Postoperative radiotherapy for locally advanced colon cancer

Background: The role of adjuvant postoperative radiotherapy for locally advanced colon cancer is not well documented. Methods: Seventy-eight patients who underwent a complete resection of B2-C colon cancer received postoperative radiotherapy. Twenty-eight patients received ⩽45 Gy; 50 patients received 50–55 Gy. Twenty-seven patients received adjuvant fluorouracil-based chemotherapy. All patients were followed for a minimum of 3 years; no patients were lost to follow-up. Results: The overall local control rate was 88%. The 5-year actuarial rate of local control was 96% after 50–55 Gy postoperative radiotherapy compared with 76% after <50 Gy (p=0.0095). Multivariate analysis of local control showed that only radiotherapy dose significantly influenced this end point. Cause-specific survival rates at 5 years were B2, 67%; B3, 90%; C1, 100%; C2, 61%; C3, 36%; and overall, 63%. Multivariate analysis of cause-specific survival showed that only stage significantly influenced this end point. Bowel obstruction caused by adhesions developed in three patients and required a laparotomy; radiation-induced sarcoma developed in one additional patient. Conclusions: Postoperative radiotherapy appears to reduce the risk of local recurrence in patients with locally advanced colon cancer. The optimal dose is probably 50–55 Gy at 1.8 Gy per fraction. Postoperative radiotherapy may improve cause-specific survival for patients with stages B3 and C2 cancers.     

Effect of norgestrel on development of mouse pre-embryos

The effect of synthetic progestins found in oral contraceptives has potential implications for developing embryos in women who receive oral contraceptives during early pregnancy. We assessed the effect of the progestin norgestrel on the developing pre-embryo. B3C6F1 mice were given 5 IU PMSG followed by 5 IU hCG 48h later. Studies were performed on pre-embryos recovered and pooled at both 24h (Group A) and 48h (Group B) post hCG. At each time period, they were randomly assigned to control or norgestrel (4.0 ng/ml) treatment. In a third study, pre-embryos collected 24h post hCG were cultured in the absence or presence of 8.0, 80.0, or 800 ng/ml norgestrel. Cultures were performed in Ham's F-10 media with 10% fetal calf serum at 37 degrees C in an atmosphere of 5% CO2, 5% O2 and 90% N2 with 15-30 embryos per 1 ml of culture fluid. At 24h, 48h and 72h post recovery, cultures were viewed, the appearance of embryos noted, and number of blastomeres recorded. Compared to control groups, analysis demonstrated no significant difference in the rate of development of control and norgestrel pre-embryos in any group at any time period (24h, 48h, or 72h post recovery). We conclude that norgestrel at the dose tested has no acute adverse morphological effects on development of mouse pre-embryos. This observation has potential clinical implications with regard to inadvertent use of norgestrel-containing oral contraceptives during early days of pregnancy, as well as consideration of the mechanism of action of norgestrel-containing "morning after" pills.     

Effect of margins of excision on recurrence after local excision of ductal carcinoma in situ of the breast

AIMS: To determine important factors influencing recurrence after local excision of duct carcinoma in situ (DCIS) of the breast. MATERIALS AND METHODS: The extent (size) in millimetres, classification (by cytonuclear grade (NHSBSP system), by extent of necrosis, and by the Van Nuys system), and excision margins of 115 cases of screen detected DCIS treated by local excision were measured. A prognostic index was calculated by the addition of the Van Nuys classification (low grade, 1; moderate grade, 2; high grade, 3), margin score (> or = 10 mm, 1; 1-9 mm, 2; < 1 mm, 3), and size score (< or = 15 mm, 1; 16-40 mm, 2; and > or = 41 mm, 3), giving a total score of 3-9. RESULTS: Classification using cytonuclear grade, extent of necrosis, or the Van Nuys system did not correlate significantly with recurrence. The excision margin (in millimetres) was associated with recurrence (p = 0.027) and if excision margin status was simplified using the scoring system (> or = 10 mm, 1; 1-9 mm, 2; < 1 mm, 3), the margin score was significantly associated with recurrence (p = 0.03). A prognostic index based on the Van Nuys score, margin status, and size was significantly associated with recurrence (p = 0.003). CONCLUSION: The results support the hypothesis that the margin of excision is the most important factor predicting the recurrence of DCIS after local excision.     

Incidence of sperm antibodies before and after vasectomy.

Sperm-agglutinating antibodies were present in three (2-6%) of 117 fertile men and in 19 (33-9%) of 56 men who had been vasectomized. Twenty-four of the 56 vasectomized men had been studied before vasectomy; sperm-agglutinating antibodies were present in one (4-3%) compared with eight (33-3%) after vasectomy. No sperm-immobilizing antibodies were detected before vasectomy but were present in 10 (17-9%) of the 56 men after vasectomy.     

Identification of non-amplifying CYP21 genes when using PCR-based diagnosis of 21-hydroxylase deficiency in congenital adrenal hyperplasia (CAH) affected pedigrees

Steroid 21-hydroxylase deficiency is among the most common inborn errors of metabolism in man. Characterization of mutations in the 21- hydroxylase gene (CYP21) has permitted genetic diagnosis, facilitated by the polymerase chain reaction (PCR). The most common mutation is conversion of an A or C at nt656 to a G in the second intron causing aberrant splicing of mRNA. Homozygosity for nt656G is associated with profoundly deficient adrenal cortisol and aldosterone synthesis, secondary hypersecretion of adrenal androgens, and a severe form of congenital adrenal hyperplasia (CAH) characterized by ambiguous genitalia and/or sodium wasting in newborns. During the course of genetic analysis of CYP21 mutations in CAH families, we and others have noticed a number of relatives genotyped as nt656G homozygotes, yet showing no clinical signs of disease. A number of lines of evidence have led us to propose that the putative asymptomatic nt656G/G individuals are incorrectly typed due to dropout of one haplotype during PCR amplification of CYP21. For prenatal diagnosis, we recommend that microsatellite typing be used as a supplement to CYP21 genotyping in order to resolve ambiguities at nt656.      

Complex mosaic structural variations in human fetal brains

Somatic mosaicism, manifesting as single nucleotide variants (SNVs), mobile element insertions, and structural changes in the DNA, is a common phenomenon in human brain cells, with potential functional consequences. Using a clonal approach, we previously detected 200–400 mosaic SNVs per cell in three human fetal brains (15–21 wk postconception). However, structural variation in the human fetal brain has not yet been investigated. Here, we discover and validate four mosaic structural variants (SVs) in the same brains and resolve their precise breakpoints. The SVs were of kilobase scale and complex, consisting of deletion(s) and rearranged genomic fragments, which sometimes originated from different chromosomes. Sequences at the breakpoints of these rearrangements had microhomologies, suggesting their origin from replication errors. One SV was found in two clones, and we timed its origin to ∼14 wk postconception. No large scale mosaic copy number variants (CNVs) were detectable in normal fetal human brains, suggesting that previously reported megabase-scale CNVs in neurons arise at later stages of development. By reanalysis of public single nuclei data from adult brain neurons, we detected an extrachromosomal circular DNA event. Our study reveals the existence of mosaic SVs in the developing human brain, likely arising from cell proliferation during mid-neurogenesis. Although relatively rare compared to SNVs and present in ∼10% of neurons, SVs in developing human brain affect a comparable number of bases in the genome (∼6200 vs. ∼4000 bp), implying that they may have similar functional consequences.

Somatic mosaicism, the presence of more than one genotype in the somatic cells of an individual, is a prominent phenomenon in the human central nervous system. Forms of mosaicism include aneuploidies and smaller copy number variants (CNVs), structural variants (SVs), mobile element insertions, indels, and single nucleotide variants (SNVs). The developing human brain exhibits high levels of aneuploidy compared to other tissues, generating genetic diversity in neurons (Pack et al. 2005; Yurov et al. 2007; Bushman and Chun 2013). Such aneuploidy was suggested to be a natural feature of neurons, rather than a distinctive feature of neurodegeneration. However, the frequency of aneuploidy in neurons has been debated, with a separate study suggesting that aneuploidies occur in only about 2.2% of mature adult neurons (Knouse et al. 2014). They hence infer that such aneuploidy could have adverse effects at the cellular and organismal levels. Additionally, analysis of single cells from normal and pathological human brains identified large, private, and likely clonal somatic CNVs in both normal and diseased brains (Gole et al. 2013; McConnell et al. 2013; Cai et al. 2014; Knouse et al. 2016; Chronister et al. 2019; Perez-Rodriguez et al. 2019), with 3%–25% of human cerebral cortical nuclei carrying megabase-scale CNVs (Chronister et al. 2019) and deletions being twice as common as duplications (McConnell et al. 2013). Given that CNVs often arise from nonhomologous recombination and replication errors, their likely time of origin is during brain development. However, when CNVs first arise in human brain development has not yet been investigated. The present work is the first to examine this question using clonal populations of neuronal progenitor cells (NPCs) obtained from fetal human brains.Detection of CNVs in single neurons is challenging, given the need to amplify DNA. Such amplification may introduce artifacts that could, in turn, be misinterpreted as CNVs. In order to address this technical limitation, Hazen et al. reprogrammed adult postmitotic neurons using somatic cell nuclear transfer (SCNT) of neuronal nuclei into enucleated oocytes (Hazen et al. 2016). These oocytes then made sufficient copies of the neuronal genome allowing for whole-genome sequencing (WGS), thus eliminating the need for amplification in vitro. Using this method, they identified a total of nine structural variants in six neurons from mice, three of which were complex rearrangements. However, it is not possible to extend such studies to humans, given the ethical issues involved, besides the technical challenges in obtaining and cloning adult neurons. To circumvent the need of single-cell DNA amplification or nuclear cloning, we examined clonal cell populations obtained from neural progenitor cells from the frontal region of the cerebral cortex (FR), parietal cortex (PA) and basal ganglia (BG) and describe here the discovery and analysis of mosaic SVs in these NPCs (Bae et al. 2018). These clones were sequenced at 30× coverage (much higher than most previous single-cell studies), allowing identification of SVs other than large deletions and duplications as well as precise breakpoint resolution.     

In vivo antigenic variation of Campylobacter flagellin.

Campylobacter coli VC167 cells producing either antigenic phase 1 (P1) or phase 2 (P2) flagellins (as determined by characteristic protein and DNA patterns) were used to infect rabbits by the removable intestinal tie-adult rabbit diarrhea (RITARD) procedure. Rabbits infected with P2 cells shed predominantly P2 cells throughout the infection; in rabbits infected with P1 cells, a transition of fecal isolates from P1 to P2 was observed.     

Hemo-De as substitute for ethyl acetate in formalin-ethyl acetate concentration technique.

In comparative studies, Hemo-De (PMP Medical Industries, Inc., Irving, Tex.) was found to be a suitable replacement for ethyl acetate in the Formalin-ethyl acetate concentration technique. With essentially equivalent recovery rates for both procedures, the Formalin-Hemo-De concentration technique is considered to be the preferred technique because Hemo-De is less toxic and less flammable and does not present disposal problems, and its cost is approximately one-fourth that of ethyl acetate.     

Coeliac disease, adenocarcinoma of jejunum and in situ squamous carcinoma of oesophagus

The development of both adenocarcinoma of the jejunum and in situ squamous carcinoma of the oesophagus in an adult coeliac patient is described. Good evidence that adenocarcinoma of jejunum occurs more frequently in patients with coeliac disease has recently become available though this association has been suggested for some time. While oesophageal carcinoma has long been associated with coeliac disease, in situ carcinoma of oesophagus has not been previously described in these circumstances. We feel that the risk of this complication, as calculated from published series, warrants a screening programme for oesophageal malignancy in adult coeliacs.