Next generation of immune checkpoint molecules in maternal-fetal immunity.

Successful pregnancy is a unique situation requires the maternal immune system to recognize and tolerate a semi-identical fetus and allow normal invasion of trophoblast cells. Although efforts have been made, the deep mechanisms of the maternal-fetal crosstalk have not yet been fully deciphered. Immune checkpoint molecules (ICMs) are a group of negative modulators of the immune response that avoid immune damage. They have been extensively studied in the fields of oncology and transplantation, while the latest evidence suggests that they are closely associated with pregnancy outcomes via multiple inhibitory mechanisms. Although studies have mostly demonstrated the regulatory role of the well-known PD-1, CTLA-4 at the maternal-fetal interface, what is unique about the newly discovered multiple ICMs remains a mystery. Here, we review the latest knowledge on ICMs, focusing on the first generation of checkpoints (PD-1, CTLA-4) and the next generation (Tim-3, Tigit, Lag-3, VISTA) highlighting their immunoregulatory roles in maternal-fetal tolerance and decidual vascular remodeling, and their involvement in pathological pregnancies. The content covers three aspects: the characteristics they possess, the dynamic expression profile of their expression at the maternal-fetal interface, and their involvement in pathological pregnancy. In immunotherapy strategies for pregnancy complications, upregulation of immune checkpoints may play a role. Meanwhile, the impact on pregnancy outcomes when using ICMs in clinical cancer treatment during pregnancy is a topic worth exploring. These may serve as a guide for future basic research and clinical applications of maternal-fetal immunity.

Calcitonin gene-related peptide: a possible biomarker in migraine patients with patent foramen ovale.

BACKGROUND: Serum CGRP has been found to increase during migraine attack. However, whether CGRP can identify MA with PFO subtypes in MA remains unknown. This study aimed to investigate the differential expression of calcitonin gene-related peptide (CGRP) between migraine (MA) patients with and without patent foramen ovale (PFO), and to evaluate the predictive value of CGRP for MA with PFO. METHODS: A total of 153 patients with MA, 51 patients with PFO and 102 patients without. Venous blood was drawn and HIT-6 score was calculated during the onset of MA, and blood routine, inflammatory indexes and serum CGRP were detected. The differences in serum markers and HIT-6 scores were compared between the two groups, and the risk factors of MA with PFO were determined by univariate and multivariate logistics regression. Furthermore, the correlation between CGRP level with right-to-left shunt (RLS) grades and headache impact test-6 (HIT-6) score in MA patients with PFO were assessed. Independent risk factors were screened out by multivariate Logistic regression analysis. We used the receiver operating characteristic (ROC) curve to analyze the diagnostic value of these risk factors in MA complicated with PFO. RESULTS: The serum CGRP level and HIT-6 scores in the MA with PFO group were significantly higher than those in the MA group (P < 0.001). Multivariate regression analysis showed that CGRP was an independent risk factor for MA with PFO (OR = 1.698, 95% CI = 1.325-2.179, P < 0.001). CGRP values ​​increased with the increase of RLS grade(Spearmen rho = 0.703, P < 0.001). Furthermore, a positive correlation between CGRP and HIT-6 scores was found (Spearmen rho = 0.227; P = 0.016). ROC curve showed that the optimal cut-off value for diagnosing MA with PFO was 79 pg/mL, the area under the curve (AUC) for predicting MA with PFO was 0.845, with 72.55% sensitivity and 78.43% specificity. CONCLUSIONS: MA patients with PFO have higher serum CGRP level. elevated CGRP concentration was associated with higher RLS grade and increased HIT-6 score. Higher serum CGRP level has certain clinical value in predicting PFO in MA patients. TRIAL REGISTRATION: This study was approved by the Ethics Committee of Zhuhai Hospital of Integrated Traditional Chinese and Western Medicine (Ethics batch number: 20,201,215,005).

Effect of C-Type Natriuretic Peptide (CNP) on Spermatozoa Maturation in Adult Rat Epididymis.

C-type natriuretic peptide (CNP) is highly expressed in male reproductive tissues, such as the epididymis. The aim of this study is to explore the role of CNP in the maturation of rat epididymal spermatozoa. First, the expression levels of CNP and its specific natriuretic peptide receptor-B (NPR-B) were detected in various tissues of rats and epididymis at different stages after birth. Then a castrated rat model was established to analyze the relationship between testosterone and CNP/NPR-B expression in the epididymis. Finally, CNP and different inhibitors (NPR-B inhibitors, cGMP inhibitors) were used to incubate epididymal sperm in vitro to examine sperm mobility and expression of sperm maturation-related factors. The results showed CNP/NPR-B mRNAs were expressed in all tissues of rats, but were extremely highly expressed in male genital ducts (seminal vesicle, prostate and epididymis). The expression of CNP/NPR-B in epididymis was the highest at birth and the fifth week after birth. In the epididymis, CNP/NPR-B were highly expressed in the caput and located in the epididymal epithelial cells. After castration, the expression of CNP/NPR-B decreased sharply and was restored quickly after testosterone supplementation. In vitro, CNP could significantly promote the acquisition of epididymal sperm motility through the NPR-B/cGMP pathway and induce the expression of sperm maturation-related factors (such as Bin1b, Catsper 1, Dnah17, Fertilin). This study shows that CNP plays a role in epididymal sperm maturation. The mechanism of CNP is to promote the acquisition of epididymal sperm fluidity through the NPR-B/cGMP signaling pathway and also to regulate sperm maturation-related genes. Moreover, the expression of CNP/NPR-B was regulated by testosterone.

Dietary total antioxidant capacity and the risk of developing asthenozoospermia: a hospital-based case-control study in China.

STUDY QUESTION: Is dietary total antioxidant capacity (DTAC) associated with the odds of developing asthenozoospermia in Chinese men? SUMMARY ANSWER: There is no statistically significant association between DTAC indices and the odds of developing asthenozoospermia. WHAT IS KNOWN ALREADY: Both diet and oxidative stress may be related to sperm quality; however, few studies have investigated the association between DTAC and sperm quality. STUDY DESIGN, SIZE, DURATION: This case-control study was conducted from June 2020 to December 2020. Those diagnosed with asthenozoospermia were assigned to the case group, whereas those with normal sperm parameters were assigned to the control group. Data from a total of 553 cases and 586 controls were included in the final analysis. PARTICIPANTS/MATERIALS, SETTING, METHODS: Men who had been referred to the infertility clinic of Shengjing Hospital of China Medical University were enrolled. Dietary intake was assessed using a validated food frequency questionnaire. DTAC was based on ferric-reducing ability of plasma (FRAP), total oxygen radical absorbance capacity (T-ORAC), hydrophilic oxygen radical absorbance capacity (H-ORAC), lipophilic oxygen radical absorbance capacity (L-ORAC), total phenolics (TP), total radical-trapping antioxidant parameter (TRAP), and Trolox equivalent antioxidant capacity (TEAC). Asthenozoospermia was defined according to the criteria published in the fifth edition of the World Health Organization laboratory manual for the examination and processing of human semen. MAIN RESULTS AND THE ROLE OF CHANCE: No significant association was observed between the DTAC indices and the odds of asthenozoospermia after multivariable adjustment (T3 vs T1, odds ratio (OR) = 0.99, 95% CI: 0.73-1.33 for FRAP; OR = 1.05, 95% CI: 0.77-1.42 for T-ORAC; OR = 0.88, 95% CI: 0.65-1.18 for H-ORAC; OR = 0.98, 95% CI: 0.71-1.34 for L-ORAC; OR = 1.03, 95% CI: 0.76-1.39 for TP; OR = 1.18, 95% CI: 0.87-1.59 for TRAP; and OR = 1.15, 95% CI: 0.85-1.55 for TEAC). Both additive and multiplicative interaction analyses suggested that smoking might modify the association of T-ORAC with the odds of developing asthenozoospermia (relative excess risk due to interaction = 0.45, 95% CI: 0.07-0.83, attributable proportion due to interaction = 0.46, 95% CI: 0.07-0.84 for additive interaction; P = 0.033 for multiplicative interaction). LIMITATIONS, REASONS FOR CAUTION: Recall bias and protopathic bias were inevitable in this retrospective case-control study. The estimation accuracy of the DTAC indices may have also affected the findings. WIDER IMPLICATIONS OF THE FINDINGS: To the best of our knowledge, this is the first study to specifically investigate whether an association exists between DTAC and the odds of developing asthenozoospermia. Although no significant association was found, this study provides novel information pertaining to the fields of nutrition and human reproduction. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the JieBangGuaShuai Project of Liaoning Province (2021JH1/10400050), the Shengjing Hospital Clinical Research Project (M0071), and the Outstanding Scientific Fund of Shengjing Hospital (M1150). All authors have no competing interests to disclose. TRIAL REGISTRATION NUMBER: N/A.

The Progress of Research on Genetic Factors of Recurrent Pregnancy Loss.

Recurrent pregnancy loss (RPL) is both mental and physical health problem affecting about 1-5% of women of childbearing age. The etiology of RPL is complex, involving chromosomal abnormalities, autoimmune diseases, metabolic disorders, and endometrial dysfunction. The causes of abortion are still unknown in more than 50% of these cases. With the development of science and technology, an increasing number of scholars focus on this field and find that genetic factors may play an essential role in unexplained RPL, such as embolism-related genes, immune factor-related genes, and chromosomal numeric, and structural variation. This review summarizes the genetic factors associated with RPL, including genetic mutations and genetic polymorphisms, chromosomal variants, and chromosomal polymorphisms. Many related genetic factors have been found to be demographically and geographically relevant, some of which can be used for risk prediction or screening for the etiology of RPL. However, it is difficult to predict and prevent RPL due to uncertain pathogenesis and highly variable clinical presentation. Therefore, the genetic factors of RPL still need plentiful research to obtain a more accurate understanding of its pathogenesis and to provide more detection means for the screening and prevention of RPL.

Low vitamin D levels in follicular fluid, but not in serum, are associated with adverse outcomes in assisted reproduction.

PURPOSE: To assess the relationship between serum/follicular fluid (FF) vitamin D (VD) status and assisted reproductive technology (ART) treatment outcomes among infertile patients. METHODS: A prospective cohort study, including 132 infertile patients scheduled for their first ART treatment cycle, was carried out in a Reproductive Medical Center. Serum and FF samples were collected to assess 25-hydroxy VD [25(OH)D] levels. Low VD level was defined as 25(OH)D concentration of less than 30 ng/mL. RESULTS: Most infertile patients had low VD levels in serum (88%) and FF (90%). We observed a moderately positive correlation between VD levels in serum and FF (r = 0.34, p < 0.0001). Compared to the group of patients with low VD levels in the FF, those with sufficient VD levels had a significantly higher number of retrieved oocytes (p = 0.03), normal fertilization (p = 0.01), and high-quality embryos (p = 0.001). Moreover, patients with sufficient VD levels in the FF also had significantly higher implantation rates than those with low VD levels (76.92% vs. 46.58%, respectively, p = 0.01) and clinical pregnancy rates (92.31% vs. 61.54%, respectively, p = 0.04). CONCLUSION: These data collectively revealed that low VD levels in serum and FF were common among infertile patients. VD levels in FF, but not in serum, were associated with embryo quality, normal fertilization, implantation rates, and clinical pregnancy rates. Further studies are mandatory to determine the molecular mechanism and VD's potential therapeutic benefits in infertile patients.

C-Type Natriuretic Peptide (CNP) Could Improve Sperm Motility and Reproductive Function of Asthenozoospermia.

This study is to analyze the effect of C-type natriuretic peptide (CNP) on sperm motility of asthenozoospermia and explore the influence mechanism of CNP on the reproductive system and sperm motility. Our results showed that the concentration of CNP in asthenospermia patients' semen was lower than in normal people's. The motility of sperm could be improved markedly by CNP and 8-Br-cGMP, while the effect of CNP was inhibited by NPR-B antagonist and KT5823. In the asthenozoospermia mouse model induced by CTX, CNP injection could improve sperm motility in the epididymis, alleviate tissue damage in the testes and epididymis, and increase testosterone levels. The asthenospermia mouse model showed high activity of MDA and proinflammatory factors (TNF-α, IL-6), as well as low expression of antioxidants (SOD, GSH-Px, CAT) in the testis and epididymis, but this situation could be significantly ameliorated after being treated with CNP. Those studies indicated that the concentration of CNP in the semen of asthenospermia patients is lower than in normal people and could significantly promote sperm motility through the NPR-B/cGMP pathway. In the asthenospermia mouse model induced by CTX, CNP can alleviate the damage of cyclophosphamide to the reproductive system and sperm motility. The mechanism may involve increasing testosterone and reducing ROS and proinflammatory factors to damage the tissue and sperm.

The Role of Mononuclear Phagocytes in the Testes and Epididymis.

The mononuclear phagocytic system (MPS) is the primary innate immune cell group in male reproductive tissues, maintaining the balance of pro-inflammatory and immune tolerance. This article aims to outline the role of mononuclear macrophages in the immune balance of the testes and epididymis, and to understand the inner immune regulation mechanism. A review of pertinent publications was performed using the PubMed and Google Scholar databases on all articles published prior to January 2021. Search terms were based on the following keywords: 'MPS', 'mononuclear phagocytes', 'testes', 'epididymis', 'macrophage', 'Mφ', 'dendritic cell', 'DC', 'TLR', 'immune', 'inflammation', and 'polarization'. Additionally, reference lists of primary and review articles were reviewed for other publications of relevance. This review concluded that MPS exhibits a precise balance in the male reproductive system. In the testes, MPS cells are mainly suppressed subtypes (M2 and cDC2) under physiological conditions, which maintain the local immune tolerance. Under pathological conditions, MPS cells will transform into M1 and cDC1, producing various cytokines, and will activate T cell specific immunity as defense to foreign pathogens or self-antigens. In the epididymis, MPS cells vary in the different segments, which express immune tolerance in the caput and pro-inflammatory condition in the cauda. Collectively, MPS is the control point for maintaining the immune tolerance of the testes and epididymis as well as for eliminating pathogens.

Saniculamin C, a new flavonoid from Sanicula lamelligera.

A new flavonoid named saniculamin C (1), together with six known compounds (2-7), were isolated from the whole plants of Sanicula lamelligera Hance. The chemical structures were elucidated by spectroscopic and physico-chemical analyses. All isolates were evaluated for in vitro cytotoxic activities against four human cancer cell lines, HepG2, SGC-7901 gastric cancer, Hela and A-549 lung cancer. Compound 1 showed potent antiproliferative activities against SGC-7901 cells with IC50 value of 7.45 µM. In addition, compound 6 exhibited weak antiproliferative activities against HepG2, SGC-7901, Hela cancer cells with IC50 values of 10.43, 8.24 and 15.32 µM, respectively.[Formula: see text].

Corilagin suppresses RANKL-induced osteoclastogenesis and inhibits oestrogen deficiency-induced bone loss via the NF-κB and PI3K/AKT signalling pathways.

Over-activated osteoclastogenesis, which is initiated by inflammation, has been implicated in osteoporosis. Corilagin, a natural compound extracted from various medicinal herbaceous plants, such as Cinnamomum cassia, has antioxidant and anti-inflammatory activities. We found that Corilagin suppressed osteoclast differentiation in a dose-dependent manner, significantly decreased osteoclast-related gene expression and impaired bone resorption by osteoclasts. Moreover, phosphorylation of members of the nuclear factor-kappaB (NF-κB) and PI3K/AKT signalling pathways was reduced by Corilagin. In a murine model of osteoporosis, Corilagin inhibited osteoclast functions in vivo and restored oestrogen deficiency-induced bone loss. In conclusion, our findings suggested that Corilagin inhibited osteoclastogenesis by down-regulating the NF-κB and PI3K/AKT signalling pathways, thus showing its potential possibility for the treatment of osteoporosis.

C-type natriuretic peptide regulates sperm capacitation by the cGMP/PKG signalling pathway via Ca2+ influx and tyrosine phosphorylation.

RESEARCH QUESTION: What is the effect of C-type natriuretic peptide (CNP) on human sperm capacitation in vitro and what is the mechanism of this effect? DESIGN: CNP/NPR-B expression in the female rat genital tract was examined by immunohistochemistry and western blot assay, and then the role of CNP in human sperm capacitation was determined. The signal transduction pathway of CNP in the process was determined to elucidate the regulation mechanism of CNP by enzyme-linked immunosorbent assay and flow cytometry. RESULTS: Both CNP and NPR-B were expressed in the genital tract of female rats, especially in the mucosa epithelium cell of the oviduct; the CNP level in the rat oviduct was higher than that in the cervix. Both CNP and NPR-B level in the rat oviduct varied during the oestrus cycle, maximal expression being observed at proestrus. Furthermore, intracellular cGMP level in spermatozoa was significantly enhanced by CNP (P < 0.01). PKG activity was detected in the spermatozoa, and it can be activated by the CNP and 8-Br-cGMP (cGMP analogue). The PKG inhibitor KT5823 inhibited the effect of CNP on sperm hyperactivation and the acrosome reaction. Finally, Ca2+ and tyrosine phosphorylation levels in spermatozoa were markedly improved by CNP and 8-Br-cGMP but significantly inhibited by the addition of KT5823 (P < 0.05). CONCLUSIONS: CNP secreted by the female genital tract might bind to NPR-B on the spermatozoa. It successively stimulated intracellular cGMP/PKG signalling, increased Ca2+ and tyrosine-phosphorylated proteins, promoted hyperactivation and induced the acrosome reaction, which ultimately facilitated sperm capacitation.

TNER: a novel background error suppression method for mutation detection in circulating tumor DNA.

BACKGROUND: Ultra-deep next-generation sequencing of circulating tumor DNA (ctDNA) holds great promise as a tool for the early detection of cancer and for monitoring disease progression and therapeutic responses. However, the low abundance of ctDNA in the bloodstream coupled with technical errors introduced during library construction and sequencing complicates mutation detection. RESULTS: To achieve high accuracy of variant calling via better distinguishing low-frequency ctDNA mutations from background errors, we introduce TNER (Tri-Nucleotide Error Reducer), a novel background error suppression method that provides a robust estimation of background noise to reduce sequencing errors. The results on both simulated data and real data from healthy subjects demonstrate that the proposed algorithm consistently outperforms a current, state-of-the-art, position-specific error polishing model, particularly when the sample size of healthy subjects is small. CONCLUSIONS: TNER significantly enhances the specificity of downstream ctDNA mutation detection without sacrificing sensitivity. The tool is publicly available at https://github.com/ctDNA/TNER .

Selective serotonin reuptake inhibitor use during early pregnancy and congenital malformations: a systematic review and meta-analysis of cohort studies of more than 9 million births.

BACKGROUND: In 2005, the FDA cautioned that exposure to paroxetine, a selective serotonin reuptake inhibitor (SSRI), during the first trimester of pregnancy may increase the risk of cardiac malformations. Since then, the association between maternal use of SSRIs during pregnancy and congenital malformations in infants has been the subject of much discussion and controversy. The aim of this study is to systematically review the associations between SSRIs use during early pregnancy and the risk of congenital malformations, with particular attention to the potential confounding by indication. METHODS: The study protocol was registered with PROSPERO (CRD42018088358). Cohort studies on congenital malformations in infants born to mothers with first-trimester exposure to SSRIs were identified via PubMed, Embase, Web of Science, and the Cochrane Library databases through 17 January 2018. Random-effects models were used to calculate summary relative risks (RRs). RESULTS: Twenty-nine cohort studies including 9,085,954 births were identified. Overall, use of SSRIs was associated with an increased risk of overall major congenital anomalies (MCAs, RR 1.11, 95% CI 1.03 to 1.19) and congenital heart defects (CHD, RR 1.24, 95% CI 1.11 to 1.37). No significantly increased risk was observed when restricted to women with a psychiatric diagnosis (MCAs, RR 1.04, 95% CI 0.95 to 1.13; CHD, RR 1.06, 95% CI 0.90 to 1.26). Similar significant associations were observed using maternal citalopram exposure (MCAs, RR 1.20, 95% CI 1.09 to 1.31; CHD, RR 1.24, 95% CI 1.02 to 1.51), fluoxetine (MCAs, RR 1.17, 95% CI 1.07 to 1.28; CHD, 1.30, 95% CI 1.12 to 1.53), and paroxetine (MCAs, RR 1.18, 95% CI 1.05 to 1.32; CHD, RR 1.17, 95% CI 0.97 to 1.41) and analyses restricted to using women with a psychiatric diagnosis were not statistically significant. Sertraline was associated with septal defects (RR 2.69, 95% CI 1.76 to 4.10), atrial septal defects (RR 2.07, 95% CI 1.26 to 3.39), and respiratory system defects (RR 2.65, 95% CI 1.32 to 5.32). CONCLUSIONS: The evidence suggests a generally small risk of congenital malformations and argues against a substantial teratogenic effect of SSRIs. Caution is advisable in making decisions about whether to continue or stop treatment with SSRIs during pregnancy.

Effects of maternal body mass index on pregnancy outcome after 8570 artificial insemination cycles with donor's sperm.

Increased body mass index (BMI) might have an adverse effect on pregnancy. However, the influence of BMI on the pregnancy outcomes after artificial insemination with donor's sperm (AID) had been rarely reported. This study aimed to investigate the correlation between BMI and AID. The pregnancy outcome of 8570 AID cycles was retrospectively analyzed. BMI was categorized as underweight (<18.5 kg/m2; group A), normal weight (18.5-23.9 kg/m2; group B), overweight (24-27.9 kg/m2; group C), and obese (≥28 kg/m2; group D). The results showed that cumulative pregnancy rate and cumulative live birth rate in groups A, B, C and D tended to decrease as BMI increased; however, abortion rate, and ectopic pregnancy rate in groups A, B, C, and D exhibited a gradual increase in the tendency. Cesarean delivery rate also increased as BMI increased. Birth defect rate in the group D were significantly higher than that in the group A. Interestingly, the pregnancy rate was gradually decreased with increasing age in groups A, B, and C, but this was not observed in the group D. The findings suggested that BMI can affect the pregnancy outcomes after AID; it is important to achieve a normal BMI prior to AID treatments.

Acquired resistance to crizotinib from a mutation in CD74-ROS1.

Crizotinib, an inhibitor of anaplastic lymphoma kinase (ALK), has also recently shown efficacy in the treatment of lung cancers with ROS1 translocations. Resistance to crizotinib developed in a patient with metastatic lung adenocarcinoma harboring a CD74-ROS1 rearrangement who had initially shown a dramatic response to treatment. We performed a biopsy of a resistant tumor and identified an acquired mutation leading to a glycine-to-arginine substitution at codon 2032 in the ROS1 kinase domain. Although this mutation does not lie at the gatekeeper residue, it confers resistance to ROS1 kinase inhibition through steric interference with drug binding. The same resistance mutation was observed at all the metastatic sites that were examined at autopsy, suggesting that this mutation was an early event in the clonal evolution of resistance. (Funded by Pfizer and others; ClinicalTrials.gov number, NCT00585195.).

Basal Follicle-Stimulating Hormone or Inhibin B Combined with Age as Predictors of Pregnancy After Treatment by Donor Sperm Insemination.

OBJECTIVE: To evaluate the clinical significance of basal reproductive hormones and basal inhibin B (INHB) combined with age on predicting the outcomes of artificial insemination with donor sperm (AID). STUDY DESIGN: A retrospective analysis was performed in 1,772 patients who underwent AID at the Department of Assisted Re- production, Tongji Medical College, from 2009-2011. We compared the age and The levels of basal menstrual cycle day 3 reproductive hormones and INHB regarding the pregnancy rates after AID treatment. RESULTS: There was a low clinical pregnancy rate in women with a basal follicle-stimulating hormone (FSH) ≥ 15 IU/L or a basal INHB < 25 ng/mL. An age-related decrease in the pregnancy rate was found also. Moreover, the pregnancy rate dropped remarkably when the FSH x age value was > 500, and it rose to 70.6% when the INHB ÷ age value was > 10. CONCLUSION: Basal FSH and basal INHB are closely correlated with clinical pregnant rates in AID treatment. Furthermore, FSH x age and INHB ÷ age could be used as an optimal tool for predicting AID outcomes.

Patterns of somatic alterations between matched primary and metastatic colorectal tumors characterized by whole-genome sequencing.

Colorectal cancer (CRC) patients have poor prognosis after formation of distant metastasis. Understanding the molecular mechanisms by which genetic changes facilitate metastasis is critical for the development of targeted therapeutic strategies aimed at controlling disease progression while minimizing toxic side effects. A comprehensive portrait of somatic alterations in CRC and the changes between primary and metastatic tumors has yet to be developed. We performed whole genome sequencing of two primary CRC tumors and their matched liver metastases. By comparing to matched germline DNA, we catalogued somatic alterations at multiple scales, including single nucleotide variations, small insertions and deletions, copy number aberrations and structural variations in both the primary and matched metastasis. We found that the majority of these somatic alterations are present in both sites. Despite the overall similarity, several de novo alterations in the metastases were predicted to be deleterious, in genes including FBXW7, DCLK1 and FAT2, which might contribute to the initiation and progression of distant metastasis. Through careful examination of the mutation prevalence among tumor cells at each site, we also proposed distinct clonal evolution patterns between primary and metastatic tumors in the two cases. These results suggest that somatic alterations may play an important role in driving the development of colorectal cancer metastasis and present challenges and opportunities when considering the choice of treatment.

Multiplexed deep sequencing analysis of ALK kinase domain identifies resistance mutations in relapsed patients following crizotinib treatment.

The recently approved ALK kinase inhibitor crizotinib has demonstrated successful treatment of metastatic and late stage ALK fusion positive non-small cell lung cancer (NSCLC). However, the median duration of clinical benefit is ~10-11months due to the emergence of multiple and simultaneous resistance mechanisms in these tumors. Mutations in the ALK kinase domain confer resistance to crizotinib in about one-third of these patients. We developed a multiplex deep sequencing method using semiconductor sequencing technology to quickly detect resistance mutations within the ALK kinase domain from tumor biopsies. By applying a base-pair specific error-weighted mutation calling algorithm (BASCA) that we developed for this assay, genomic DNA analysis from thirteen relapsed patients revealed three known crizotinib resistance mutations, C1156Y, L1196M and G1269A. Our assay demonstrates robust and sensitive detection of ALK kinase mutations in NSCLC tumor samples and aids in the elucidation of resistance mechanisms pertinent to the clinical setting.