P53 loss of heterozygosity (LOH) in formalin-fixed paraffin-embedded leiomyosarcoma (LMS): a novel report.

BACKGROUND: The occurrence of p53 loss of heterozygosity (LOH) is a common genetic event in malignancy. LOH occurs when a heterozygous locus loses one of its two parental alleles, becoming homozygous at that locus, by either copy number loss (CNL-LOH) or by becoming copy number neutral (CNN-LOH). A role for CNL-LOH (cnLOH) has been postulated in cancer aetiology. Loss of heterozygosity (LOH) results in irreversible genetic loss. AIMS: LOH was determined in DNA extracted from formalin-fixed paraffin-embedded (FFPE) leiomyosarcoma (LMS) specimens in a retrospective study from 30 patients, to assess the prognostic significance of LOH. The findings were analysed and their validity assessed. LOH was an adverse prognostic factor in LMS. Prospective uniform standardisation of formalin-fixation techniques is required. METHODS: DNA was extracted from 169 formalin-fixed paraffin blocks of 30 patients with LMS, following extensive tissue microdissection. Genomic DNA was amplified using the polymerase chain reaction (PCR) technique. Fluorescence-based microsatellite PCR was used to detect and quantitate heterozygosity loss. RESULTS: LOH was detected at gene locus 17p13 in 16 LMS (Four grade 2 and 12 grade 3 LMS). LOH was not detected in 14 LMS cases (one grade 1, five grade 2 and eight grade 3 LMS). LOH was associated with shorter patient survival. CONCLUSIONS: The results reported herein endorse the value of utilizing FFPE DNA in identifying LOH as a prognostic factor in LMS. The results have implications for tumour biobanking and precision medicine in patients with sarcomas.

Increased spontaneous apoptosis, but not survivin expression, is associated with histomorphologic response to neoadjuvant chemoradiation in rectal cancer.

PURPOSE: Survivin has been shown to be an important mediator of cellular radioresistance in vitro. This study aims to compare survivin expression and apoptosis to histomorphologic responses to neoadjuvant radiochemotherapy (RCT) in rectal cancer. MATERIALS AND METHODS: Thirty-six pre-treatment biopsies were studied. Survivin mRNA and protein expression plus TUNEL staining for apoptosis was performed. Response to treatment was assessed using a 5-point tumour regression grade. RESULTS: Survivin expression was not found to be predictive of response to RCT (p = NS). In contrast, spontaneous apoptosis was significantly (p = 0.0051) associated with subsequent response to RCT. However, no association between survivin expression and levels of apoptosis could be identified. CONCLUSIONS: This in vivo study failed to support in vitro studies showing an association between survivin and response to chemotherapy and radiation therapy. These results caution against the translation of the in vitro properties of survivin into a clinical setting.

COX-2 overexpression in pretreatment biopsies predicts response of rectal cancers to neoadjuvant radiochemotherapy.

PURPOSE: To determine the utility of COX-2 expression as a response predictor for patients with rectal cancer who are undergoing neoadjuvant radiochemotherapy (RCT). METHODS AND MATERIALS: Pretreatment biopsies (PTB) from 49 patients who underwent RCT were included. COX-2 and proliferation in PTB were assessed by immunohistochemistry (IHC) and apoptosis was detected by TUNEL stain. Response to treatment was assessed by a 5-point tumor-regression grade (TRG) based on the ratio of residual tumor to fibrosis. RESULTS: Good response (TRG 1+2), moderate response (TRG 3), and poor response (TRG 4+5) were seen in 21 patients (42%), 11 patients (22%), and 17 patients (34%), respectively. Patients with COX-2 overexpression in PTB were more likely to demonstrate moderate or poor response (TRG 3+4) to treatment than were those with normal COX-2 expression (p=0.026, chi-square test). Similarly, poor response was more likely if patients had low levels of spontaneous apoptosis in PTBs (p=0.0007, chi-square test). CONCLUSIONS: COX-2 overexpression and reduced apoptosis in PTB can predict poor response of rectal cancer to RCT. As COX-2 inhibitors are commercially available, their administration to patients who overexpress COX-2 warrants assessment in clinical trials in an attempt to increase overall response rates.

The human side of cancer biobanking.

The future success of translational research is critically dependent on the procurement and availability of high-quality tissue specimens linked to accurate histopathologic and clinical information about the individual banked specimen. The international community has awakened to this critical need only recently. Three major roadblocks have hindered the success of previous biobank consortiums: (1) Ethical issues surrounding patient consent and ownership of intellectual property, (2) Failure to properly preserve the molecular content of the tissue, and failure to reliably document clinical data linked to the specimen, and (3) Management issues: inadequate funding, competition for use of the tissue, inadequate personnel and facilities, and absence of dedicated database software. This chapter reviews these critical roadblocks and discusses international efforts to provide strategies to implement high-quality biobanks.

The human face of biobank networks for translational research.

The biobanking literature frequently addresses donor and societal issues surrounding biobanking, but the biobanker's perspective is rarely highlighted. While not comprehensive, this article offers an overview of the human aspects of biobanking from the viewpoint of biobank personnel-from biobank formation, through the process, and in addressing post-biobanking issues. As every biobank and biobank network may differ, such factors may vary. Before biobanking can commence, the purpose of the biobank network must be defined, and buy-in achieved from many stakeholders. An attitude of trust and sharing is essential, as is good communication. Developing a biobank is time consuming and laborious. Forming a network requires significantly more time due to the need for cross-institutional harmonization of policies, procedures, information technology considerations, and ethics. Circumstances may dictate whether development occurs top-down and/or bottom-up, as well as whether network management may be independent or by personnel from participating biobanks. Funding tends to be a prominent issue for biobanks and networks alike. In particular, networks function optimally with some level of government support, particularly for personnel. Quality biospecimen collection involves meticulously documented coordination with a network of medical and nursing staff. Examining and sampling operative specimens requires timely collaboration between the surgical and pathology teams. "Catch rates" for samples may be difficult to predict and may occur at a frequency less than anticipated due to factors related to the institution, staff, or specimen. These factors may affect specimen quality, and have a downstream effect on competition for specimens for research. Thus, release of samples requires a fair, carefully constructed sample access policy, usually incorporating an incentive for researchers, and an encouragement to form collaborations. Finally, the public and patient groups should aim to understand the benefits of a biobank network, so that patient care is improved through coordinated biobanking activity.

Maintaining Breast Cancer Specimen Integrity and Individual or Simultaneous Extraction of Quality DNA, RNA, and Proteins from Allprotect-Stabilized and Nonstabilized Tissue Samples.

The Saint James's Hospital Biobank was established in 2008, to develop a high-quality breast tissue BioResource, as a part of the breast cancer clinical care pathway. The aims of this work were: (1) to ascertain the quality of RNA, DNA, and protein in biobanked carcinomas and normal breast tissues, (2) to assess the efficacy of AllPrep(®) (Qiagen) in isolating RNA, DNA, and protein simultaneously, (3) to compare AllPrep with RNEasy(®) and QIAamp(®) (both Qiagen), and (4) to examine the effectiveness of Allprotect(®) (Qiagen), a new tissue stabilization medium in preserving DNA, RNA, and proteins. One hundred eleven frozen samples of carcinoma and normal breast tissue were analyzed. Tumor and normal tissue morphology were confirmed by frozen sections. Tissue type, tissue treatment (Allprotect vs. no Allprotect), extraction kit, and nucleic acid quantification were analyzed by utilizing a 4 factorial design (SPSS PASW 18 Statistics Software(®)). QIAamp (DNA isolation), AllPrep (DNA, RNA, and Protein isolation), and RNeasy (RNA isolation) kits were assessed and compared. Mean DNA yield and A(260/280) values using QIAamp were 33.2 ng/µL and 1.86, respectively, and using AllPrep were 23.2 ng/µL and 1.94. Mean RNA yield and RNA Integrity Number (RIN) values with RNeasy were 73.4 ng/µL and 8.16, respectively, and with AllPrep were 74.8 ng/µL and 7.92. Allprotect-treated tissues produced higher RIN values of borderline significance (P=0.055). No discernible loss of RNA stability was detected after 6 h incubation of stabilized or nonstabilized tissues at room temperature or 4°C or in 9 freeze-thaw cycles. Allprotect requires further detailed evaluation, but we consider AllPrep to be an excellent option for the simultaneous extraction of RNA, DNA, and protein from tumor and normal breast tissues. The essential presampling procedures that maintain the diagnostic integrity of pathology specimens do not appear to compromise the quality of molecular isolates.