Douglas P. Clark

Biospecimen Reporting for Improved Study Quality (BRISQ)

Human biospecimens are subject to a number of different collection, processing, and storage factors that can significantly alter their molecular composition and consistency. These biospecimen preanalytical factors, in turn, influence experimental outcomes and the ability to reproduce scientific results. Currently, the extent and type of information specific to the biospecimen preanalytical conditions reported in scientific publications and regulatory submissions varies widely. To improve the quality of research utilizing human tissues, it is critical that information regarding the handling of biospecimens be reported in a thorough, accurate, and standardized manner. The Biospecimen Reporting for Improved Study Quality (BRISQ) recommendations outlined herein are intended to apply to any study in which human biospecimens are used. The purpose of reporting these details is to supply others, from researchers to regulators, with more consistent and standardized information to better evaluate, interpret, compare, and reproduce the experimental results. The BRISQ guidelines are proposed as an important and timely resource tool to strengthen communication and publications around biospecimen-related research and help reassure patient contributors and the advocacy community that the contributions are valued and respected.

Cryptosporidiosis in Patients with AIDS: Correlates of Disease and Survival

Although 10%-15% of patients with AIDS in the United States may acquire cryptosporidium infection, little data exist on clinical or histological characteristics that differentiate clinical outcomes. A case-control study of 83 HIV-positive adult patients with cryptosporidiosis was conducted, as was a histopathologic review of data on gastrointestinal biopsy specimens from 30 patients. Four clinical syndromes were identified: chronic diarrhea (36% of patients), choleralike disease (33%), transient diarrhea (15%), and relapsing illness (15%). A multivariate analysis of data for cases and controls revealed that acquiring cryptosporidiosis was associated with the presence of candidal esophagitis (odds ratio [OR], 2.53; P < .002) and Caucasian race (OR, 6.71; P = .0001) but not with sexual orientation. Cases had a significantly shorter duration of survival from the time of diagnosis than did controls (240 vs. 666 days, respectively; P = .0004), which was independent of sex, race, or or injection drug use. Antiretroviral use was protective against disease (OR, 0.072; P = .0001). All four clinical syndromes were represented among the histological data. There was no statistically significant correlation between histological intensity of infection and clinical severity of illness.

Cryptosporidium parvum Induces Host Cell Actin Accumulation at the Host-Parasite Interface

ABSTRACT Cryptosporidium parvum is an intracellular protozoan parasite that causes a severe diarrheal illness in humans and animals. Previous ultrastructural studies have shown thatCryptosporidium resides in a unique intracellular compartment in the apical region of the host cell. The mechanisms by which Cryptosporidium invades host intestinal epithelial cells and establishes this compartment are poorly understood. The parasite is separated from the host cell by a unique electron-dense structure of unknown composition. We have used indirect immunofluorescence microscopy and confocal laser scanning microscopy to characterize this structure. These studies indicate that host filamentous actin is assembled into a plaque-like structure at the host-parasite interface during parasite invasion and persists during parasite development. The actin-binding protein α-actinin is also present in this plaque early in parasite development but is lost as the parasite matures. Other actin-associated proteins, including vinculin, talin, and ezrin, are not present. We have found no evidence of tyrosine phosphorylation within this structure. Molecules known to link actin filaments to membrane were also examined, including α-catenin, β-catenin, plakoglobin, and zyxin, but none was identified at the host-parasite junction. Thus, Cryptosporidium induces rearrangement of the host cell cytoskeleton and incorporates host cell actin and α-actinin into a host-parasite junctional complex.

A clinicopathologic analysis of AIDS-related cryptosporidiosis

Objective:To characterize the histology of AIDS-associated cryptosporidiosis and identify features that explain the clinical variability. Design:A retrospective analysis of HIV-positive individuals with cryptosporidiosis who underwent endoscopy at the Johns Hopkins Hospital between 1985 and 1996. Methods:The histologic features (intensity of Cryptosporidium infection, inflammation, mucosal damage, copathogens) of gastrointestinal biopsies from 37 HIV-positive individuals with cryptosporidiosis were systematically graded. These histologic features were correlated with the severity of the diarrheal illness obtained from a patient chart review. Results:Histologic features associated with Cryptosporidium infection include a neutrophilic infiltrate in the stomach, villus blunting in the duodenum, cryptitis and epithelial apoptosis in the colon, and reactive epithelial changes in the stomach and duodenum. The nature and intensity of the inflammatory response varied widely; however, duodenal biopsies from a subset of patients (37%) revealed marked acute inflammation that was associated with concomitant cytomegalovirus infection. Although duodenal infection was common (93% of individuals), infection of other sites was variable (gastric cryptosporidiosis in 40% and colonic cryptosporidiosis in 74%). Widespread infection of the intestinal tract, which included both the large and small intestine, was associated with the most severe diarrheal illness. Conclusions:Cryptosporidium infection produces histologic evidence of gastrointestinal mucosal injury. The inflammatory response to the infection is variable, and may be modified by copathogens such as cytomegalovirus. The clinical manifestations are influenced, in part, by the anatomic distribution of the infection, with extensive infections involving both small and large intestines producing the most severe illness.

Seize the opportunity: underutilization of fine-needle aspiration biopsy to inform targeted cancer therapy decisions.

Two years ago, Krishnamurthy presented a comprehensive review on the applications of molecular techniques to fine-needle aspiration (FNA) biopsy samples, noting ‘‘the excellent potential of using material procured from FNAB for almost any type of molecular test.’’ She further observed that ‘whereas few of these tests alone are used for patient care, some of them have the potential for clinical use in the near future’’ and ‘‘most of the molecular tests using FNAB specimens are currently investigational.’’ This article will provide an update on the current utilization of FNA-based tests to inform targeted cancer therapy decisions, making the case that FNAs are underutilized for such testing. The emphasis will be on solid tumors rather than sarcomas or lymphoma. Some of the major impediments to the widespread clinical utilization of FNAs for molecular tests will also be identified and possible solutions will be presented.

The pathogenesis of cryptosporidiosis

Human infection with the protozoan parasite Cryptosporidium parvum has recently emerged as a global public health problem. Although infection is unrelenting in patients classically regarded as immunocompromised, a tantalizing observation is that infection with this parasite results in both acute self-limited as well as chronic diarrhea in young children. Recent data have begun to elucidate multiple potential mechanisms by which parasitism of the intestinal epithelium may yield an intestinal secretory response. However, a central issue for future studies is to understand how Cryptosporidium infection in young children results in such a broad spectrum of clinical presentation. An answer to this question is likely to result through a dual understanding of how systemic or enteric immunity impacts on intestinal secretory responses and how intra-cellular parasitism alters intestinal epithelial cell function and signals the submucosal intestinal compartment. The virulence factors of Cryptosporidium mediating these events need to be identified. Douglas Clark and Cynthia Sears here review the current understanding of the pathogenesis of intestinal secretion in response to Cryptosporidium infection, and discuss key questions requiring additional study.

Practice patterns in cervical cancer screening and human papillomavirus testing.

The use of human papillomavirus DNA testing plus Papanicolaou (Pap) testing (cotesting) for cervical cancer screening in women 30 years and older has been recommended since 2006. However, few studies have detailed the adoption of such cotesting in clinical practice. We examined the trends in monthly percentage of Pap tests ordered as cotests in our laboratory over a 2.5-year period and used joinpoint regression to identify periods in which there was a change in the average monthly proportion of cotests. Cotesting of patients 30 years and older increased from 15.9% in January 2008 to 39.4% in June 2010. In patients aged 18 to 29 years, cotesting initially increased, but showed a downward trend in the last 14 months of the study, ending at 7.7% in June 2010. Our study highlights increased adoption of age-appropriate cotesting as well as the persistence of age-inappropriate cotesting.

Multiple Cycles of Dose-Intensive Cyclophosphamide, Etoposide, and Cisplatinum (DICEP) Produce Durable Responses in Refractory Non-Hodgkin's Lymphoma

Patients whose lymphoma is resistant to standard treatment regimens continue to do poorly, with only an occasional patient achieving long-term remission even with bone marrow transplantation. Twenty-three patients with primarily refractory (11), or refractory relapsed (12) non-Hodgkins lymphoma were treated with repeated cycles of dose-intensive cyclophosphamide, etoposide, and cisplatin (DICEP) without bone marrow transplantation. Each cycle of DICEP consisted of cyclophosphamide (2500 mg/m2/day, days 1-2), etoposide (500 mg/m2/day, days 1-3), and cisplatin (50 mg/m2/day, days 1-3). Twelve patients (52%) have achieved a complete response and 6 (26%)r a partial response. Three of the complete responders remain continuously free of disease for 19, 29, and 32 months, and 3 more are disease-free at 58, 59, and 65 months after receiving further therapy. Three-year survival for all patients is 45%. Patients with a good initial performance status (Zubrod 0 or 1) had a 58% complete response rate and a 2-year survival rate of 53%. One of 19 patients with an initial performance status of 0 or 1 had treatment-related mortality. Repeated cycles of DICEP can produce long-term responses in patients with refractory non-Hodgkins lymphoma.

Molecular diagnostics on thyroid fine-needle aspirations: The pathway to value creation

In this issue of Cancer Cytopathology, Ohori et al describe the application of a complex molecular test to thyroid fine-needle aspirations (FNA) to further stratify the category of follicular lesion of undetermined significance/atypia of undetermined significance into high-risk and low-risk categories. This represents a fortunate convergence of the new Bethesda System for Reporting Thyroid Cytopathology (BSRTC) with the power of molecular diagnostics. Both represent a model for the future of cytopathology in which cytomorphologic criteria are defined, evidence-based reporting guidelines are established, and then biologically based biomarkers are used to add further diagnostic value. But what is the best pathway to develop thyroid FNA biomarkers, and where are we now on that path? The National Cancer Institute has defined one such pathway for biospecimen-based assay development. On the basis of these recommendations, several key hurdles need to be crossed before thyroid FNA molecular diagnosis will be widely adopted. First, the intended use for a thyroid FNA molecular test must be clearly and specifically defined. Which thyroid FNAs should be tested and how should the results impact clinical management? One answer lies in the categories defined by the BSRTC. The performance of morphology-based thyroid cytopathology, when limited to definitively benign or malignant diagnoses, is excellent, at least at the large medical centers where the retrospective studies have taken place. However, the cases that reside in the diagnostic ‘‘gray zone’’ between benign and malignant have far inferior performance, with malignancy rates ranging from 5% to 75%. The percentage of cases that lie in this gray zone varies between pathologists and institutions, but it may be as high as 25%. One could take 2 different approaches to refine the gray zone. One approach, analogous to high-risk HPV testing in atypical squamous cells of undetermined significance (ASCUS) in cervical cancer screening, uses a molecular test with a very high negative predictive value (NPV) to identify women who do not need colposcopy. Because we do not yet have a candidate biomarker or biomarker panel that will detect most thyroid carcinomas, a thyroid FNA molecular test that identifies patients who do not need further follow-up or surgery does not seem within our immediate grasp. Conversely, a test with a high positive predictive value (PPV) might enable the identification of patients with gray zone FNA diagnoses who need surgery, perhaps even those patients who need a total thyroidectomy. It is even possible that prognostic markers exist that could identify patients who would benefit from a central neck lymph node dissection, in addition to a total thyroidectomy. The PPV andNPV statistics given byOhori et al were for a relatively small number of patients and may not represent the overall performance characteristics of their test. Also, the high percentage of follicular lesion of undetermined significance/atypia of undetermined significance cases in their population raises questions about the underlying malignancy rate in this particular group at their institutions. Next, thyroid FNA specimen procurement and processing must be standardized and optimized for molecular diagnostics. As stated by the National Cancer Institute (NCI) Office of Biorepositories and Biospecimen Research: ‘‘The

Functional Profiling of Live Melanoma Samples Using a Novel Automated Platform

Aims This proof-of-concept study was designed to determine if functional, pharmacodynamic profiles relevant to targeted therapy could be derived from live human melanoma samples using a novel automated platform. Methods A series of 13 melanoma cell lines was briefly exposed to a BRAF inhibitor (PLX-4720) on a platform employing automated fluidics for sample processing. Levels of the phosphoprotein p-ERK in the mitogen-activated protein kinase (MAPK) pathway from treated and untreated sample aliquots were determined using a bead-based immunoassay. Comparison of these levels provided a determination of the pharmacodynamic effect of the drug on the MAPK pathway. A similar ex vivo analysis was performed on fine needle aspiration (FNA) biopsy samples from four murine xenograft models of metastatic melanoma, as well as 12 FNA samples from patients with metastatic melanoma. Results Melanoma cell lines with known sensitivity to BRAF inhibitors displayed marked suppression of the MAPK pathway in this system, while most BRAF inhibitor-resistant cell lines showed intact MAPK pathway activity despite exposure to a BRAF inhibitor (PLX-4720). FNA samples from melanoma xenografts showed comparable ex vivo MAPK activity as their respective cell lines in this system. FNA samples from patients with metastatic melanoma successfully yielded three categories of functional profiles including: MAPK pathway suppression; MAPK pathway reactivation; MAPK pathway stimulation. These profiles correlated with the anticipated MAPK activity, based on the known BRAF mutation status, as well as observed clinical responses to BRAF inhibitor therapy. Conclusion Pharmacodynamic information regarding the ex vivo effect of BRAF inhibitors on the MAPK pathway in live human melanoma samples can be reproducibly determined using a novel automated platform. Such information may be useful in preclinical and clinical drug development, as well as predicting response to targeted therapy in individual patients.