 eyes were CIN and 79 eyes were SCC. Males were affected in 65.5%. The mean age was 58.8 ± 16.8 (29–99) years. In most cases (60.3%), the tumors were located at the limbus. The most common clinical characteristic was papilliform appearance (46.2%). Human immunodeficiency virus (HIV) infection was found in 37 (22.0%) patients with a mean age of 40.5 ± 7.7 years. The treatments and outcomes were evaluated in 136 eyes whose main initial treatment was wide excision with adjunctive cryotherapy (47.8%), followed by topical mitomycin C (30.9%). The mean follow-up time after treatment was 20.8 ± 2.2 (3–110) months and the recurrence occurred in 18 eyes (13.2%) during the follow-up period. The mean recurrence-free time (months) for CIN was significantly longer than that of SCC (81.3 ± 10.0 [95%CI 61.5 – 101.1] vs 33.2 ± 4.6 [95%CI 24.0 – 42.3], p = 0.030). SCC was the only significant risk factor that influences the recurrence of the tumors with the adjusted hazard ratio of 5.69 (p = 0.005). Conclusion OSSN in Northern Thailand usually involved a limbal area and presented as a papilliform mass. HIV infection should be suspected in young patients. CIN had better outcomes after treatments than invasive SCC.

Background: The Indian Ayurvedic herbal formula Triphala (TPL) is known for its pharmacological properties for immunomodulation, anti-inflammation, antioxidant, and anti-cancer. This study aimed to investigate the acute and chronic toxicities of the Triphala recipe in a rat model. Methods: To assess the acute toxicities, 5000 mg/kg of TPL was orally administered to Sprague-Dawley rats. For chronic toxicities, different dose levels of TPL at 600, 1200, and 2400 mg/kg/day were given daily for 270 days. General health and behaviors and the body and organ weights of the rats were monitored. At the end of the experiment, blood samples were evaluated for hematology and biochemistry profiles. The evaluation of the internal organs’ appurtenance and necropsy was performed to confirm the tissue histopathology. Results: The results showed that there was no sign of acute toxicity in the TPL group with a decrease in sex organ weights. No significant differences in the rats’ behaviors, physical health, body, or organ weights were found between the controls and the rats receiving the 270/day of oral Triphala at 600, 1200, and 2400 mg/kg/day. However, some alterations in blood chemistries and hematology, including glucose, BUN, red blood cells, Hb, HCT, and MCV, were observed without abnormalities in histopathology. Conclusions: It has been demonstrated that the long-term use of TPL in rat models is safe. No toxic effects were found, suggesting possible safety for long-term use in humans.

Thailand is the country with highest incidence and prevalence of cholangiocarcinoma (CCA) in the world. Due to the frequently late diagnosis that is associated with this disease, most CCA patients are prescribed chemotherapy as a form of treatment. However, CCA is able to resist the presently available chemotherapy, so to the prognosis of this disease is still very poor. In this study, we investigated the anticancer potential of a Thai herbal recipe, Benja Amarit (BJA) against CCA and the relevant mechanisms of action that are involved. We found that BJA inhibited CCA cell viability in a dose-dependent manner, especially in highly invasive KKU-213 cells. The extract induced mitochondrial- and caspase-dependent apoptosis in CCA cells by regulating the nuclear factor-κB (NF-κB) signaling pathway. BJA also triggered autophagy in CCA cells. Nonetheless, the inhibition of autophagy enhanced BJA-induced CCA cell death via apoptosis. An in vivo xenograft model revealed the growth-inhibiting and death-inducing effects of BJA against CCA by targeting apoptosis. However, general toxicity to blood cells, kidneys and the liver, as well as changes in body weight, did not appear. Our findings suggest that the herbal recipe BJA might be used as a potentially new and effective treatment for cholangiocarcinoma patients.

Abstract Primary liver cancer (PLC), which includes intrahepatic cholangiocarcinoma (iCCA) and hepatocellular carcinoma (HCC), has the highest incidence of all cancer types in Thailand. Known etiological factors, such as viral hepatitis and chronic liver disease do not fully account for the country’s unusually high incidence. However, the gut-liver axis, which contributes to carcinogenesis and disease progression, is influenced by the gut microbiome. To investigate this relationship, fecal matter from 44 Thai PLC patients and 76 healthy controls were subjected to whole-genome metagenomic shotgun sequencing and then analyzed by marker gene-based and assembly based methods. Results revealed greater gut microbiome heterogeneity in iCCA compared to HCC and healthy controls. Two Veillonella species were found to be more abundant in iCCA samples and could distinguish iCCA from HCC and healthy controls. Conversely, Ruminococcus gnavus was depleted in iCCA patients and could distinguish HCC from iCCA samples. High Veillonella genus counts in the iCCA group were associated with enriched amino acid biosynthesis and glycolysis pathways, while enriched phospholipid and thiamine metabolism pathways characterized the HCC group with high Blautia genus counts. These findings reveal distinct landscapes of gut dysbiosis among Thai iCCA and HCC patients and warrant further investigation as potential biomarkers.

Abstract Programmed death-ligand 1 (PD-L1) expression has now been implicated in gastric cancer (GC). This study was conducted to determine the impact of clinicopathological characteristics on PD-L1 expression and its association with survival in GC patients receiving standard-of-care. In total, 268 GC patients receiving upfront surgery were enrolled at Chiang Mai University Hospital. PD-L1 expression was assayed by immunohistochemistry staining using the Dako 22C3 pharmDx. The rates of PD-L1 positivity by combined positive score (CPS) at a cutoff value of 1 and 5 were 22% and 7%. PD-L1 positivity was significantly higher in patients younger than 55 than those older than 55 (32.6% vs. 16.5%, p = 0.003; 11.6% vs. 4.4%, p = 0.027). PD-L1 positivity was observed more frequently in GC with metastases than without (25.2% vs. 17.1%, p = 0.112; 7.2% vs. 6.7%, p = 0.673). Patients with PD-L1 positive had a significantly shorter median overall survival than those with PD-L1 negative (32.7 vs. 41.6 months, p = 0.042, 27.6 vs. 40.8 months, p = 0.038). In conclusion, PD-L1 expression has been associated with young age, short survival, and metastases, although unrelated to the tumor stage. For GC patients, PD-L1 testing is recommended, especially among young patients with metastases.

In Thai traditional medicine, Pikad Tri-phol-sa-mut-than has long been used to alleviate gastrointestinal symptoms, renal disease, inflammation-related disorders, and severe malady. This recipe is composed of dried fruits of Morinda citrifolia L., Coriandrum sativum L., and Aegle marmelos (L.) Corrêa. The aim of this study was to assess the anti-gastric ulcer property of the water extract of Pikad Tri-phol-sa-mut-than (TS), using various animal models with different inducers, including restraint water immersion stress, indomethacin, and ethanol/hydrochloric acid (EtOH/HCl). Its mechanisms of anti-gastric ulcer actions were also elucidated using both in vitro and in vivo experiments. When compared with the control groups, the oral pretreatment of TS at the doses of 150, 300 and 600 mg/kg significantly reduced the gastric ulcer formation in all models. It was also found that TS at the dose of 600 mg/kg could increase gastric wall mucus in rats but could not produce the significant reduction of the gastric volume or total acidity of gastric content. Results from hematoxylin and eosin (H&E) and Periodic acid-Schiff (PAS) staining examinations of gastric tissues confirmed that TS visibly reduced gastric mucosal damage, while immunohistochemistry revealed that TS remarkably suppressed the protein expression of Bcl-2-associated X (BAX), a regulator of apoptosis, compared to those of the control group. The DPPH, ABTS, and FRAP assays showed antioxidant effects of TS. All of these findings demonstrated that TS has gastroprotective effects, which may be related to the increase in the gastric wall mucus secretion, not anti-secretory activity, as well as its antioxidant and antiapoptotic activities.

We report a case of conjunctival intraepithelial neoplasia (CIN) in a patient presenting with the pigmented conjunctival lesion.This study involved a 56-year-old woman that presented with right eye irritation for 1 month. She noticed brownish pigmentation arising from her right nasal conjunctiva and growing slowly over time. Biomicroscopic examination showed a gelatinous pigmented conjunctival mass with feeder vessels. Conjunctival impression cytology (CIC) was done and reported as CIN. Treatment was started with 0.02% mitomycin-C eye drops. The conjunctival lesion responded well to medication. This report shows that CIN can manifest as a pigmented tumor, resembling melanoma. CIC plays a role in the diagnosis of this condition. This tumor responded well with 0.02% mitomycin-C eye drops.

Background: Bacopa monnieri is a medicinal plant which has long been used in Ayurvedic medicines to augment brain function and to improve memory. The purpose of our study was to identify and evaluate possible toxic effects of B. monnieri extract in rats by assessing hematological, biochemical, and histopathological parameters. Methods: Acute oral toxicity of Bacopa monnieri extract was studied in female rats by giving a single orally administered dose at a level of 5,000 mg/kg. The rats were monitored for toxic signs for 14 days. In the chronic toxicity test, groups of both female and male rats were given daily oral doses of B. monnieri extract at dose levels of either 30, 60, 300 or 1,500 mg/kg for 270 days. The behavior and health of the animals was then monitored. At the end of the observation period, the body and organ weights of the rats in each group were measured. Blood was collected and necropsy was performed to evaluate their hematology, blood clinical chemistry, and microanatomy. Results: The acute toxicity test found no significant differences between the experimental and the control group rats. In the chronic toxicity test, animal behavior and health of the experimental groups were normal, just as in the control rats. All values of other parameters assessed remained within the normal range. Conclusion: A single oral administration of B. monnieri extract at the dose of 5,000 mg/kg did not cause any serious undesirable effects. B. monnieri extract at doses of 30, 60, 300 and 1,500 mg/kg given for 270 days did not produce any toxicity in rats. [ABSTRACT FROM AUTHOR]

The water extract from Harrisonia perforata Merr. was studied for acute and subchronic toxicities. The extract at a single dose of 5,000 mg/kg was administered orally to female and male rats (5 males, 5 females). After 14 days, signs and behavioral changes, mortality, gross and histopathological changes of internal organs were examined. The body weight of the male treated rats was significantly decreased when compared to the control group. The extract did not produce signs of toxicity. For the subchronic toxicity test, the extract at doses of 300, 600 and 1,200 mg/kg body weight were orally administeredto rats daily for 90 days (10 males, 10 females). Observation of signs, behavior and health status showed no abnormality in the test groups as compared with the controls. However, the body weight of all male treated rats was significantly decreased when compared to the control group. At the end of the study, necropsy and histopathology examination were performed in all animals in the control group, the test groups and the satellite group in which the extract was discontinued for another 28 days. Body and organ weights, hematological and blood clinical chemistry were also examined. The results suggest that the water extract of Harrisonia perforata Merr. does not cause acute and subchronic toxicities in rats.

The study was carried out to evaluate acute and subchronic toxicities of the water extract from Tiliacora triandra(Colebr.) Diels. A single oral administration of the extract at a dose of 5,000 mg/kg body weight (5 males, 5 females) did not produce signs of toxicity, behavioral changes, mortality, changes on gross appearance or histopathological changes of internal organs. The subchronic toxicity was determined by oral feeding both male and female rats (10 males, 10 females) daily with the test substance at the doses of 300, 600 and 1,200 mg/kg body weight continuously for 90 days.The examinations of signs, animal behavior and health monitoring showed no abnormalities in the test groups as compared to the controls. The test and control groups (on the 90th day) and the satellite group (on the 118th day) were analyzed by measuring their final body and organweights, taking necropsy, and examining hematology, blood clinical chemistry and histopathology. The results suggest that thewater extract from the T. triandra does not cause acute or subchronic toxicities in either male or female rats.

Acute and subchronic toxicities of the water extract from the root of Sida rhombifolia Linn. were studied in both male and female rats. Oral administration of the extract at a single dose of 5,000 mg/kg body weight (5 males, 5 females) did not produce signs of toxicity, behavioral changes, mortality or differences on gross appearance of internal organs. The subchronic toxicity was determined by oral feeding the test substance at the doses of 300, 600 and 1,200 mg/kg body weight for 90 days (10 males, 10 females). The examinations of signs, animal behavior and health monitoring showed no signs of abnormalities in the test groups as compared to the controls. The test and control groups (on the 90th day) and the satellite group (on the 118th day) were analyzed by measuring their final body and organ weights, taking necropsy, and examining hematological parameters, blood clinical chemistry and histopathology features. The results suggest that S. rhombifolia administered orally did not cause acute or subchronic toxicities to male and female rats.

The study was carried out to evaluate acute and subchronic toxicities of the water extract from the dried fruits of Piper nigrum L. A single oral administration of the extract at a dose of 5,000 mg/kg body weight (5 male, 5 female) did not produce signs of toxicity, behavioral changes, mortality, changes on gross appearance or histopathological changes of internal organs. The subchronic toxicity was determined by oral feeding both male and female rats (10 male, 10 female) daily with the test substance at the doses of 300, 600 and 1,200 mg/kg body weight continuously for 90 days. The examinations of signs, animal behavior and health monitoring showed no abnormalities in the test groups as compared to the controls. The test and control groups (on the 90th day) and the satellite group (on the 118th day) were analyzed by measuring their final body and organ weights, taking necropsy, and examining hematology, blood clinical chemistry and histopathology. The results suggest that the water extract from the dried fruits of P. nigrum does not cause acute or subchronic toxicities in either male or female rats.

Acute and subchronic toxicities of the water extract from the roots of Citrus aurantifolia were studied in both male and female rats. Oral administration of the extract at a single dose of 5,000 mg/kg body weight (5 male, 5 female) did not produce signs of toxicity, behavioral changes, mortality or differences on gross appearance of internal organs. The subchronic toxicity was determined by oral feeding the test substance at the doses of 300, 600 and 1,200 mg/kg body weight for 90 days (10 male, 10 female). The examinations of signs, animal behavior and health monitoring showed no signs of abnormalities in the test groups as compared to the controls. The test and control groups (on the 90th day) and the satellite group (on the 118th day) were analyzed by measuring their final body and organ weights, taking necropsy, and examining hematological parameters, blood clinical chemistry and histopathology features. The oral administration of 1,200 mg/kg/ day of the extract of C. aurantifolia in male and female rats caused a significant increase in the liver enzymes, which remained within the normal range, but did not produce a significant histopathological change in the internal organs. In conclusion, the extract from the roots of C. aurantifolia administered orally did not cause acute or subchronic toxicities to male and female rats.

The water extract from root of Imperata cylindrica (Linn.) Raeusch. was studied for acute and subchronic toxicities. The extract at a single dose of 5,000 mg/kg was administered orally to female and male rats (5 male, 5 female). After 14 days, signs and behavioral changes, mortality, gross and histopathological changes of internal organs were examined. The extract did not produce signs of toxicity. For the subchronic toxicity test, the extract at doses of 300, 600 and 1,200 mg/kg body weight was orally administered to rats daily for 90 days (10 male, 10 female). The observation of signs, behavior and health status showed no abnormality in the test groups as compared with the controls. At the end of the study, necropsy and histopathology examination were performed in all animals in the control group, the test groups and the satellite group in which the extract was discontinued for another 28 days. Body and organ weights, hematological and blood clinical chemistry were also examined. The results suggest that the water extract of Imperata cylindrica (Linn.) Raeusch does not cause acute and subchronic toxicities in rats.