Effectiveness of Postoperative Single-shot and Continuous Transverse Abdominis Plane Block Compared to Conventional Analgesia in Hand-assisted Laparoscopic Live-donor Nephrectomy

BackgroundFew studies have evaluated the efficacy of transverse abdominis plane (TAP) block in patients undergoing hand-assisted laparoscopic live-donor nephrectomy (HALN). We aimed to evaluate the analgesic effectiveness of TAP block as part of a multimodal pain management regimen in patients undergoing HALN.MethodsWe retrospectively reviewed the medical records of living kidney donors at our center between June 2016 and February 2020. HALNs were performed via a transperitoneal approach through a suprapubic incision. Additional laparoscopic ports were used in the upper midabdomen. In consenting donors, TAP block was performed postoperatively under ultrasound guidance with either a single-shot or continuous infusion of long-acting local anesthetic (0.2%-0.5% ropivacaine). All the patients received postoperative around-the-clock ketorolac and acetaminophen.ResultsOverall, 72 donors received the block (block group, 38 single-shot, 34 continuous), whereas 86 donors did not receive the block (control group). Baseline characteristics were comparable between the groups except for body weight (control: 71.8 ± 13.3 versus block: 77.8 ± 17.3 kg; P = 0.01) and intraoperative opioid dose (32.1 ± 9.6 versus 26.6 ± 10.7 morphine milligram equivalents; P < 0.001). After adjusting for baseline differences, postoperative opioid requirements were similar between the groups. When the baseline pain scale was adjusted for, there was no difference in the overall pain scale scores between the groups (P = 0.242). Subgroup analyses comparing single-shot or continuous TAP versus control did not show any differences.ConclusionsWith the caveat of the retrospective nature of the study, the adjunctive effect of TAP block after transabdominal HALN was limited when other multimodal analgesia was used

RNA-Seq of Circulating Tumor Cells in Stage II-III Breast Cancer.

BackgroundWe characterized the whole transcriptome of circulating tumor cells (CTCs) in stage II-III breast cancer to evaluate correlations with primary tumor biology.MethodsCTCs were isolated from peripheral blood (PB) via immunomagnetic enrichment followed by fluorescence-activated cell sorting (IE/FACS). CTCs, PB, and fresh tumors were profiled using RNA-seq. Formalin-fixed, paraffin-embedded (FFPE) tumors were subjected to RNA-seq and NanoString PAM50 assays with risk of recurrence (ROR) scores.ResultsCTCs were detected in 29/33 (88%) patients. We selected 21 cases to attempt RNA-seq (median number of CTCs = 9). Sixteen CTC samples yielded results that passed quality-control metrics, and these samples had a median of 4,311,255 uniquely mapped reads (less than PB or tumors). Intrinsic subtype predicted by comparing estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) versus PAM50 for FFPE tumors was 85% concordant. However, CTC RNA-seq subtype assessed by the PAM50 classification genes was highly discordant, both with the subtype predicted by ER/PR/HER2 and by PAM50 tumors. Two patients died of metastatic disease, both of whom had high ROR scores and high CTC counts. We identified significant genes, canonical pathways, upstream regulators, and molecular interaction networks comparing CTCs by various clinical factors. We also identified a 75-gene signature with highest expression in CTCs and tumors taken together that was prognostic in The Cancer Genome Atlas and Molecular Taxonomy of Breast Cancer International Consortium datasets.ConclusionIt is feasible to use RNA-seq of CTCs in non-metastatic patients to discover novel tumor biology characteristics

EpCAM based capture detects and recovers circulating tumor cells from all subtypes of breast cancer except claudin-low.

PurposeThe potential utility of circulating tumor cells (CTCs) as liquid biopsies is of great interest. We hypothesized that CTC capture using EpCAM based gating is feasible for most breast cancer subtypes.ResultsCancer cells could be recovered from all intrinsic subtypes of breast cancer with IE/FACS, however, claudin-low cell lines showed very low capture rates compared to the four other groups (p = 0.03). IE/FACS detection of CTC mimic cells was time sensitive, emphasizing controlling for pre-analytic variables in CTC studies. Median fluorescent intensity for flow cytometry and RNA flow cell type characterization were highly correlated, predicting for CTC isolation across molecular subtypes. RNA-Seq of IE/FACS sorted single cell equivalents showed high correlation compared to bulk cell lines, and distinct gene expression signatures compared to PB.Materials and methodsTen cell lines representing all major subtypes of breast cancer were spiked (as CTC mimics) into and recovered from peripheral blood (PB) using immunomagnetic enrichment followed by fluorescence-activated cell sorting (IE/FACS). Flow cytometry and RNA flow were used to quantify the expression of multiple breast cancer related markers of interest. Two different RNA-Seq technologies were used to analyze global gene expression of recovered sorted cells compared to bulk cell lines and PB.ConclusionsEpCAM based IE/FACS detected and captured a portion of spiked cells from each of the 10 cell lines representing all breast cancer subtypes, including basal-like but not claudin-low cancers. The assay allows for the isolation of high quality RNA suitable for accurate RNA-Seq of heterogeneous rare cell populations

Expression profiling of circulating tumor cells in metastatic breast cancer.

Circulating tumor cells (CTCs) are prognostic in all stages of breast cancer. However, since they are extremely rare, little is known about the molecular nature of these cells. We report a novel strategy for the isolation and expression profiling of pure populations of CTCs derived from peripheral blood. We developed a method to isolate CTCs based on immunomagnetic capture followed by fluorescence-activated cell sorting (IE/FACS). After assay validation using the BT474 cell line spiked into blood samples in vitro, RNA from CTCs isolated from the blood of five metastatic breast cancer (MBC) patients was linearly amplified and subjected to gene expression profiling via cDNA microarrays. We isolated a range of 9-993 captured CTCs from five MBC patients' blood and profiled their RNA in comparison to a diverse panel of primary breast tumors (n = 55). Unsupervised hierarchical clustering revealed that CTC profiles clustered with more aggressive subtypes of primary breast tumors and were readily distinguishable from peripheral blood (PB) and normal epithelium. Differential expression analysis revealed CTCs to have downregulated apoptosis, and they were distinguishable from PB by the relative absence of immune-related signals. As expected, CTCs from MBC had significantly higher risk of recurrence scores than primary tumors (p = 0.0073). This study demonstrates that it is feasible to isolate CTCs from PB with high purity through IE/FACS and profile them via gene expression analysis. Our approach may inform the discovery of therapeutic predictors and be useful for real-time identification of emerging resistance mechanisms in MBC patients