Case Study: Diffuse Large B-Cell Lymphoma Arising in Ovarian Mature Cystic Teratoma.

We describe an unexpected finding of diffuse large B-cell lymphoma associated with mature cystic teratoma of the ovary. A 68-yr-old woman with a complex left ovarian cystic mass on imaging underwent bilateral salpingo-oophorectomy, lymphadenectomy, appendicectomy, and omentectomy. Histopathologic examination revealed nodules of malignant non-Hodgkin lymphoma within the teratoma. A diagnosis of diffuse large B-cell lymphoma, germinal center cell subtype by Hans criteria was made after immunostaining and molecular studies. The patient was treated with R-CHOP chemotherapy and remains disease-free at 14-mo follow-up.

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.

What GDPR and the Health Research Regulations (HRRs) mean for Ireland: a research perspective.

BACKGROUND: Irish Health Research Regulations (HRRs) were introduced following the European Union (EU) General Data Protection Regulation (GDPR) in 2018. The HRRs described specific supplementary regulatory requirements for research regarding governance, processes and procedure that impact on several facets of research. The numerous problems that the HRRs and particularly "explicit consent" inadvertently created were presented under the auspices of the Irish Academy of Medical Sciences (IAMS) on November 25, 2019, at the Royal College of Surgeons in Ireland. AIMS: The objective of this review was to obtain feedback and to examine the impact of GDPR and the HRRs on health research in Ireland in order to determine whether the preliminary feedback, presented at the IAMS meetings, was reflected at a national level. METHODS: Individuals from the research community were invited to provide feedback on the impact, if any, of the HRRs on health research. Retrospective patient recruitment and consent outside a hospital setting for a multi-institutional Breast Predict study (funded by the Irish Cancer Society) were also analysed. RESULTS: Feedback replicated the issues presented at the IAMS with additional concerns identified. Only 20% of the original target population (n = 1987) could be included in the Breast Predict study. CONCLUSIONS: Our results confirm that the HRRs have had a significantly negative impact on health research in Ireland. Urgent meaningful engagement between patient advocate groups, the research community and legislators would help ameliorate these impacts.

What GDPR and the Health Research Regulations (HRRs) mean for Ireland: "explicit consent"-a legal analysis.

BACKGROUND: Irish Health Research Regulations (HRRs) were introduced following the commencement of the General Data Protection Regulation (GDPR) in 2018. The HRRs set out supplementary regulatory requirements for research. A legal analysis presented under the auspices of the Irish Academy of Medical Sciences (IAMS) on April 8 and November 25, 2019 at the Royal College of Surgeons in Ireland welcomed the introduction of GDPR and the HRRs. The analysis found the GDPR "explicit consent" introduced by the HRRs is problematic. A call was made to regulate informed consent in line with the common law as an achievable alternative safeguard, bringing Ireland in line with other EU Member States. AIMS: This article aims to review academic papers, legal opinion, EU opinion and advice and data protection law in relation to research and explicit consent, in order to examine the legal burden of GDPR and the HRRs on health research in Ireland and to determine whether the analysis presented at the IAMS meetings is reflected more widely in legal text. METHODS: Legal literature review of academic papers, legal opinion, EU opinion and advice and data protection legislation. RESULTS: The legal literature review overwhelmingly supports the concerns raised. CONCLUSIONS: Our results confirm the GDPR explicit consent requirement of the HRRs is having had a significantly negative and far-reaching impact on the conduct of health research in Ireland. Urgent review of the HRRs and meaningful engagement between the health research community and legislators in healthcare is required.

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.

RET/PTC rearrangement occurring in primary peritoneal carcinoma.

RET/PTC rearrangements are initiating events in the development of a significant proportion of papillary thyroid carcinomas. Activated RET/PTC mutations are thought to be restricted to thyroid disease, but this study proposes that these events may also occur in nonthyroid tumors. A total of 57 nonthyroid papillary tumors were examined for RET/PTC rearrangements using interphase fluorescence in situ hybridization, Taqman reverse transcriptase polymerase chain reaction, and immunohistochemistry. Taqman single nucleotide polymorphism detection was used to analyze for expression of mutated BRAF T1799A. In all, 20% (3/15) of primary peritoneal carcinoma had detectable RET/PTC1 rearrangements by all 3 methodologies. A further case of similar histotype had an alternate RET/ PTC rearrangement. No RET/PTC1 rearrangements were detected in the remaining tumor cohort. All 57 tumors were homozygous for wild-type BRAF. The results indicate that RET/PTC rearrangements occur in a small subset of nonthyroid papillary tumors. These rearrangements may not be directly implicated in tumor growth; rather representing "passenger" mutations reflecting RET instability in secondary tumor subclones.

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.

Development and progress of Ireland's biobank network: Ethical, legal, and social implications (ELSI), standardized documentation, sample and data release, and international perspective.

Biobank Ireland Trust (BIT) was established in 2004 to promote and develop an Irish biobank network to benefit patients, researchers, industry, and the economy. The network commenced in 2008 with two hospital biobanks and currently consists of biobanks in the four main cancer hospitals in Ireland. The St. James's Hospital (SJH) Biobank coordinates the network. Procedures, based on ISBER and NCI guidelines, are standardized across the network. Policies and documents-Patient Consent Policy, Patient Information Sheet, Biobank Consent Form, Sample and Data Access Policy (SAP), and Sample Application Form have been agreed upon (after robust discussion) for use in each hospital. An optimum sequence for document preparation and submission for review is outlined. Once consensus is reached among the participating biobanks, the SJH biobank liaises with the Research and Ethics Committees, the Office of the Data Protection Commissioner, The National Cancer Registry (NCR), patient advocate groups, researchers, and other stakeholders. The NCR provides de-identified data from its database for researchers via unique biobank codes. ELSI issues discussed include the introduction of prospective consent across the network and the return of significant research results to patients. Only 4 of 363 patients opted to be re-contacted and re-consented on each occasion that their samples are included in a new project. It was decided, after multidisciplinary discussion, that results will not be returned to patients. The SAP is modeled on those of several international networks. Biobank Ireland is affiliated with international biobanking groups-Marble Arch International Working Group, ISBER, and ESBB. The Irish government continues to deliberate on how to fund and implement biobanking nationally. Meanwhile BIT uses every opportunity to promote awareness of the benefits of biobanking in events and in the media.

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.

RET protein expression in papillary renal cell carcinoma.

OBJECTIVE: To examine the role of RET in renal malignancy, in particular papillary renal cell carcinoma (RCC). MATERIALS AND METHODS: A cohort of 111 archival renal samples was used consisting of 94 renal cancers (66 papillary RCC, 18 conventional clear cell carcinoma, 10 chromophobe RCC), 4 benign oncocytomas, and 13 normal kidney tissues. RET protein expression was examined by immunohistochemistry and expression levels were correlated with clinicopathologic and patient survival data. RESULTS: Positive RET staining was seen in 34/66 (52%) papillary RCCs, 4/10 (40%) chromophobe carcinomas, 4/4 (100%) oncocytomas, and 11/13 (85%) normal kidney samples. All 18 cases of conventional clear cell carcinoma had negative RET staining. RET expression was associated with low Fuhrman nuclear grade. CONCLUSIONS: RET protein may be contributing in part to an adaptation of a papillary growth pattern in certain renal malignancies. Given the possible therapeutic benefit of small molecule inhibitors of RET activation, further work needs to be done to highlight the functional relevance of RET protein expression in papillary RCC.

One-step preservation of phosphoproteins and tissue morphology at room temperature for diagnostic and research specimens.

BACKGROUND: There is an urgent need to measure phosphorylated cell signaling proteins in cancer tissue for the individualization of molecular targeted kinase inhibitor therapy. However, phosphoproteins fluctuate rapidly following tissue procurement. Snap-freezing preserves phosphoproteins, but is unavailable in most clinics and compromises diagnostic morphology. Formalin fixation preserves tissue histomorphology, but penetrates tissue slowly, and is unsuitable for stabilizing phosphoproteins. We originated and evaluated a novel one-step biomarker and histology preservative (BHP) chemistry that stabilizes signaling protein phosphorylation and retains formalin-like tissue histomorphology with equivalent immunohistochemistry in a single paraffin block. RESULTS: Total protein yield extracted from BHP-fixed, routine paraffin-embedded mouse liver was 100% compared to snap-frozen tissue. The abundance of 14 phosphorylated proteins was found to be stable over extended fixation times in BHP fixed paraffin embedded human colon mucosa. Compared to matched snap-frozen tissue, 8 phosphoproteins were equally preserved in mouse liver, while AMPKß1 Ser108 was slightly elevated after BHP fixation. More than 25 tissues from mouse, cat and human specimens were evaluated for preservation of histomorphology. Selected tissues were evaluated in a multi-site, independent pathology review. Tissue fixed with BHP showed equivalent preservation of cytoplasmic and membrane cytomorphology, with significantly better nuclear chromatin preservation by BHP compared to formalin. Immunohistochemical staining of 13 non-phosphorylated proteins, including estrogen receptor alpha, progesterone receptor, Ki-67 and Her2, was equal to or stronger in BHP compared to formalin. BHP demonstrated significantly improved immunohistochemical detection of phosphorylated proteins ERK Thr202/Tyr204, GSK3-α/ß Ser21/Ser9, p38-MAPK Thr180/Tyr182, eIF4G Ser1108 and Acetyl-CoA Carboxylase Ser79. CONCLUSION: In a single paraffin block BHP preserved the phosphorylation state of several signaling proteins at a level comparable to snap-freezing, while maintaining the full diagnostic immunohistochemical and histomorphologic detail of formalin fixation. This new tissue fixative has the potential to greatly facilitate personalized medicine, biobanking, and phospho-proteomic research.

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.

Spontaneous resolution of lung opacities reported as metastases in a patient with a history of leiomyosarcoma.

This is a case of a 69-year-old woman who was diagnosed as having metastatic leiomyosarcoma. She presented with multiple lung opacities on plain films and CT scans following resection of a high-grade leiomyosarcoma of the uterus 16 months earlier. The radiological, oncology, gynaecology and respiratory teams diagnosed metastatic cancer on the basis of the clinical presentation and radiological appearance. Surprisingly, despite no treatment, there was gradual resolution of the lung lesions on plain films. A CT scan 18 months later showed full resolution of the lesions. The consultants from the various specialities involved in this case were unable to give a satisfactory explanation as to why these lesions resolved.

Sentinel nodes are identifiable in formalin-fixed specimens after surgeon-performed ex vivo sentinel lymph node mapping in colorectal cancer.

BACKGROUND: In recent years, the technique of sentinel lymph node (SLN) mapping has been applied to colorectal cancer. One aim was to ultrastage patients who were deemed node negative by routine pathologic processing but who went on to develop systemic disease. Such a group may benefit from adjuvant chemotherapy. METHODS: With fully informed consent and ethical approval, 37 patients with primary colorectal cancer and 3 patients with large adenomas were prospectively mapped. Isosulfan blue dye (1 to 2 mL) was injected around tumors within 5 to 10 minutes of resection. After gentle massage to recreate in vivo lymph flow, specimens were placed directly into formalin. During routine pathologic analysis, all nodes were bivalved, and blue-staining nodes were noted. These later underwent multilevel step sectioning with hematoxylin and eosin and cytokeratin staining. RESULTS: SLNs were found in 39 of 40 patients (98% sensitivity), with an average of 4.1 SLNs per patient (range, 1-8). In 14 of 16 (88% specificity) patients with nodal metastases on routine reporting, SLN status was in accordance. Focused examination of SLNs identified occult tumor deposits in 6 (29%) of 21 node-negative patients. No metastatic cells were found in SLNs draining the three adenomas. CONCLUSIONS: The ability to identify SLNs after formalin fixation increases the ease and applicability of SLN mapping in colorectal cancer. Furthermore, the sensitivity and specificity of this simple ex vivo method for establishing regional lymph node status were directly comparable to those in previously published reports.

Keratinising squamous metaplasia of the bladder: natural history and rationalization of management based on review of 54 years experience.

OBJECTIVE: To review our experience of keratinising squamous metaplasia of the bladder as a predictor for the development of cancer and other complications, and formulate a policy for its management. MATERIALS AND METHODS: A retrospective review (1945-1999) identified 34 patients with histologically proven keratinising squamous metaplasia (27 males and 7 females, average age 50 years, range 13-80 years). The histological criteria used to diagnose keratinising squamous metaplasia were squamous metaplasia of the urothelium with keratinisation and/or hyperkeratosis and/or acanthosis. Female patients with non-keratinising squamous metaplasia (vaginal metaplasia) were excluded. RESULTS: Four patients had synchronous bladder carcinoma (three advanced with early death; one localised, cured by cystectomy). Another 14 patients had extensive metaplasia (Group A, >50% of mucosal involvement). Three cases had cystectomy and cure. Six cases (out of 11) developed subsequent cancer (4 advanced and early death, two localised and cured by cystectomy). One other case died of obstructive uropathy secondary to squamous metaplasia. Two cases died of unrelated causes. Sixteen patients had limited squamous metaplasia (Group B, <50% involvement mucosal surface). Twelve patients had endoscopic resection, extraction bladder calculus etc. with no further complications. Another two patients underwent urinary diversion. Two patients (out of 16) developed subsequent cancer both with advanced disease and early death. CONCLUSION: Keratinising squamous metaplasia of the bladder is a significant risk factor for vesical carcinoma and complications, such as bladder contracture and ureteral obstruction. This risk of complications increases with more extensive bladder mucosal involvement. The wide variation in lag time to the development of complications necessitates indefinite follow-up. Selected patients with extensive bladder involvement and long life expectancy should be offered cystectomy.