Diagnosis of Ovarian Carcinoma Homologous Recombination DNA Repair Deficiency From Targeted Gene Capture Oncology Assays.

PURPOSE: Homologous recombination DNA repair deficiency (HRD) is a therapeutic biomarker for sensitivity to platinum and poly(ADP-ribose) polymerase inhibitor therapies in breast and ovarian cancers. Several molecular phenotypes and diagnostic strategies have been developed to assess HRD; however, their clinical implementation remains both technically challenging and methodologically unstandardized. METHODS: We developed and validated an efficient and cost-effective strategy for HRD determination on the basis of calculation of a genome-wide loss of heterozygosity (LOH) score through targeted, hybridization capture and next-generation DNA sequencing augmented with 3,000 common, polymorphic single-nucleotide polymorphism (SNP) sites distributed genome-wide. This approach requires minimal sequence reads and can be readily integrated into targeted gene capture workflows already in use for molecular oncology. We interrogated 99 ovarian neoplasm-normal pairs using this method and compared results with patient mutational genotypes and orthologous predictors of HRD derived from whole-genome mutational signatures. RESULTS: LOH scores of ≥11% had >86% sensitivity for identifying tumors with HRD-causing mutations in an independent validation set (90.6% sensitivity for all specimens). We found strong agreement of our analytic approach with genome-wide mutational signature assays for determining HRD, yielding an estimated 96.7% sensitivity and 50% specificity. We observed poor concordance with mutational signatures inferred using only mutations detected by the targeted gene capture panel, suggesting inadequacy of the latter approach. LOH score did not significantly correlate with treatment outcomes. CONCLUSION: Targeted sequencing of genome-wide polymorphic SNP sites can be used to infer LOH events and subsequently diagnose HRD in ovarian tumors. The methods presented here are readily generalizable to other targeted gene oncology assays and could be adapted for HRD diagnosis in other tumor types.

Less is more: Detecting clinical deterioration in the hospital with machine learning using only age, heart rate, and respiratory rate.

AIM: We sought to develop a machine learning analytic (eCART Lite) for predicting clinical deterioration using only age, heart rate, and respiratory data, which can be pulled in real time from patient monitors and updated continuously without need for additional inputs or cumbersome electronic health record integrations. METHODS: We utilized a multicenter dataset of adult admissions from five hospitals. We trained a gradient boosted machine model using only current and 24-hour trended heart rate, respiratory rate, and patient age to predict the probability of intensive care unit (ICU) transfer, death, or the combined outcome of ICU transfer or death. The area under the receiver operating characteristic curve (AUC) was calculated in the validation cohort and compared to those for the Modified Early Warning Score (MEWS), National Early Warning Score (NEWS), and eCARTv2, a previously-described, 27-variable, cubic spline, logistic regression model without trends. RESULTS: Of the 556,848 included admissions, 19,509 (3.5%) were transferred to an ICU and 5764 (1.0%) died within 24 hours of a ward observation. eCART Lite significantly outperformed the MEWS, NEWS, and eCART v2 for predicting ICU transfer (0.79 vs 0.71, 0.74, and 0.78, respectively; p < 0.01) and the combined outcome (0.80 vs 0.72, 0.76, and 0.79, respectively; p < 0.01). Two of the strongest predictors were respiratory rate and heart rate. CONCLUSION: Using only three inputs, we developed a tool for predicting clinical deterioration that is similarly or more accurate than commonly-used algorithms, with potential for use in inpatient settings with limited resources or in scenarios where low-cost tools are needed.

Heparin-induced haemorrhagic bullous dermatosis.

BACKGROUND: Heparin-induced haemorrhagic bullous dermatosis (HBD) is a rare but probably underdiagnosed reaction to heparin, with 26 cases reported in the English literature. Currently, there is no consensus regarding the treatment. AIM: To assess our new cases of HBD and review the previously reported cases, in order to draw conclusions about this adverse skin reaction to heparin. METHODS: A PubMed search was performed for articles containing the terms '(heparin-induced AND (blister OR bulla OR bullae)) OR (hemorrhagic bullous dermatosis AND heparin) OR heparin bullous dermatosis'. Descriptive statistical data analysis was performed using Microsoft Excel. RESULTS: We assessed five new cases of HBD. In addition, our literature search revealed 26 previously reported patients. Combining these, we found that the mean ± SD age of patients with HBD was 71.4 ± 14 years. HBD affects men more commonly than women (men 22/31; P = 0.02). Patients develop tense bullae most frequently on the extremities, approximately 8 days (mean ± SD 7.5 ± 6.4 days) after starting treatment with a heparin product, usually enoxaparin. CONCLUSIONS: The typical clinical course is spontaneous resolution within days to weeks irrespective of continuation of heparin therapy. Because of its self-limiting nature, interruption of heparin therapy may not be required.

Epigenetic memory via concordant DNA methylation is inversely correlated to developmental potential of mammalian cells.

In storing and transmitting epigenetic information, organisms must balance the need to maintain information about past conditions with the capacity to respond to information in their current and future environments. Some of this information is encoded by DNA methylation, which can be transmitted with variable fidelity from parent to daughter strand. High fidelity confers strong pattern matching between the strands of individual DNA molecules and thus pattern stability over rounds of DNA replication; lower fidelity confers reduced pattern matching, and thus greater flexibility. Here, we present a new conceptual framework, Ratio of Concordance Preference (RCP), that uses double-stranded methylation data to quantify the flexibility and stability of the system that gave rise to a given set of patterns. We find that differentiated mammalian cells operate with high DNA methylation stability, consistent with earlier reports. Stem cells in culture and in embryos, in contrast, operate with reduced, albeit significant, methylation stability. We conclude that preference for concordant DNA methylation is a consistent mode of information transfer, and thus provides epigenetic stability across cell divisions, even in stem cells and those undergoing developmental transitions. Broader application of our RCP framework will permit comparison of epigenetic-information systems across cells, developmental stages, and organisms whose methylation machineries differ substantially or are not yet well understood.

Co-option of an Ancestral Hox-Regulated Network Underlies a Recently Evolved Morphological Novelty.

The evolutionary origins of complex morphological structures such as the vertebrate eye or insect wing remain one of the greatest mysteries of biology. Recent comparative studies of gene expression imply that new structures are not built from scratch, but rather form by co-opting preexisting gene networks. A key prediction of this model is that upstream factors within the network will activate their preexisting targets (i.e., enhancers) to form novel anatomies. Here, we show how a recently derived morphological novelty present in the genitalia of D. melanogaster employs an ancestral Hox-regulated network deployed in the embryo to generate the larval posterior spiracle. We demonstrate how transcriptional enhancers and constituent transcription factor binding sites are used in both ancestral and novel contexts. These results illustrate network co-option at the level of individual connections between regulatory genes and highlight how morphological novelty may originate through the co-option of networks controlling seemingly unrelated structures.

Genetic Changes to a Transcriptional Silencer Element Confers Phenotypic Diversity within and between Drosophila Species.

The modification of transcriptional regulation has become increasingly appreciated as a major contributor to morphological evolution. However, the role of negative-acting control elements (e.g. silencers) in generating morphological diversity has been generally overlooked relative to positive-acting "enhancer" elements. The highly variable body coloration patterns among Drosophilid insects represents a powerful model system in which the molecular alterations that underlie phenotypic diversity can be defined. In a survey of pigment phenotypes among geographically disparate Japanese populations of Drosophila auraria, we discovered a remarkable degree of variation in male-specific abdominal coloration. In testing the expression patterns of the major pigment-producing enzymes, we found that phenotypes uniquely correlated with differences in the expression of ebony, a gene required for yellow-colored cuticle. Assays of ebony's transcriptional control region indicated that a lightly pigmented strain harbored cis-regulatory mutations that caused correlated changes in its expression. Through a series of chimeric reporter constructs between light and dark strain alleles, we localized function-altering mutations to a conserved silencer that mediates a male-specific pattern of ebony repression. This suggests that the light allele was derived through the loss of this silencer's activity. Furthermore, examination of the ebony gene of D. serrata, a close relative of D. auraria which secondarily lost male-specific pigmentation revealed the parallel loss of this silencer element. These results demonstrate how loss-of-function mutations in a silencer element resulted in increased gene expression. We propose that the mutational inactivation of silencer elements may represent a favored path to evolve gene expression, impacting morphological traits.

Errors in the bisulfite conversion of DNA: modulating inappropriate- and failed-conversion frequencies.

Bisulfite treatment can be used to ascertain the methylation states of individual cytosines in DNA. Ideally, bisulfite treatment deaminates unmethylated cytosines to uracils, and leaves 5-methylcytosines unchanged. Two types of bisulfite-conversion error occur: inappropriate conversion of 5-methylcytosine to thymine, and failure to convert unmethylated cytosine to uracil. Conventional bisulfite treatment requires hours of exposure to low-molarity, low-temperature bisulfite ('LowMT') and, sometimes, thermal denaturation. An alternate, high-molarity, high-temperature ('HighMT') protocol has been reported to accelerate conversion and to reduce inappropriate conversion. We used molecular encoding to obtain validated, individual-molecule data on failed- and inappropriate-conversion frequencies for LowMT and HighMT treatments of both single-stranded and hairpin-linked oligonucleotides. After accounting for bisulfite-independent error, we found that: (i) inappropriate-conversion events accrue predominantly on molecules exposed to bisulfite after they have attained complete or near-complete conversion; (ii) the HighMT treatment is preferable because it yields greater homogeneity among sites and among molecules in conversion rates, and thus yields more reliable data; (iii) different durations of bisulfite treatment will yield data appropriate to address different experimental questions; and (iv) conversion errors can be used to assess the validity of methylation data collected without the benefit of molecular encoding.

Computational modeling of a stereoselective epoxidation: reaction of carene with peroxyformic acid.

Electronic structure theory was used to model the epoxidation of 3-carene by peroxyformic acid. Reactants, products, and transition states were optimized at the B3LYP/6-31G* level of theory, followed by B3LYP/6-311+G** and MP2/6-311+G** single point calculations. The reaction pathway yielding the trans-epoxide product was found to have a significantly lower reaction barrier (7.8 kcal/mol) than that leading to the cis-epoxide product (9.4 kcal/mol), in agreement with expectations. Magnetic shieldings of the two isomeric carene epoxides were also calculated, using the GIAO method, and compared to experimental (1)H and (13)C NMR spectra. Although the calculated carbon spectra proved inconclusive, the proton shieldings calculated for the trans-epoxide correlated much more closely to the experimental values for the major epoxidation product than did the shieldings calculated for the cis-epoxide, serving to verify the identity of the major product.

A prospective comparison of scalpel versus bipolar scissors in the elevation of radial forearm fasciocutaneous free flaps.

OBJECTIVES/HYPOTHESIS: The radial forearm fasciocutaneous free flap has become the workhorse for reconstruction of head and neck ablative defects. A location distal to the head and neck allows this flap to be elevated concurrent with the ablation. Most commonly, the flap is elevated under tourniquet control. This involves a primary ischemic insult with a certain amount of hemorrhage after the tourniquet is released. Bipolar scissors are a new method of dissection. They may allow for a speedier dissection with a concomitant decrease in ischemic time. Their hemostatic properties may control hemorrhage after use of the tourniquet. STUDY DESIGN: A prospective study of patients undergoing radial forearm free flaps over a 12-month period at a tertiary care referral center was undertaken. RESULTS: Forty patients were entered into the study, flaps were elevated with scalpel (20) and with bipolar scissors (20). Mean time under tourniquet was 39 minutes (range, 30-56 min) with scalpel compared with 27 minutes (range, 21-31 min) with bipolar scissors (P <.001). Total mean time of elevation (including control of hemostasis and pedicle dissection) for scalpel elevation was 50 minutes (range, 35-61 min) compared with 32 minutes (range, 20-41 min) for bipolar scissors elevation (P <.001). Mean blood loss was 46 mL (range, 15-110 mL) in the scalpel elevation group compared with 14 mL (range, 0-50 mL) in the bipolar scissors elevation group (P <.001). Complications at the donor site were equal between groups. CONCLUSIONS: Bipolar scissors are a safe, efficient method for elevating radial forearm free flaps.

Effect of anemia on the fasciocutaneous flap survival in a rat model.

OBJECTIVE/HYPOTHESIS: Ablative surgery for head and neck cancer that creates large composite defects often results in a significant decrease in the hematocrit level. These defects are best reconstructed with a microvascular free tissue transfer. Effect of the decreased hematocrit on microvascular flap survival is controversial. The purpose of this study was to assess the effect of isovolemic anemia on flap survival in a rat model. STUDY DESIGN: Prospective analysis. METHODS: Ninety rats were used (30 control and 60 experimental animals). Experimental animals were rendered anemic by blood draw and volume resuscitated with either a colloid (30 animals) or crystalloid (30 animals) solution. In all animals a ventral fasciocutaneous flap was raised. A vascular clamp was applied to the arteriovenous pedicle, and different ischemic times were allowed to elapse before clamp removal. Flap survival was assessed at 5 days. Probit analysis was performed for the three animal groups. RESULTS: A significantly increased probability of the flap survival was found in the anemic animals compared with the control group (P < or = .05). No difference was found between the colloid and crystalloid resuscitation groups. CONCLUSIONS: A decreased hematocrit level increases fasciocutaneous flap tolerance to ischemia and significantly increases the primary ischemic time in the ventral flap clamp model in rats. Fluid replacement with either crystalloid or colloid produces identical results.

Osteomyelitis of the temporomandibular joint.

Osteomyelitis of the temporomandibular joint is very uncommon, and osteomyelitis as a result of Aspergillus niger infection has not previously been reported. A case report of skull base and condylar osteomyelitis is presented. Previously reported cases of temporomandibular joint osteomyelitis are reviewed, and management is discussed. Because of the significant morbidity possible with infections in this region, otolaryngologists should be familiar with the anatomy, diagnostic modalities, and therapeutic options. [Editorial comment: This unusual case presents unique aspects of the pathophysiology of osteomyelitis of the skull base.]

Dysphonia and dysphagia following the anterior approach to the cervical spine.

BACKGROUND: Speech and swallowing dysfunctions are common following the anterior approach to the cervical spine. Despite functional morbidity and legal implications, the incidence and etiologic factors of these complications have not been adequately elucidated. OBJECTIVE: To better define speech and swallowing dysfunction both in the quantitative and qualitative sense. METHODS: A questionnaire was mailed to 497 patients who had undergone anterior cervical fusion or anterior cervical discectomy at a university hospital (study group). One hundred fifty questionnaires were sent to a control group. RESULTS: The study group response rate was 46%; the control group response was 51%. The incidence of hoarseness in the study group was 51%; the incidence in the control group was 19%. The difference was statistically significant (P<.01). Dysphagia was present in 60% of study group patients vs 23% of control group patients (P<.01). Qualitative questions revealed that constant hoarseness, pain with talking, difficulty eating solid foods, and odynophagia were significantly more common following the anterior approach to the cervical spine. CONCLUSIONS: Our findings show a much higher incidence than previously reported of both voice and swallowing impairment following the anterior approach to the cervical spine. Hoarseness and dysphagia may adversely affect recovery and the patient's sense of well-being. Preoperative counseling and postoperative evaluation are essential.

Pursestring closure of radial forearm fasciocutaneous donor sites.

BACKGROUND: The radial forearm fasciocutaneous free flap has become one of the most common methods of reconstructing defects after head and neck ablative surgery. The fasciocutaneous flap is an excellent replacement for the tissue that has been removed. Unfortunately, donor site morbidity remains a problem. Donor site morbidity is primarily related to poor skin graft take, cosmesis, and neural dysfunction. Decreasing the size of the donor site defect may allow for improved cosmesis with decreased morbidity. MATERIAL AND METHODS: Prospective evaluation of a pursestring closure of radial forearm fasciocutaneous donor sites over a 16-month period. RESULTS: Seventy-one radial forearm free flaps were used. Sixty-seven had a pursestring closure of the donor site. After flap elevation the mean size of the defect was 61 cm2 (range, 28-140 cm2). Pursestring closure decreased the mean of the defect to 34 cm2 (range, 10-104 cm2) (P <.0001). Defect size was decreased by a mean of 44.5% (range, 24.5%-66.7%) (P <.0001). COMPLICATIONS: The rate of skin graft loss (>25%) (9% of patients) was less than that reported in the literature. No patient required a second surgical procedure. Neural morbidity was equal in both groups. Cosmesis was much improved. CONCLUSIONS: Pursestring closure allowed for a significantly decreased donor defect, associated with better cosmesis and less skin graft loss.

Bipolar scissors in facial plastic surgery.

BACKGROUND: Many methods are available to the facial plastic surgeon for elevating and separating tissue, from cold steel to monopolar cautery to various laser-cutting technologies. Bipolar cautery has replaced monopolar cautery as the optimal hemostatic technique because of its decreased tissue damage and improved capabilities. Bipolar scissors concurrently offer a dissecting technique with hemostatic capability. Little exists in the otolaryngology literature on the use of bipolar scissors for soft tissue dissection. OBJECTIVE: To describe our experience using bipolar scissors in a variety of facial plastic and reconstructive procedures. SETTING: Tertiary care referral academic center. DESIGN: We retrospectively reviewed 78 procedures performed using bipolar scissors between June 1997 and August 1999. In facial plastic cosmetic surgery, bipolar scissors were used 31 times for deep plane face-lifts and 16 times for endoscopic browlifts. In facial plastic reconstructive surgery, bipolar scissors were used in 15 radial forearm free flaps, 10 fibula osteocutaneous flaps, 3 rectus abdominis free flaps, and 3 latissimus dorsi myocutaneous free flaps. RESULTS: In all procedures, bipolar scissors facilitated the dissection. By allowing for a drier field, less time was required to elevate the flap and obtain hemostasis. Complications were not increased compared with historical controls. In the harvesting of fibula osteocutaneous free flaps, use of bipolar scissors allowed harvesting without use of a tourniquet. CONCLUSION: Bipolar scissors, a new technology in facial plastic surgery, allow the same control as sharp dissection and provide simultaneous hemostasis.

A retrospective analysis of temporomandibular joint reconstruction with free fibula microvascular flap.

OBJECTIVES: The temporomandibular joint is occasionally encountered in extirpative surgery of the head and neck. It presents a difficult management issue. Little has been reported on functional outcomes after resection and reconstruction of the temporomandibular joint. DESIGN: A retrospective analysis consisting of chart reviews and phone interviews was performed on 17 patients who underwent fibular free flap reconstruction of the temporomandibular joint from 1993 to 1998. RESULTS: Mean follow-up in surviving patients (10) was 41.3 months. Mean age of the group was 62; male-to-female ratio was 11:6. Average hospital stay was 11.6 days. Four patients had no radiation therapy, 2 had preoperative and 11 had postoperative treatment. Five patients had one osteotomy, seven had two, one had three, and one had four. Ten patients could chew, one could not, and none were recorded for the remaining. Diet consisted of regular food for two patients, soft food for seven, full liquids for four, and tube feeds for four. Cosmesis was judged as excellent by eight patients, acceptable by two, and unacceptable by two. Five patients did not describe cosmesis. Most patients stated that bony contour was excellent, but that the soft tissue defect was noticeable. Speech was judged as intelligible by seven and moderately understandable by one. Nine patients did not describe speech. Two patients had postoperative displacement of the fibular head out of the fossa. CONCLUSION: Primary reconstruction of the temporomandibular joint with microvascular fibular flaps is a viable and effective means of restoring function. The majority of patients are able to resume oral feeds, obtain excellent or pleasing cosmetic results, and maintain intelligible speech.