Analysis of 72 patients with colorectal high-grade neuroendocrine neoplasms from three Chinese hospitals.

BACKGROUND: Colorectal high-grade neuroendocrine neoplasms (HGNENs) are rare and constitute less than 1% of all colorectal malignancies. Based on their morphological differentiation and proliferation identity, these neoplasms present heterogeneous clinicopathologic features. Opinions regarding treatment strategies for and improvement of the clinical outcomes of these patients remain controversial. AIM: To delineate the clinicopathologic features of and explore the prognostic factors for this rare malignancy. METHODS: This observational study reviewed the data of 72 consecutive patients with colorectal HGNENs from three Chinese hospitals between 2000 and 2019. The clinicopathologic characteristics and follow-up data were carefully collected from their medical records, outpatient reexaminations, and telephone interviews. A survival analysis was conducted to evaluate their outcomes and to identify the prognostic factors for this disease. RESULTS: According to the latest recommendations for the classification and nomenclature of colorectal HGNENs, 61 (84.7%) patients in our cohort had poorly differentiated neoplasms, which were categorized as high-grade neuroendocrine carcinomas (HGNECs), and the remaining 11 (15.3%) patients had well differentiated neoplasms, which were categorized as high-grade neuroendocrine tumors (HGNETs). Most of the neoplasms (63.9%) were located at the rectum. More than half of the patients (51.4%) presented with distant metastasis at the date of diagnosis. All patients were followed for a median duration of 15.5 mo. In the entire cohort, the median survival time was 31 mo, and the 3-year and 5-year survival rates were 44.3% and 36.3%, respectively. Both the univariate and multivariate analyses demonstrated that increasing age, HGNEC type, and distant metastasis were risk factors for poor clinical outcomes. CONCLUSION: Colorectal HGNENs are rare and aggressive malignancies with poor clinical outcomes. However, patients with younger age, good morphological differentiation, and without metastatic disease can have a relatively favorable prognosis.

Efficient Delivery and Nuclear Uptake Is Not Sufficient to Detect Gene Editing in CD34+ Cells Directed by a Ribonucleoprotein Complex.

CD34+ cells are prime targets for therapeutic strategies for gene editing, because modified progenitor cells have the capacity to differentiate through an erythropoietic lineage. Although experimental advances have been reported, the associated experimental protocols have largely been less than clear or robust. As such, we evaluated the relationships among cellular delivery; nuclear uptake, often viewed as the benchmark metric of successful gene editing; and single base repair. We took a combinatorial approach using single-stranded oligonucleotide and a CRISPR/Cas9 ribonucleoprotein to convert wild-type HBB into the sickle cell genotype by evaluating conditions for two common delivery strategies of gene editing tools into CD34+ cells. Confocal microscopy data show that the CRISPR/Cas9 ribonucleoprotein tends to accumulate at the outer membrane of the CD34+ cell nucleus when the Neon Transfection System is employed, while the ribonucleoproteins do pass into the cell nucleus when nucleofection is used. Despite the high efficiency of cellular transformation, and the traditional view of success in efficient nuclear uptake, neither delivery methodology enabled gene editing activity. Our results indicate that more stringent criteria must be established to facilitate the clinical translation and scientific robustness of gene editing for sickle cell disease.

Analyses of point mutation repair and allelic heterogeneity generated by CRISPR/Cas9 and single-stranded DNA oligonucleotides.

The repair of a point mutation can be facilitated by combined activity of a single-stranded oligonucleotide and a CRISPR/Cas9 system. While the mechanism of action of combinatorial gene editing remains to be elucidated, the regulatory circuitry of nucleotide exchange executed by oligonucleotides alone has been largely defined. The presence of the appropriate CRISPR/Cas9 system leads to an enhancement in the frequency of gene editing directed by single-stranded DNA oligonucleotides. While CRISPR/Cas9 executes double-stranded DNA cleavage efficiently, closure of the broken chromosomes is dynamic, as varying degrees of heterogeneity of the cleavage products appear to accompany the emergence of the corrected base pair. We provide a detailed analysis of allelic variance at and surrounding the target site. In one particular case, we report sequence alteration directed by a distinct member of the same gene family. Our data suggests that single-stranded DNA molecules may influence DNA junction heterogeneity created by CRISPR/Cas9.