An Optimised Protocol Harnessing Laser Capture Microdissection for Transcriptomic Analysis on Matched Primary and Metastatic Colorectal Tumours.

Generation of large amounts of genomic data is now feasible and cost-effective with improvements in next generation sequencing (NGS) technology. Ribonucleic acid sequencing (RNA-Seq) is becoming the preferred method for comprehensively characterising global transcriptome activity. Unique to cytoreductive surgery (CRS), multiple spatially discrete tumour specimens could be systematically harvested for genomic analysis. To facilitate such downstream analyses, laser capture microdissection (LCM) could be utilized to obtain pure cell populations. The aim of this protocol study was to develop a methodology to obtain high-quality expression data from matched primary tumours and metastases by utilizing LCM to isolate pure cellular populations. We demonstrate an optimized LCM protocol which reproducibly delivered intact RNA used for RNA sequencing and quantitative polymerase chain reaction (qPCR). After pathologic annotation of normal epithelial, tumour and stromal components, LCM coupled with cDNA library generation provided for successful RNA sequencing. To illustrate our framework's potential to identify targets that would otherwise be missed with conventional bulk tumour sequencing, we performed qPCR and immunohistochemical technical validation to show that the genes identified were truly expressed only in certain sub-components. This study suggests that the combination of matched tissue specimens with tissue microdissection and NGS provides a viable platform to unmask hidden biomarkers and provides insight into tumour biology at a higher resolution.

Hematological toxicities associated with cytoreductive surgery and hyperthermic intraperitoneal chemotherapy.

INTRODUCTION: Cytoreductive surgery (CRS) and hyperthermic intraperitoneal chemotherapy (HIPEC) have been found to prolong survival in selected patients with peritoneal disease, but the extent of cytoreduction and chemoperfusion can result in systemic toxicities. We evaluate the incidence of perioperative hematological complications and its associated risk factors. METHODS: Retrospective analysis of a prospectively collected database of CRS-HIPEC cases between April 2001 and October 2016 was performed. Patients were stratified based on the clinicopathological characteristics, perioperative incidence, grade, and duration of leukopenia (white blood cells < 4000/mm3 ), neutropenia (absolute neutrophils < 2000/mm3 ), and thrombocytopenia (platelets < 140 000/mm3 ). RESULTS: Two hundred and thirty-five CRS-HIPEC were performed in 220 patients with peritoneal metastasis of colorectal, ovarian, primary peritoneal, appendiceal, or mesothelioma origins. The incidences of leukopenia, neutropenia, and thrombocytopenia were 15.3%, 3.8%, and 37.9%, respectively. Median time to onset was 1 day (0-16 days), 0 day (0-2 days), and 1 day (1-2 days), respectively, after operation. Median duration of leukopenia, neutropenia, and thrombocytopenia was 1 day (1-3 days), 1 day (1-2days), and 3 days (range 0-16 days), respectively. Age > 60 (odds ratio [OR] 0.229 [95% CI: 0.105-0.502], P < .001) and the use of prior chemotherapy (OR 2.46 [95% CI: 1.24, 4.83], P = .010) were independent risk factors for thrombocytopenia on multivariable logistic regression. CONCLUSION: Hematological toxicities are common after hyperthermic intraperitoneal chemotherapy with thrombocytopenia being most common. Patients with age > 60, and who have undergone chemotherapy, are at risk of these toxicities and should be closely monitored post CRS-HIPEC.

Clinical patterns and management of primary mucosal melanoma: a single centre experience.

BACKGROUND: Primary mucosal melanomas (MM) are rare neoplasms arising from melanocytes in mucosal membranes. Delayed diagnosis and aggressive disease biology contribute to a poorer prognosis. The clinical patterns of MMs treated in a large tertiary centre, and the differences between MMs in the head and neck versus other anatomical sites are described. METHODS: A retrospective review of 43 patients diagnosed with MM in the head and neck, urogenital, esophageal and anorectal sites from 1993 to 2015 was conducted. RESULTS: Distribution of head and neck, urogenital and gastrointestinal MM were 42, 30 and 28% respectively. Disease extent was local in 44%, regional in 40% and distal in 12% at diagnosis. Head and neck MMs were more likely to be diagnosed at an earlier stage as compared to other sites (P = 0.04). Surgery was performed with curative intent in 72%, while 2% had palliative surgery for symptom control. Of the remaining patients who did not undergo surgery, four had palliative chemotherapy and/or radiotherapy. Median disease-free survival was 13 months (1-179 months). There was a significantly longer time to locoregional recurrence in head and neck MM (16 months) compared to other sites (11 months) (P = 0.03). The 2-year overall survival was also significantly higher in head and neck MM (P = 0.003). CONCLUSION: MM of the head and neck is diagnosed at an earlier stage and associated with a longer time to locoregional recurrence. Surgical resection is the mainstay of treatment and may offer long-term survival benefit in selected patients.

Morbidity and mortality of elderly patients following cytoreductive surgery and Hyperthermic Intraperitoneal Chemotherapy (HIPEC).

AIM: Cytoreductive surgery (CRS) and Hyperthermic Intraperitoneal Chemotherapy (HIPEC) is known to improve survival in selected patients with peritoneal metastasis. However, there is limited data supporting the role of CRS and HIPEC in elderly patients (≥65 years old). METHODS: A retrospective review of a prospectively maintained database of patients who underwent CRS-HIPEC between April 2001 and July 2015 from a single institution was performed. Patients were divided into two groups non-elderly (<65 years old), and elderly (≥65 years old). Clinico- pathological parameters, morbidity and overall (OS) and disease-free survival (DFS) of the patients were compared. RESULTS: A total of 177 patients (median age 52, range 9-74) underwent CRS-HIPEC with curative intent. There were 159 non-elderly patients and 18 elderly patients. Median PCI scores were 12 (0-39) for the non- elderly patients and 11 (1-29) for the elderly patients (p=0.77). High-grade complications occurred in 39 non-elderly patients (24.5%) and 8 elderly patients (44.4%) (p=0.79), while 58 non-elderly patients (38.7%) and 7 elderly patients (41.2%) stayed in ICU for more than 1 day (p=0.69). There was no difference in the 30-day mortality between the two groups (0% vs. 0%, p=1). After a median follow-up of 16 months for all patients, there was no difference in 5-years OS (51.0% vs. 59.6%, p=0.88) and 5-years DFS (23.3% vs. 53.3%, p=0.60) between non-elderly and elderly patients. CONCLUSIONS: Surgical outcomes after CRS-HIPEC do not differ significantly between non-elderly and elderly patients. Hence, age should not be a contraindication in selecting patients for CRS and HIPEC.

Repeat Cytoreductive Surgery and HIPEC for Peritoneal Surface Malignancy and Peritoneal Carcinomatosis.

BACKGROUND: Peritoneal-based malignancy (PBM), especially peritoneal carcinomatosis from gastrointestinal malignancies traditionally carries a poor prognosis. Cytoreductive surgery (CRS) and hyperthermic intra-peritoneal chemotherapy (HIPEC) have been shown to attain long median survival of 34-92 months and 5 year survival of 29-59% in patients with favorable histopathological subtypes. Recurrence after CRS and HIPEC poses a management dilemma. This paper evaluates our institution's experience with repeat CRS and HIPEC, its associated morbidity and outcomes. METHODS: One-hundred and thirty underwent CRS and HIPEC for PBM from April 2001 to June 2013. 49 had peritoneal recurrences, of which 24 had peritoneal only recurrence. 7 out of the 24 underwent a second CRS and HIPEC. RESULTS: Five females and two males with median age of 51 (37-63), underwent a second CRS and HIPEC. The primary malignancies were: 1 peritoneal mesothelioma, 3 appendiceal, 2 ovarian, and 1 colorectal cancers. Median peritoneal cancer indices for the initial and second CRS were 19 and 12, respectively. Completeness of cytoreduction score of 0 was achieved for all patients. Median hospitalization after second CRS and HIPEC was 12 days (7-60). 1 out of 7 (14%) experienced grade 3 or 4 post-operative complications. There was no 30-day or inpatient mortality. Median follow-up was 13 months (1-97). Median disease-free interval between the first CRS and HIPEC to peritoneal recurrence was 20 months (14-87). Median disease-free survival of 6 months (1-97) was achieved after the second CRS and HIPEC. Six patients remained alive without disease and one passed away with disease. Two had recurrences at 12 and 71 months after second CRS and HIPEC, 1 died and the other, still alive, went on to have a third CRS. CONCLUSION: Repeat CRS and HIPEC can achieve prolonged survival in selected patients with peritoneal-based malignancies, and can be performed with acceptable morbidity and mortality.

Cytoreductive surgery (CRS) and hyperthermic intraperitoneal chemotherapy (HIPEC) for peritoneal mesothelioma.

INTRODUCTION: Peritoneal mesothelioma is a rare neoplasm. Due to the limited understanding of its biology and behaviour, peritoneal mesothelioma poses a diagnostic and management challenge. The management of peritoneal mesothelioma has been controversial; systemic chemotherapy, palliative surgery and cytoreductive surgery (CRS) with intraoperative hyperthermic intraperitoneal chemotherapy (HIPEC) have been described. MATERIALS AND METHODS: This study shares our experience with cytoreductive surgery and HIPEC for 5 out of the 6 cases of peritoneal mesotheliomas treated surgically, at a single institution in Singapore over the past 2 years. Computed tomography (CT) scans, positron emission tomography (PET)-CT scans and tumour markers were performed preoperatively but were not conclusive for the disease. All 6 cases presented to the Department of Surgical Oncology at National Cancer Centre Singapore, were diagnosed by histology of intraoperative biopsies. The combination of aggressive cytoreductive surgery and HIPEC was performed in 5 patients, with abandonment of procedure in 1 with extensive disease, who was treated with systemic chemotherapy instead. RESULTS: Median duration of surgery, median length of hospital stay, and median follow-up duration were 7.04 hours, 11 days, and 15 months respectively. One postoperative morbidity relating to chemical peritonitis required exploratory laparotomy with good outcome. There were no mortality. All patients are alive at the last follow-up with no evidence of recurrences at 4 to 31 months from the time of their surgery. CONCLUSION: Peritoneal mesothelioma is a rare disease that requires early diagnosis and can be effectively treated by CRS and HIPEC in selected group of patients.