Stem cells-derived natural killer cells for cancer immunotherapy: current protocols, feasibility, and benefits of ex vivo generated natural killer cells in treatment of advanced solid tumors.

Nowadays, natural killer (NK) cell-based immunotherapy provides a practical therapeutic strategy for patients with advanced solid tumors (STs). This approach is adaptively conducted by the autologous and identical NK cells after in vitro expansion and overnight activation. However, the NK cell-based cancer immunotherapy has been faced with some fundamental and technical limitations. Moreover, the desirable outcomes of the NK cell therapy may not be achieved due to the complex tumor microenvironment by inhibition of intra-tumoral polarization and cytotoxicity of implanted NK cells. Currently, stem cells (SCs) technology provides a powerful opportunity to generate more effective and universal sources of the NK cells. Till now, several strategies have been developed to differentiate types of the pluripotent and adult SCs into the mature NK cells, with both feeder layer-dependent and/or feeder laye-free strategies. Higher cytokine production and intra-tumoral polarization capabilities as well as stronger anti-tumor properties are the main features of these SCs-derived NK cells. The present review article focuses on the principal barriers through the conventional NK cell immunotherapies for patients with advanced STs. It also provides a comprehensive resource of protocols regarding the generation of SCs-derived NK cells in an ex vivo condition.

The Iranian Study of Opium and Cancer (IROPICAN): Rationale, Design, and Initial Findings.

BACKGROUND: The International Agency for Research on Cancer (IARC) recently classified opium use as a Group 1 carcinogen. However, much remains to be studied on the relation between opium and cancer. We designed the Iranian Opium and Cancer (IROPICAN) study to further investigate the association of opium use and cancers of the head and neck, bladder, lung, and colon and rectum. In this paper, we describe the rationale, design, and some initial results of the IROPICAN Study. METHODS: The IROPICAN is a multi-center case-control study conducted in 10 provinces of Iran. The cases were all histologically confirmed and the controls were selected from hospital visitors who were free of cancer, were not family members or friends of the cancer patients, and were visiting the hospital for reasons other than their own ailment. The questionnaires included detailed questions on opium use (including age at initiation, duration, frequency, typical amount, and route), and potential confounders, such as tobacco use (e.g., cigarettes, nass and water-pipe), and dietary factors. Biological samples, including blood and saliva, were also collected. RESULTS: The validation and pilot phases showed reasonably good validity, with sensitivities of 70% and 69% for the cases and controls, respectively, in reporting opium use. The results also showed excellent reliability, with intra-class correlation coefficients of 0.96 for ever opium use and 0.88 (95% CI: 0.80, 0.92) for regular opium use. In the main phase, we recruited 3299 cancer cases (99% response rate) and 3477 hospital visitor controls (89% response rate). The proportion of ever-use of opium was 40% among cases and 18% among controls. CONCLUSION: The IROPICAN study will serve as a major resource in studies addressing the effect of opium on risk of cancers of the head and neck, bladder, lung, and colon and rectum.

Opium use and the risk of head and neck squamous cell carcinoma.

Scant evidence exists to support the association of opium use with head and neck cancer, limited to the larynx and oral cavity. In a multicenter case-control study-Iran Opium and Cancer study, we recruited 633 cases of head and neck squamous cell carcinoma (HNSCC) (254 lip and oral cavity, 54 pharynx, 327 larynx and 28 other subsites within the head and neck) and 3065 frequency-matched controls from April 2016 to April 2019. Odds ratios (ORs) for opium use and 95% confidence intervals (95% CIs) were obtained using mixed-effects logistic regression because of heterogeneity among centers. The adjusted OR (95% CI) for regular opium use was 3.76 (2.96-4.79) for all HNSCC combined. Strong dose-response effects were observed by frequency or amount of use, and duration of use. Regular opium uses significantly increased the risk of HNSCC of the pharynx, larynx and other subsites within the head and neck with OR (95% CI) of 2.90 (1.40-6.02), 6.55 (4.69-9.13) and 5.95 (2.41-14.71), respectively. The observed associations were significant even among never tobacco smokers (including cigarette and water-pipe smoking). Moreover, by the multiplicative interaction scale, the effect of opium use could be varied by cigarette smoking on HNSCC, 8.16 (6.20-10.74). For the first time, the current study showed opium users have an increased risk of several anatomic subsites of HNSCC.

Association between mitochondrial DNA content and opium exposure.

To date, not much study has been done to investigate the mitochondrial DNA (mtDNA) copy number as the potential biomarker for opium exposure. Here, we conducted a cross-sectional study to determine the relative mtDNA content as the potential biomarker for opium exposure. Quantitative real-time PCR was performed to investigate the mtDNA copy number variation across 205 individuals, including blood samples of 45 opium users, 41 cigarette users, 47 dual users, and 72 never users of any product. We found a significantly higher mtDNA content among the opium-only users (adjusted OR: 3.21; 95% CI: [1.34, 7.66]; P = .009) and dual users (adjusted OR: 2.64; 95% CI: [1.15, 6.1]; P = .02) compared to that in never users even after adjustment for confounding factors, age, and sex. Discordantly, analysis of mitochondrial DNA in cigarette smokers revealed an indirect association between cigarette smoking and mtDNA content although it was not statistically significant. The reason behind the increased mitochondrial DNA is unclear. The possible hypothesis is that there might be a way to compensate for the oxidative damage induced by opium consumption. Taken together, our findings indicated that the mtDNA copy number may alter during opium exposure. Since changes in the mitochondrial DNA copy number was associated with the etiology of many diseases including cancer, further investigations on the mtDNA copy number may shed light on the carcinogenicity of opium consumption and means for early detection among the populations who have been exposed to opium and its products.

Gene Panel Testing in Hereditary Breast Cancer.

BACKGROUND: Breast cancer (BC) is a highly complex, heterogeneous and multifactorial disease and is the most commonly diagnosed cancer and the leading cause of cancer-related mortality in women worldwide. Family history and genetic mutations are important risk factors for BC. While studies in twins have estimated that about 10%-30% of BC are due to hereditary factors, only 4%-5% of them are due to mutations in BRCA1 or BRCA2 genes. Our aim was to investigate the role of other BC genes in familial BC among the Iranian population. METHODS: We selected 61 BC patients who were wild-type for BRCA1 and BRCA2 mutations but who met the criteria for hereditary BC based on the American College of Medical Genetics and Genomics (ACMG) and the National Comprehensive Cancer Network (NCCN) guidelines. We performed targeted sequencing covering the exons of 130 known cancer susceptibility genes based on the Cancer Gene Census list. RESULTS: We found seven mutations in seven known BC susceptibility genes (RAD50, PTEN, TP53, POLH, DKC1, WRN and CHEK2) in seven patients including two pathogenic frameshift variants in RAD50 and WRN genes, four pathogenic missense variants in TP53, PTEN, POLH, and DKC1 genes and a pathogenic splice donor variant in the CHEK2 gene. The presence of all these variants was confirmed by Sanger sequencing and Gap reverse transcription-polymerase chain reaction (RT-PCR) for the splice variant. In silico analysis of all of these variants predicted them to be pathogenic. CONCLUSION: Panel testing of BC patients who met the established criteria for hereditary BC but who were negative for BRCA1/2 mutations provided additional relevant clinical information for approximately 11.5% of the families. Our findings indicate that next generation sequencing (NGS) is a powerful tool to investigative putative mutagenic variants among patients who meet the criteria for hereditary BC, but with negative results on BRCA1/2 testing.

The NCCN Criterion "Young Age at Onset" Alone is Not an Indicator of Hereditary Breast Cancer in Iranian Population.

Because the contribution of genetic factors to the burden of breast cancer is not well investigated in Iran, we aimed to examine the prevalence of mutations in breast cancer susceptibility genes, BRCA1/2 and PALB2, and to investigate the predictive potential of hereditary breast cancer risk criteria for genetic testing in Iranian population. Next-generation sequencing was conducted on a population consisting of 299 and 125 patients with breast cancer, with and without hereditary cancer risk criteria for genetic testing, respectively. The pathogenic mutation frequency rate was 10.7% in patients with hereditary cancer criteria versus 1.6% in no criteria group (P = 0.0017). None of the 107 tested patients with only young age at onset (<40) criterion had a pathogenic mutation. Patients who had only a single heritable risk criterion [OR, 6.15; 95% confidence interval (CI), 1.26-58.59; P = 0.009] and patients with multiple heritable risk criteria (OR, 22.5; 95% CI, 5.19-201.31; P < 0.0001) had higher probabilities of carrying a mutation compared with no criteria group. Our results showed that young age at onset alone is not an indicator of hereditary breast cancer at least in the Iranian population. This is while women with multiple hereditary breast cancer risk criteria were enriched for BRCA1/2 mutations. Given such high risk of identification of a disease-causing mutation, multiple hereditary criteria should be regarded as a strong predictor for a hereditary breast cancer syndrome. These findings are important concerning the optimization of genetic counseling and furthermore establishing criteria for BRCA1/2 testing of the Iranian population.

The first successful application of preimplantation genetic diagnosis for hearing loss in Iran.

Hearing impairment (HI) caused by mutations in the connexin-26 gene (GJB2) accounts for the majority of cases with inherited, nonsyndromic sensorineural hearing loss. Due to the illegality of the abortion of deaf fetuses in Islamic countries, preimplantation genetic diagnosis (PGD) is a possible solution for afflicted families to have a healthy offspring. This study describes the first use of PGD for GJB2 associated non-syndromic deafness in Iran. GJB2 donor splicing site IVS1+1G>A mutation analysis was performed using Sanger sequencing for a total of 71 Iranian families with at least 1 deaf child diagnosed with non-syndromic deafness. In Vitro Fertilization (IVF) was performed, followed by PGD for a cousin couple with a 50% chance of having an affected child. Bi-allelic pathogenic mutations were found in a total of 12 families (~17 %); of which a couple was a PGD volunteer. The deaf woman in this family was homozygous and her husband was a carrier of the IVS1+1G>A gene mutation. Among 8 biopsied embryos, two healthy embryos were implanted which resulted in a single pregnancy and subsequent birth of a healthy baby boy. This is the first report of a successful application of PGD for hearing loss in Iran. Having a baby with a severe hearing impairment often imposes families with long-term disease burden and heavy therapy costs. Today PGD has provided an opportunity for high-risk individuals to avoid the birth of a deaf child.

Mitochondrial DNA copy number instability in ERBB2-amplified breast cancer tumors.

Increase in the copy number of ERBB2, a Tyrosine Kinase Receptor (TKR) leads to the overexpression of oncogene product and consequently uncontrolled cell proliferation which has been reported in different aggressive cancers with mitochondrial malfunctions. Although, amplification of ERBB2 has been reported in different studies; however, the association between changes in mitochondrial DNA content and the ERBB2 gene copy number is poorly understood. The relative mitochondrial DNA content of breast cancer tumor tissues of 70 patients who were referred to Imam Khomeini Hospital Complex was determined using quantitative Real-time PCR. Multiplex ligation-dependent probe amplification (MLPA) was conducted to evaluate the ERBB2 gene copy number variation and finally, two-sample Wilcoxon rank-sum (Mann-Whitney) test was used to investigate the possible association between mitochondrial DNA (mtDNA) content and the ERBB2 gene amplification. Seventeen out of 70 breast cancer tumor tissues were found with ERBB2 gene amplification. Comparison of the mitochondrial DNA content of the aforementioned samples with the rest of the cases showed a significant decrease in the mitochondrial DNA content of the ERBB2-amplified samples (P=0.01). Our data provided evidence that ERBB2 have the potential to have a regulatory role over mitochondrial activity by controlling the mtDNA content.

CYP17 MspA1 Gene Polymorphism and Breast Cancer Patients According to Age of Onset in Cancer Institute of Iran.

BACKGROUND: Exposure to endogenous hormones such as estrogen is known as a lifetime Breast Cancer (BC) risk factor. Polymorphisms in genes that are involved in the steroidogenic process, such as Cytochrome P450c17alpha (CYP17), affect individuals' susceptibility to BC. In Iran, the highest incident of BC is among young women. This study aimed to find prevalence of Single Nucleotide Polymorphisms (SNPs) in genes such as CYP17 and significant correlation with age-oriented group of breast cancer. METHODS: In 2016, a case series study was conducted on a total population of 205 patients suffering from breast cancer referred to Cancer Institute, Imam Khomeini Hospital Complex, Tehran, Iran. This population consisted of 104 cases less than 40 yr old and 101 cases over 40. The genotype variants of CYP17 MspA1 were determined using PCR, followed by RFLP. The association of CYP17 MspA1 polymorphisms with the risk of BC in two different age groups was evaluated by calculating odds ratio and 95% confidence intervals using unconditional logistic regression. RESULTS: Carriers of at least one A2 allele may have higher risk of developing breast cancer at younger age compared to patients with A1/A1 genotype (Odds Ratio: 1.99, 95% Confidence Interval: 1.11-3.57, P=0.02). CONCLUSION: CYP17gene polymorphisms may have influence on the early onset of breast cancer.

BIRC5 Genomic Copy Number Variation in Early-Onset Breast Cancer.

BACKGROUND: Baculoviral inhibitor of apoptosis repeat-containing 5 (BIRC5) gene is an inhibitor of apoptosis that expresses in human embryonic tissues but it is absent in most healthy adult tissues. The copy number of BIRC5 has been indicated to be highly increased in tumor tissues; however, its association with the age of onset in breast cancer is not well understood. METHODS: Forty tumor tissues of breast cancer were obtained from Tumor Bank of Cancer Institute, Imam Khomeini Hospital, Tehran, Iran. BIRC5 gene copy number variation (CNV) was evaluated using Multiplex Ligation-dependent Probe Amplification (MLPA) and then compared with the age of onset for each patient. RESULTS: BIRC5 amplification was seen in 17.5% of cases. Also, a significant association was observed between BIRC5 gene amplification and individuals under 40 years of age (P=0.04). CONCLUSION: BIRC5 gene has the potential to be a marker for the detection and prognosis of cancer at an early age.