Evolving or immutable - phase I solid tumor trials in the era of precision oncology.

In the era of precision oncology (PO), systemic therapies for patients (pts) with solid tumors have shifted from chemotherapy (CT) to targeted therapy (TT) and immunotherapy (IO). This systematic survey describes features of trials enrolling between 2010 and 2020, focusing on inclusion criteria, type of dose escalation scheme (DES) utilized, and use of expansion cohorts (ECs). A literature search identified phase I studies in adults with solid tumors published January 1, 2000- December 31, 2020 from 12 journals. We included only studies enrolling between 2010 and 2020 to better capture the PO era. Two reviewers abstracted data; a third established concordance. Of 10,744 studies, 10,195 were non-topical or enrolled prior to 2010; 437 studies were included. The most common drug classes were TT (47.6%), IO (22%), and CT (6.9%). In studies which reported race, patients were predominantly white (61.7%) or Asian (25.7%), followed by black (6.5%) or other (6.1%). Heterogeneity was observed in the reporting and specification of study inclusion criteria. Only 40.1% of studies utilized ECs, and among the studies which used ECS, 46.6% were defined by genomic selection. Rule-based DES were used in 89% of trials; a 3+3 design was used in 80.5%. Of all drugs tested, 37.5% advanced to phase II, while 10.3% garnered regulatory licensure (for an indication tested in phase I). In the era of PO, TT and IO have emerged as the most studied agents in phase I trials. Rule-based DES, which are more relevant for escalating CT, are still chiefly utilized.

Carcinoid Heart Disease Management: A Multi-Disciplinary Collaboration.

Carcinoid heart disease (CaHD) is an important complication among patients with metastatic neuroendocrine tumors and carcinoid syndrome (CS). CS patients (25%-65%) eventually develop CaHD; these patients face a significantly increased risk of morbidity and mortality. Guidance papers (eg, clinical practice guidelines, consensus guidelines, and expert statements) have been established by major organizations across the disciplines of cardiology and oncology; however, these recommendations are not routinely implemented. The aim of this article is to encourage the integration of current recommendations from national societies into clinical practice. Early screening upon recognition of CS and prior to the development of CaHD symptoms is paramount, as no existing therapies are approved to reverse the fibrotic damage to the heart once it occurs. Valvular replacement is the only definitive treatment for CaHD once it has developed. When patients are noted to have urinary 5-hydroxyindoleacetic acid (5-HIAA) levels ≥300 µmol/24 h and/or serum N-terminal pro B-type natriuretic peptide (NT-proBNP) levels >260 pg/mL, echocardiography is recommended. Systemic approaches to control tumor growth and hormonal secretion include somatostatin analogs (SSAs), followed by options including peptide receptor radiotherapy (PRRT), everolimus and liver embolization. Telotristat is the primary choice for control of diarrhea refractory to SSA. Diuretics are the mainstay of heart failure symptom management for patients who develop CaHD. Considerations for future research are discussed, including the ongoing TELEHEART (TELotristat Ethyl in a HEART biomarker study) trial involving telotristat and not yet activated CHARRT (Carcinoid Heart disease And peptide Receptor Radiotargetted Therapy) study involving PRRT with lutetium 177 (177Lu) dotatate.

18F-FDG PET/CT improves diagnostic certainty in native and prosthetic valve Infective Endocarditis over the modified Duke Criteria.

BACKGROUND: International guidance recognizes the shortcomings of the modified Duke Criteria (mDC) in diagnosing infective endocarditis (IE) when transoesophageal echocardiography (TOE) is equivocal. 18F-FDG PET/CT (PET) has proven benefit in prosthetic valve endocarditis (PVE), but is restricted to extracardiac manifestations in native disease (NVE). We investigated the incremental benefit of PET over the mDC in NVE. METHODS: Dual-center retrospective study (2010-2018) of patients undergoing myocardial suppression PET for NVE and PVE. Cases were classified by mDC pre- and post-PET, and evaluated against discharge diagnosis. Receiver Operating Characteristic (ROC) analysis and net reclassification index (NRI) assessed diagnostic performance. Valve standardized uptake value (SUV) was recorded. RESULTS: 69/88 PET studies were evaluated across 668 patients. At discharge, 20/32 had confirmed NVE, 22/37 PVE, and 19/69 patients required surgery. PET accurately re-classified patients from possible, to definite or rejected (NRI: NVE 0.89; PVE 0.90), with significant incremental benefit in both NVE (AUC 0.883 vs 0.750) and PVE (0.877 vs 0.633). Sensitivity and specificity were 75% and 92% in NVE; 87% and 86% in PVE. Duration of antibiotics and C-reactive Protein level did not impact performance. No diagnostic SUV cut-off was identified. CONCLUSION: PET improves diagnostic certainty when combined with mDC in NVE and PVE.

All in the Levels-Programmed Death-Ligand 1 Expression as a Biomarker for Immune Checkpoint Inhibitor Response in Patients with Gastrointestinal Cancer.

Immune checkpoint inhibitors (ICIs) benefit patients with rare subsets of gastrointestinal (GI) cancer. Significant interest exists to identify predictive biomarkers that may increase the applicability of ICI therapy for these patients. Programmed death ligand 1 (PD-L1) is one such candidate; however, this biomarker has well-chronicled limitations. Combined positive score (CPS) ≥1 is the minimum PD-L1 expression threshold necessary for patients with gastric or gastroesophageal junction (GEJ) cancer to qualify for treatment with pembrolizumab; however, studies suggest that patients with higher CPS scores may derive greater benefit. We present the cases of two patients, both with low tumor mutational burden, microsatellite stable, and CPS ≥70 GI tumors (cholangiocarcinoma and GEJ cancer), who have achieved excellent tumor control with pembrolizumab. We postulate that, by testing for CPS in all patients with GI cancer and identifying a CPS threshold predictive of ICI response, PD-L1 expression could identify the patiets with GI cancer, in tissue agnostic fashion, who could benefit from ICI therapy.

Mismatch Repair (MMR) Gene Alteration and BRAF V600E Mutation Are Potential Predictive Biomarkers of Immune Checkpoint Inhibitors in MMR-Deficient Colorectal Cancer.

BACKGROUND: Immune checkpoint inhibitor (ICI) therapy is highly effective in metastatic mismatch repair-deficient (MMR-D) colorectal cancer (CRC). In this study, we evaluated molecular and clinical predictors of ICI response in MMR-D CRC. MATERIALS AND METHODS: Patient databases at four cancer institutions were queried. The Fisher exact test was performed to test the association of clinical and molecular markers. The Kaplan-Meier method was used to estimate progression-free survival (PFS) and compared by the log-rank test. Twelve- and 24-month PFS rates were compared by the Z test. RESULTS: A total of 60 patients with CRC with MMR-D/microsatellite instability-high who previously received ICIs were identified. Patients with liver metastasis had a lower overall response rate as compared with other sites of metastasis (36.4% vs. 68.7%; p = .081). Patients with MLH1/PMS2 loss had worse 1-year and 2-year PFS rates compared with patients with MSH2/MSH6 loss (84.2% vs. 57.8% and 78.2% vs. 54.2%, respectively; p < .001). There were improved 1-year and 2-year PFS rates in patients with wild-type BRAF when compared with patients with BRAF V600E mutation (73.3% vs. 40%, and 73.3% vs. 26.7%; respectively; p < .001). Patients aged >65 had significantly worse PFS rates as compared with patients aged ≤65 (p < .001). CONCLUSION: BRAF V600E mutation, MLH1 and/or PMS2 loss, as well as age >65 years and liver metastasis, may be predictive of duration of ICI response in patients with MMR-D CRC. Larger cohorts are needed to confirm our findings. IMPLICATIONS FOR PRACTICE: The results of this study reveal clinically important biomarkers that potentially predict immune checkpoint inhibitor response in patients with mismatch repair-deficient colorectal cancer.

Can an amino acid mixture alleviate gastrointestinal symptoms in neuroendocrine tumor patients?

BACKGROUND: Neuroendocrine tumors, although relatively rare in incidence, are now the second most prevalent gastrointestinal neoplasm owing to indolent disease biology. A small but significant sub-group of neuroendocrine tumor patients suffer from diarrhea. This is usually secondary to carcinoid syndrome but can also be a result of short gut syndrome, bile acid excess or iatrogenic etiologies. Recently, an amino acid based oral rehydration solution (enterade® Advanced Oncology Formula) was found to have anti-diarrheal properties in preclinical models. METHODS: A retrospective chart review of all NET patients treated with enterade® AO was performed after IRB approval. RESULTS: Ninety-eight NET patients who had received enterade® AO at our clinic from May 2017 through June 2019 were included. Patients (N = 49 of 98) with follow up data on bowel movements (BMs) were included for final analysis. Eighty-four percent of patients (41/49) had fewer BMs after taking enterade® AO and 66% (27/41) reported more than 50% reduction in BM frequency. The mean number of daily BMs was 6.6 (range, 3-20) at baseline before initiation of therapy, while the mean number of BMs at 1 week time point post enterade® AO was 2.9 (range, 0-11). CONCLUSIONS: Our retrospective observations are encouraging and support prospective validation with appropriate controls in NET patients. This is first published report of the potential anti-diarrheal activity of enterade® AO in NET patients.

Neuroendocrine and Adrenal Tumors, Version 2.2021, NCCN Clinical Practice Guidelines in Oncology.

The NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines) for Neuroendocrine and Adrenal Gland Tumors focus on the diagnosis, treatment, and management of patients with neuroendocrine tumors (NETs), adrenal tumors, pheochromocytomas, paragangliomas, and multiple endocrine neoplasia. NETs are generally subclassified by site of origin, stage, and histologic characteristics. Appropriate diagnosis and treatment of NETs often involves collaboration between specialists in multiple disciplines, using specific biochemical, radiologic, and surgical methods. Specialists include pathologists, endocrinologists, radiologists (including nuclear medicine specialists), and medical, radiation, and surgical oncologists. These guidelines discuss the diagnosis and management of both sporadic and hereditary neuroendocrine and adrenal tumors and are intended to assist with clinical decision-making. This article is focused on the 2021 NCCN Guidelines principles of genetic risk assessment and counseling and recommendations for well-differentiated grade 3 NETs, poorly differentiated neuroendocrine carcinomas, adrenal tumors, pheochromocytomas, and paragangliomas.

Epidemiology, Incidence, and Prevalence of Neuroendocrine Neoplasms: Are There Global Differences?

PURPOSE OF REVIEW: The purpose of our review is to explore global epidemiologic trends of gastroenteropancreatic (GEP) neuroendocrine tumors (NETs). Specifically, we sought to examine whether there are differences in incidence, prevalence, distribution (by primary tumor site, tumor grade, tumor stage at presentation), and overall survival of GEP NETs between different regions of the world. RECENT FINDINGS: GEP NET incidence rates are rising steadily in North America, Asia, and Europe, though this rise appears to be most profound in North America. The distribution of GEP NETs differs regionally as in North America small intestinal and rectal NETs are most prevalent, in Asia rectal and pancreatic NETs are most prevalent, and in Europe small intestinal and pancreatic NETs are most prevalent. Overall survival for patients with GEP NETs appears to be improving with time. Some of the global increase in GEP NET incidence can be explained by increased health care utilization. This factor alone, however, does not explain the rise completely. Population-based studies utilizing uniform data collection instruments and a standard pathologic grading system are needed to identify other factors which may be contributing to this phenomenon.

Molecular biohydrogen production by dark and photo fermentation from wastes containing starch: recent advancement and future perspective.

Changing lifestyle is increasing the energy demand. Fossil fuel is unable to deliver such huge energy. Clean energy from renewable source can solve this problem. Hydrogen is a clean and energy-efficient fuel and used for electricity generation by fuel cells or can be used in combustion engine. Easy availability of starch wastes from different industrial food processing wastes makes it a potential source for hydrogen (H2) generation. Among various processes such as steam reforming, electrolysis, biophotolysis of water and anaerobic fermentation, anaerobic fermentation technique is environmentally friendly and requires less external energy, making it a preferred process for H2 generation. Dark fermentation process can use wide range of substrates including agricultural and industrial starchy waste with low level of undesirable compounds. Application of both anaerobic dark and photofermentation can improve H2 yield and production rate. H2 production from wastes containing starch serves dual benefit of waste reduction and energy generation. As starch is a polymer and all hydrogen-producing bacteria cannot produce amylase to hydrolyze it, a pretreatment step is required to convert starch into glucose and maltose. In this present review paper, we have summarized: (i) potential of various types of starch-containing wastes as feedstock, (ii) various fermentation techniques, (iii) optimization of external process parameter, (iv) application of bioreactor and simulation in fermentation technique and (v) advancement in H2 production from starchy wastes.

Association of Vegetation Size With Valve Destruction, Embolism and Mortality.

AIM: The mortality of patients with infective endocarditis (IE) is high. The management of patients with large vegetations is controversial. This study sought to investigate the association of vegetation size on outcomes including valve destruction, embolism and mortality. METHODS AND RESULTS: One hundred and forty-two (142) patients with definite IE and transoesophageal echocardiography (TEE) imaging available for analysis were identified and data retrospectively reviewed. Vegetation length, width and area were measured. Severe valve destruction was defined as the composite of one or more of severe valve regurgitation, abscess, pseudoaneurysm, perforation or fistula. Associations with 6-month mortality were identified by Cox regression analysis. Eighty (80) (56.3%) patients had evidence of valve destruction on TEE. Vegetation length ≥10 mm and vegetation area ≥50 mm2 were significantly associated with increased risk of valve destruction, (both odds ratio OR 1.21, p=0.03 and p=0.02 respectively). Thirty-nine (39) (72.2%) patients who had an embolic event, did so prior initiation of antibiotics. Six (6)-month mortality was 18.3%. In the surgically managed group, vegetation size was not associated with mortality. In the medically managed group, vegetation area (mm2) was associated with increased mortality (HR 1.01, p<0.01) along with age (HR 1.06, p=0.03). CONCLUSION: Vegetation length ≥10 mm or area ≥50 mm2 are associated with increased risk of valve destruction. Vegetation size may also predict mortality in medically managed but not surgically managed patients with IE. Further studies to evaluate whether surgery in patients with large vegetation size improves outcomes is warranted.

Epoxyazadiradione exhibit activities in head and neck squamous cell carcinoma by targeting multiple pathways.

The head and neck squamous cell carcinoma (HNSCC) constitute about 90% of all head and neck cancers. HNSCC falls in the top 10 cancers in men globally. Epoxyazadiradione (EPA) and Azadiradione (AZA) are the limonoids derived from the medicinal plant Azadirachta indica (popularly known as Neem). Whether or not the limonoids exhibit activities against HNSCC and the associated mechanism remains elusive. Herein, we demonstrate that EPA exhibits stronger activity in HNSCC in comparison to AZA. The limonoids obeyed the Lipinski's rule of 5. EPA exhibited activities in a variety of HNSCC lines like suppression of the proliferation and the induction of apoptosis. The limonoid suppressed the level of proteins associated with anti-apoptosis (survivin, Bcl-2, Bcl-xL), proliferation (cyclin D1), and invasion (MMP-9). Further, the expression of proapoptotic Bax and caspase-9 cleavage was induced by the limonoid. Exposure of EPA induced reactive oxygen species (ROS) generation in the FaDu cells. N-acetyl-L-cysteine (ROS scavenger) abrogated the down-regulation of tumorigenic proteins caused by EPA exposure. EPA induced NOX-5 while suppressing the expression of programmed death-ligand 1 (PD-L1). Further, hydrogen peroxide induced NF-κB-p65 nuclear translocation and EPA inhibited the translocation. Finally, EPA modulated the expression of lncRNAs in HNSCC lines. Overall, these results have shown that EPA exhibit activities against HNSCC by targeting multiple cancer related signalling molecules. Currently, we are evaluating the efficacy of this molecule in mice models.

Immune-Related Adverse Events and Immune Checkpoint Inhibitor Efficacy in Patients with Gastrointestinal Cancer with Food and Drug Administration-Approved Indications for Immunotherapy.

INTRODUCTION: Immune-related adverse event (IRAE) onset may represent a clinical biomarker for anti-programmed cell death protein 1 (PD-1) antibody response based on emerging evidence from patients with various advanced malignancies. This phenomenon has not been previously reported in a multidisease cohort of patients with gastrointestinal (GI) cancer with Food and Drug Administration (FDA)-approved indications to receive immune checkpoint inhibitor therapy. MATERIALS AND METHODS: The study was a multicenter retrospective cohort analysis of 76 patients with GI cancer who had received anti-PD-1 antibodies for FDA-approved indications. The primary and secondary outcomes of the study were progression-free survival (PFS) and overall survival (OS) in patients based upon IRAE presence, respectively. PFS and OS were estimated by the Kaplan-Meier method; a Cox proportional-hazards model adjusted for IRAE onset, patient age, and enrolling institution was used to analyze outcomes. RESULTS: Median PFS and OS were prolonged in patients who experienced IRAEs compared with those who did not experience them (PFS: not reached [NR] vs. 3.9 months [hazard ratio (HR) 0.13, 95% confidence interval (CI) 0.05-0.3, p < .001]; OS: NR vs. 7.4 months [HR 0.11, 95% CI 0.03-0.36, p < .001]). Among patients who experienced IRAEs, there were no significant differences in PFS and OS by either initial IRAE severity, management, or time to onset. CONCLUSION: Patients with gastrointestinal cancer who experienced IRAEs while on anti-PD-1 antibodies demonstrated significant improvements in PFS and OS compared with their counterparts who did not develop IRAEs. Although these findings add to results from studies in other tumor types, larger prospective studies are needed prior to clinical adoption of IRAE onset as a biomarker for immune checkpoint inhibitor response. IMPLICATIONS FOR PRACTICE: Predictive clinical biomarkers for immune checkpoint inhibitor response have been understudied in the field of immuno-oncology. Immune-related adverse event onset appears to be one such biomarker. Across tumor types, immune-related adverse event onset has been associated with response to anti-programmed cell death protein 1 (PD-1) antibodies. The results of this study demonstrate this for the first time in patients with gastrointestinal cancer receiving anti-PD-1 antibodies. Before immune-related adverse event onset can be adopted clinically as a predictive biomarker for immune checkpoint inhibitor response, however, larger prospective studies are needed to better understand the nuances between immune-related adverse event characteristics (severity, site, management, timing of onset) and immune checkpoint inhibitor effectiveness.

Targeting DNA Damage Repair Pathways in Pancreatic Adenocarcinoma.

OPINION STATEMENT: Metastatic (and locally advanced) pancreatic adenocarcinoma (mPDA) represents a major challenge for the oncology community given the rising mortality burden from the disease and the preponderance of patients diagnosed with unresectable disease. Although systemic therapies have become more potent with the development of fluorouracil, irinotecan, and oxaliplatin (FOLFIRINOX) and gemcitabine plus nab-paclitaxel as first-line treatments, the median overall survival for patients treated with either of these regimens remains just above 1 year. A significant need exists to build upon the effectiveness of first-line regimens, incorporate tolerable maintenance treatments, and add effective later-line options for patients with this disease. We believe every newly diagnosed mPDA patient should undergo next-generation sequencing (NGS) testing, preferably from tumor tissue, to assess for the presence of DNA damage repair (DDR) defects, microsatellite instability, and other possible actionable molecular alterations (such as neurotrophic tropomysin receptor kinase (NTRK) fusions, anaplastic lymphoma kinase (ALK) rearrangements, or human epidermal growth factor receptor 2 (HER2) amplification). Existing clinical data suggests that patients, whose tumors harbor DDR defects, benefit from treatment with platinum-based chemotherapy and poly (ADP-ribose) polymerase (PARP) inhibitors. Preclinically, inhibitors of other critical players in DDR such as ataxia-telangiectasia and Rad3 related (ATR), ataxia-telangiectasia mutated (ATM), DNA-dependent protein kinase (DNA-PK), and WEE1 have demonstrated promising anti-tumor activity in PDA cell lines and xenografts. How to move forward the preclinical promise of these newer DDR-targeting therapies into rational clinical trial combinations and sequence PARP inhibitors in relation to platinum chemotherapy remain areas of tremendous clinical research interest. We believe clinical trials should be considered early for mPDA patients, in all treatment lines, so that novel therapies may be added to the treatment armamentarium for patients with this disease. Beyond NGS testing from tumor tissue, we believe it is important to consider germline genetic testing for all patients diagnosed with PDA given recent data suggesting a much stronger hereditary component of the disease than previously understood, and the potential screening implications for family members.

Harnessing the Immune System in Pancreatic Cancer.

OPINION STATEMENT: Managing patients with metastatic pancreatic adenocarcinoma (mPDA) is a challenging proposition for any treating oncologist. Although the potency of first-line therapies has improved with the approvals of FOLFIRINOX and gemcitabine plus nab-paclitaxel, many patients are unable to derive significant benefit from later lines of therapy upon progression. Enrollment on clinical trials remains among the best options for patients with mPDA in all lines of therapy. At our institution, we routinely check for microsatellite instability (MSI-H) and perform next-generation sequencing (NGS) at the time of diagnosis in all good performance status mPDA patients. Although MSI-H status is only found in 1% of patients with mPDA, given pembrolizumab's tissue-agnostic approval for MSI-H tumors in later-line settings, it is a viable option when deciding on subsequent lines of therapy. Any use of immune therapy in mPDA is investigational outside the MSI-H setting. NGS can identify BRCA or other DNA damage response (DDR) defects in patients which can predict sensitivity to platinum-based therapies and influence choice of both initial and later lines of therapy. It can also identify rare actionable genomic alterations such as HER2 (2%) and TRK fusions (0.1%) and offer patients the option of enrollment on clinical trials with agents targeting these or other identified alterations. We believe enrolling mPDA patients on clinical trials with immune-modulating agents is critical to determine if there are other patient subsets, outside of the MSI-H setting, who would benefit from these approaches. Immunotherapy's general tolerability and potential to generate durable responses make it particularly appealing for mPDA patients. Although single-modality immunotherapy such as checkpoint inhibitors or vaccines have not demonstrated efficacy in this disease, combinatorial strategies targeting unique aspects of PDA including the tumor microenvironment and desmoplastic stroma have shown preclinical or early-phase success. Validating these treatments with later-phase prospective studies is essential to making immunotherapy a routine component of the treatment armamentarium for mPDA patients.

Promising New Agents for Colorectal Cancer.

OPINION STATEMENT: Choosing the optimal treatment approach for patients with metastatic colorectal cancer (mCRC) demands that oncologists assess both clinical and genomic variables and individualize care based upon the findings. Clinically, choices depend on assessing the side of the colon in which the primary tumor originates, the sites and burden of metastatic disease, the patient's performance status, and their individual comorbidities. Genomic assessment of the tumor to discern the mutational status of genes such as RAS/RAF, HER2, and TRK, as well as assessing whether tumors have defective mismatch repair (dMMR) or high microsatellite instability (MSI-H), all factor in to potential treatment options and can determine clinical trial eligibility. Metastasectomy may be an option for patients with a low burden of disease and accessible liver- or lung-limited metastases. In some unresectable cases, systemic therapy with a FOLFOX- or FOLFIRI-based regimen with or without a biologic agent can lead to sufficient disease reduction to make a patient eligible for resection of metastatic disease. Tumor sidedness and RAS mutational status guide which biologic we add to the initial chemotherapy backbone, with patients with left-sided, RAS wild-type (WT) tumors receiving anti-epidermal growth factor receptor (EGFR)-directed therapy and patients with right-sided tumors or those with RAS mutations receiving bevacizumab. In patients with tumors that manifest microsatellite instability or deficient mismatch repair, we typically administer checkpoint inhibitors such as pembrolizumab or nivolumab after progression on irinotecan- or oxaliplatin-based therapies. In patients with progressive disease, we routinely send tumor tissue for next generation sequencing (NGS) to assess for the presence of actionable genomic alterations such as HER2, BRAF, and TRK fusions and offer them the option of enrollment on clinical trials with agents targeting those or other identified alterations.

Dynamics of regulatory T cells (Tregs ) in patients with oral squamous cell carcinoma.

BACKGROUND AND OBJECTIVES: The immune dysfunction in oral squamous cell carcinoma (OSCC) patients is one of the major factors for growth and dissemination of tumor affecting disease-free survival. METHODS: The phenotypic and functional characteristics of Regulatory T (Treg ) CD4+ CD25+ FoxP3+ subsets in OSCC patients were assessed by multicolor flow cytometry and its effector component (TGF-ß) by Western blot and qRT-PCR. RESULTS: An increased (P < 0.05) prevalence of Treg phenotypes (CD4+ CD25+ , CD4+ FoxP3+ , CD8+ FoxP3+ , CD4+ CD25+ FoxP3+ ) was observed in the peripheral circulation of OSCC patients that positively correlated with clinicopathological features. The increased frequency of CD4+ CD8+ CD25+ FoxP3+ , a unique T cell subset, CTLA-4+ , GITR+ , NrP1+ , HLA-DR+ , CD127+ , Tbet+ , TGF-ß+ , and granzyme B+ (GzmB) Tregs also showed a significantly higher prevalence in OSCC patients. Functionally, CD4+ FoxP3+ Tregs showed skewed expression of IL-2, IL-10, and IL-35 in patients as compared with the normal controls. Further, enhanced expression of CCR5 and CCR7 on Tregs with up regulation of their ligands (CCL5, CCL19, and CCL21) in tumor cells indicates efficient recruitment and trafficking of Tregs to the tumor site. CONCLUSION: It seems reasonable to assume that modulation of functional dynamics of selective Treg subsets may be useful in developing immunotherapeutic strategy for OSCC patients.

Phenotypic and Functional Characteristics of Th17 (CD4+IL17A+) Cells in Human Oral Squamous Cell Carcinoma and Its Clinical Relevance.

Recent studies have suggested an important role of T helper 17 (Th17) cells in tumor biology however, their phenotypic and functional aspects are poorly understood in context with oral cancer. We therefore, investigated the various phenotypic and functional markers of Th17 cells elucidating their relevance in oral squamous cell carcinoma (OSCC). Multi-color flow cytometry (FACs) was used to analyze the frequency and different markers of circulating Th17 cells ex vivo in peripheral blood mono-nuclear cells (PBMCs) from 69 OSCC patients and 35 healthy controls. Percent Mean ± SEM of different types of cells were compared between the two groups using Mann-Whitney U test. We found significantly (p < 0.0001) increased frequency of Th17 cells in patients as compared to controls. These cells were found to express CCR6 profoundly but not CXCR4, CD62L, and CCR7 as chemokine receptors. Additionally, it expressed HLA-DR, CD69, and CD25 moderately but CD28 and CD161 highly. The cytokine profiling revealed 3 subsets namely Th17/1 (IL17A+IFNγ+), Th17/inflammatory (IL17A+IL8+), and Th17/2 (IL17A+IL4+) which were found to be elevated in patients as compared to controls. The early stage patients had a shift toward Th17/1 type and vice versa. Our results suggest that Th17 cells may have effector immune functions in oral cancer immunity through CCR6, CD161, HLA-DR, CD69, CD28 receptors and inducing Th17/1 type of cells expressing polyfunctional antitumor IFNγ cytokine. Thus, novel immune-boosting regimens based on enhancement of Th17 cells in oral cancer patients may provide therapeutic benefits in them.

Garcinol inhibits tumour cell proliferation, angiogenesis, cell cycle progression and induces apoptosis via NF-κB inhibition in oral cancer.

Garcinol, a polyisoprenylated benzophenone is extracted from the rind of the fruit of Garcinia indica, a plant found extensively in tropical regions. Its ability to inhibit tumour growth has been demonstrated in certain cancers. In this study, we evaluated the potential anti-tumour effects of garcinol on oral squamous cell carcinoma (OSCC) cells. Three OSCC cell lines (SCC-4, SCC-9 and SCC-25) were treated with garcinol for 48 h and its effect on growth and proliferation, clonogenic survival, cell cycle and apoptosis was studied by MTT, clonogenic assay, propidium iodide (PI) staining and annexin-V binding assay, respectively. The alteration in expression of NF-κB and COX-2 was studied by western blot analysis and that of VEGF by ELISA. Garcinol treatment significantly (p < 0.001) inhibited the growth and proliferation and colony formation of OSCC cells with a concomitant induction of apoptosis and cell cycle arrest. It did not show toxic effect on normal cells. It significantly (p < 0.05) reduced the expression of NK-κB and COX-2 expression in treated cells as compared to untreated controls besides inhibiting VEGF expression. It appears that garcinol exerts anti-proliferative, pro-apoptotic, cell-cycle regulatory and anti-angiogenic effects on oral cancer cells through inhibition of NF-κB and COX-2. Thus, garcinol may be developed as a potential chemopreventive and/or chemotherapeutic agent for treatment of oral squamous cell carcinoma.

Prognostic significance of cytoplasmic p27 in oral squamous cell carcinoma.

OBJECTIVE: p27 is a cell cycle-dependent kinase inhibitor whose presence in nucleus is associated with good prognosis. Recent studies propose that when localized to cytoplasm, it functions as an oncogene and confers a poorer prognosis. This study aimed at analysing the subcellular localization of p27 and its prognostic significance in oral squamous cell carcinomas (OSCCs). METHODS: Immunohistochemistry for p27 was carried out on 60 cases of OSCC (30 cases each of those with lymph node metastasis [LN+ve SCC] and without lymph node metastasis [LN-ve SCC]) and 30 normal mucosa. The relationship between p27 localization and prognosis was analysed statistically. RESULTS: Nuclear immunopositivity was seen in 15%, 23%, 7% and 60%, while cytoplasmic immunopositivity was seen in 80%, 63%, 97% and 43% of all SCC, LN+ve OSCC, LN-ve SCC cases and normal mucosa, respectively. There was a significant inverse correlation between nuclear and cytoplasmic p27 immunopositivity (P = 0.001). Nodal status and tumour stage were the only two parameters that correlated with disease-free survival (DFS) in OSCC cases. However, in LN+ve SCC, a significantly shortened DFS was seen in cases with cytoplasmic p27 expression compared to those without (P = 0.02). Conversely, LN+ve SCC with nuclear p27 had longer DFS on comparison with those without (P = 0.04). CONCLUSIONS: To the best of our knowledge, this is the first study to analyse cytoplasmic localization of p27 in OSCC and correlate with prognosis. Cytoplasmic localization is associated with poor prognosis in OSCC with lymph node metastasis allowing the consideration of cytoplasmic p27 in predicting prognosis and targeted therapeutic approaches.

6-Gingerol Mediates its Anti Tumor Activities in Human Oral and Cervical Cancer Cell Lines through Apoptosis and Cell Cycle Arrest.

6-Gingerol, a potent nutraceutical, has been shown to have antitumor activity in different tumors, although its mechanism of action is not well understood. In this study, we evaluated antitumor activities of 6-gingerol on human oral (SCC4, KB) and cervical cancer (HeLa) cell lines with or without wortmannin, rapamycin, and cisplatin. Tumor cell proliferation was observed using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H tetrazolium, inner salt assay, cell cycle analysis by propidium iodide labeling and flow cytometry, apoptosis by Annexin-V binding assay, and caspase activity by chemiluminescence assay. 6-Gingerol showed dose-dependent cytotoxicity in all three cell lines. Combinations of 6-gingerol with wortmannin and cisplatin showed additive effects, while with rapamycin, it showed 50% cytotoxicity that was equivalent to IC50 of 6-gingerol alone. Treatment with 6-gingerol resulted in G2-phase arrest in KB and HeLa cells and S-phase arrest in SCC4 cells. 6-Gingerol, wortmannin, and rapamycin treatment showed almost two-fold higher expression of caspase 3 in all cell lines. The results imply that 6-gingerol either alone or in combination with PI-3 K inhibitor and cisplatin may provide better therapeutic effects in oral and cervical carcinoma. Thus, 6-gingerol appears to be a safe and potent chemotherapeutic/chemopreventive compound acting through cell cycle arrest and induction of apoptosis in human oral and cervical tumor cells.