July 2005
Advances in Laboratory Diagnosis: BNP Testing and Proteomics
New assays and methodologies are continually being introduced, similar to new drugs and other medications. The challenge for the laboratory is to evaluate and choose wisely among them. What we bring to our labs and to the patients and physicians we serve must be economically and technically feasible, as well as diagnostically useful.
At Pathology Today, the annual meeting of the American Society for Clinical Pathology, the topic of the Clinical Pathology Symposium was "New Tests Coming To Your Lab." The speakers discussed important technologies and assays that are either available now or will soon be so. Each of the assays has significant or potentially significant clinical and diagnostic implications.
Brain Natriuretic Peptide and Congestive Heart Failure
BNP is a hormone with vasodilator and natriuretic properties that is useful in the diagnosis of CHF. First discovered in pigs' brains in 1988, BNP is 1 of 4 natriuretic peptides described in humans. It is preferentially secreted by cardiac ventricular cells as the active form of the hormone. The inactive form, NT-proBNP, is secreted in equimolar amounts. BNP has a plasma half-life of approximately 20 or 21 minutes, NT-proBNP of approximately 90 minutes. BNP is cleared by the type C natriuretic peptide receptor and by neutral endopeptidase. Clearance by the kidney is minimal. The clearance mechanism of NT-proBNP is not known but is thought to be renal. Elevated BNP and NT-proBNP levels have been found to be highly specific for elevated left ventricular filling pressures in patients with left ventricular dysfunction.
Congestive heart failure (CHF) is a tremendous
health problem. In 2002, it was present in 4.9 million Americans, or 2.3% of
the total
Elizabeth Sykes, MD, of the Department of
Clinical Pathology at
The clinical diagnosis of CHF is difficult to make and the validity of such diagnosis is notably poor. One reason is that objective evidence of cardiac dysfunction is required. To that end, making an earlier and accurate diagnosis of symptomatic CHF could be beneficial both to the patient and to the healthcare system. Improving the reliability of diagnosis is important because determining the etiology and stage of heart failure leads to different management choices. In one study, B-type natriuretic peptide (BNP) testing contributed to cost-effective management, reducing the overall cost of treatment of patients who presented to the emergency room with acute dyspnea by 26%.
All of the assays for BNP and NT-proBNP commercially available in the
To determine the prognostic value of plasma BNP testing, the investigators compared BNP levels to the primary physician's diagnosis in 428 clinical cases.
"In 154 cases diagnosed with CHF, BNP levels were between about 1,000 and 4,000 µg/mL," said Dr. Figurasin. "BNP was useful in differentiating dyspnea secondary to cardiovascular conditions from dyspnea secondary to pulmonary conditions.... Those with pulmonary conditions had BNP levels below 100 µg/mL."
BNP values of 4,000 µg/mL or greater were directly related to a diagnosis of CHF, the researchers found. Values between 1,000 µg/mL and 4,000 µg/mL were associated with CHF and precursor conditions such as hypertension and diabetes mellitus.
"BNP levels between 101 and 999 [µg/mL] deserve equal analysis because they exceed the normal BNP level, and yet no diagnosis of CHF was made," the authors write, adding that they may be helpful in analyzing precursor conditions and the conditions that mimic CHF.
Proteomics
Lance A. Liotta, MD, PhD, Director of the Laboratory of Pathology at the National Cancer Institute (NCI) of the National Institutes of Health (NIH) in Bethesda, Maryland, presented "Proteomics: The Next Revolution in Molecular Medicine." Dr. Liotta began by defining proteomics and then elucidated the opportunities it offers for improved detection and treatment of cancers. Proteomics is the characterization of biologic processes by quantitative and qualitative assessment of protein expression patterns. It provides a dynamic picture of normal and abnormal cellular physiology. Since proteins are responsible for all controlled biologic functions, they can be considered as the determinants of the malignant phenotype. Proteomic investigations can detect posttranslational modifications such as glycosylation, phosphorylation, cellular trafficking, and degradation, all of which can affect protein function. With proteomics one can identify and study patterns of proteins and changes in protein patterns in response to disease or treatment.
How does this knowledge translate into what Dr. Liotta termed "personalized molecular medicine?" The application of protein arrays to map signal transduction pathways in cancer biopsies enables physicians to potentially customize therapy for individual patients. Specifically, this means that a combination of drugs is used to treat multiple points in the aberrant tumor pathways for a specific patient. At a functional level, cancer is a proteomic disease driven by dominant or defective protein signal pathways. By determining a cancer cell's molecular profile, combination therapy can be tailored to an individual patient. Such a profile may identify multiple targets along the length of key signal transduction pathways, both intracellular and extracellular, allowing for multimodality therapy. Further, direct monitoring of cellular targets before, during, and after therapy may allow for real-time assessments of efficacy and toxicity.
The technical process of identifying these aberrant pathways requires laser capture microdissection (LCM) to isolate pure cellular populations. This technique requires visualization of a tissue section via light microscopy to first procure desired cells (cancer cells). Next, by activating an infrared laser beam the previously identified cells are "captured." Intact DNA, RNA, and protein is then extracted from the tissue and analyzed by conventional methods. One advantage of LCM is that binding properties of proteins are preserved. The 7 manufacturers offering LCM technology are: Arcturus Bioscience, Inc. (Mountain View, California); Bio-Rad Laboratories, Inc. (Hercules, California); Cell Robotics, Inc. (Albuquerque, New Mexico); Eppendorf AG (Hamburg, Germany); Leica Microsystems AG (Wetzlar, Germany); MMI Molecular Machines & Industries Inc. (Manchester, New Hampshire), and P.A.L.M. Microlaser Technologies AG (Bernried, Germany). Although the methodologies differ somewhat, each requires either frozen or paraffin-embedded tissue sections and all are compatible with most staining techniques, including hematoxylin and eosin (H&E), Diff-Quick and fluorescent dyes, as well as with in situ hybridization. The cells selected from the biopsy specimen yield a protein microarray (PMA) and this is used to examine the state of phosphorylated cell signaling proteins.
PMAs can be either forward-phase or reverse-phase. The advantage of reverse-phase PMAs is that there is only 1 antibody-binding event, whereas forward-phase PMAs require 2 antibody-binding events. The reverse-phase protein arrays offer "a robust new method of quantitatively assessing expression levels and the activation status of a panel of proteins." Information on proteomics, including published papers, datasets, and a listing of available antibodies and phospho-antibodies for profiling are available through the Web site of the joint Clinical Proteomics Program of the NCI/Center for Cancer Research and Food and Drug Administration/Center for Biologics Evaluation and Research at http://home.ccr.cancer.gov/ncifdaproteomics/. The potential value of PMA profiling includes a real-time assessment of therapeutic effects on targets; the discovery of novel pathway interconnections that could lead to new therapeutic strategies; individualization of therapy based on a molecular network profile; and personalized combination therapy for higher efficacy and reduced toxicity.
Dr. Liotta enumerated some of the proteomic molecular profiling clinical trials ongoing at NCI/NIH, including trials for ovarian, cervical, prostate, lung, and breast carcinomas and for melanoma. One such trial measures biochemical changes in response to imatinib in recurrent ovarian carcinoma. The clinical objective is to determine the clinical activity and toxicity profile of imatinib. The translational objectives are to describe tumor cell signaling pathways and their modification by imatinib and to correlate with outcome. Different phosphorylating events and signaling pathways are analyzed to create a decision tree that is subsequently used to separate responders to chemotherapy from nonresponders. Other translational objectives include investigating the antiangiogenic activity of imatinib and investigating other potential targets of imatinib.
A recent editorial in The Economist summarized well the state of genomic-based medicine: "Achieving the great promise of this new approach will not be easy. Doctors will have to change the way they diagnose and treat many diseases. Even more wrenching will be the changes needed in how the drug industry operates. From the patient's point of view, this move to personalized medicine should bring huge benefits. But today's drug industry is ill-equipped to deliver them... The difficulties are real, but they are not insurmountable... If personalized molecular treatments are able to score their first victory against... cancer, there will be little doubt about their immense potential in other areas."
Powers Peterson
Pathology Today:
American Society for Clinical Pathology 2004 Annual Meeting
Critical Values
in Surgical Pathology
Telma C. Pereira, Yulin Liu, Jan F. Silverman
Analogous to critical values (CVs) in clinical pathology, occasional diagnoses in surgical pathology could require immediate contact of the physician to rapidly initiate treatment. However, there are no established CV guidelines in surgical pathology.
Authors studied the incidence of CVs in surgical pathology by retrospective review of 2,659 surgical pathology reports and surveyed the perceptions of 5 clinicians and 11 pathologists about CVs in surgical pathology. Authors identified 13 CV cases (0.49%); 4 of the 13 reports documented phone calls to the clinician (most at least 1 day before final sign-out), and in 2 other reports the requisition included clinical history that implied previous knowledge of the diagnosis. The survey results indicated that for most diagnoses there was a range of opinions about whether immediate treatment was necessary and the need for a stat phone call.
CVs occur in surgical pathology, but often there is no documented phone call in the surgical pathology report. Because there is little agreement about which diagnoses require a phone call and the degree of urgency, a consensus conference might prove useful for establishing surgical pathology CV guidelines and could represent a practice improvement.
Introduction
The concept of critical value (CV) was introduced by Lundberg in 1972 as "pathophysiological derangements at such variance with normal as to be life-threatening if therapy is not instituted immediately." An example of a clinical pathology CV would be an extremely low or high potassium level, which could cause a life-threatening arrhythmia. Since the introduction of the concept, the practice of notifying physicians of critical values has become a standard of practice in clinical pathology, with well-established guidelines for which laboratory results require the technologist to immediately contact the clinician or nurse responsible for the patient.
In surgical pathology, although the results are not measured in numbers, there are certain diagnoses that could require immediate treatment or prompt evaluation of the patient. An example would be the presence of fragments of adipose tissue in an endometrial biopsy specimen, a finding that almost always represents omentum or extrauterine pelvic soft tissue and, therefore, indicates perforation of the uterus. Currently in these surgical pathology situations, common sense and personal experience of the pathologist determine when an immediate contact to the clinician is needed because there are no established guidelines to help the pathologist.
The objectives of the present study were to study the incidence and pathology management of CVs in surgical pathology and to survey the perceptions of clinicians and pathologists about CVs in surgical pathology.
American Journal of
Clinical Pathology, Vol 142,August
2005
Histopathological features of
liver and its relation to serum transaminase levels
in 91 cases of anti-HBe-positive chronic hepatitis B
Hasanjani Roushan
MR, Hajiahmadi M, Shafaie S.
For assessing the histopathological features and its relation to alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in anti-HBe-positive chronic hepatitis B in Babol, north of Iran, liver biopsy samples of 91 anti-HBe-positive chronic hepatitis B virus (HBV) infection with raised ALT were scored according to histological hepatitis activity index (HAI) from March 2000 to July 2003. Seventy-seven males and 14 females with a mean age of 32.24 (+/-11.92) years were studied. Mean ALT and AST levels were 112.5 (+/-98.9) and 69.57 (+/-60.6) IU/l, respectively. Minimal, mild, moderate and severe chronic hepatitis were seen in 35 (38.5%), 49 (53.8%), seven (7.7%) and 0 cases, respectively. There was not any significant difference between HAI and ALT levels of <60, between 60-79 and >/=80 IU/l (p = 0.299), but with regard to AST levels, this difference was significant (p = 0.005). This study showed that more than 90% of our cases are in the early stages of the liver disease. Although both serum ALT and AST levels are associated with the activity of hepatitis in HBV carriers, the AST is a better laboratory screening test for finding the severity of liver injury than ALT.
Int J Clin Pract.
59(7):791-4., 2005 Jul.
The pathology of
sudden cardiac death in patients with ischemic heart disease—arrhythmology for anatomic pathologists
Jeffrey
E. Saffitz
The goal of this review is to help anatomic pathologists interpret the significance of pathologic changes in the hearts of patients with coronary artery disease who died suddenly of spontaneous ventricular arrhythmias. Attention is focused on dynamic interactions between triggering events, such as acute ischemia, and stable anatomic substrates of arrhythmias, such as healed myocardial infarcts. A basic knowledge of arrhythmia mechanisms is necessary to understand the role of pathologic anatomy in the pathophysiology of sudden death.
Cardiovascular
Pathology,14:195-203, July-August 2005
Modern diagnosis
and treatment of primary eosinophilia
Tefferi A.
The recent discovery of an eosinophilia-specific, imatinib-sensitive, karyotypically
occult but fluorescence in situ hybridization-apparent molecular lesion in a
subset of patients with blood eosinophilia has
transformed the diagnostic as well as treatment approach to eosinophilic
disorders. Primary (i.e. nonreactive) eosinophilia is considered either "clonal"
or "idiopathic" based on the presence or absence, respectively, of
either a molecular or bone marrow histological evidence for a myeloid neoplasm.
Clonal eosinophilia might accompany
a spectrum of clinicopathological entities, the
minority of whom are molecularly characterized; Fip1-like-1-platelet-derived
growth factor receptor alpha (FIP1L1-PDGFRA(+)) systemic mastocytosis,
platelet-derived growth factor receptor beta (PDGFRB)-rearranged atypical myeloproliferative disorder, chronic myeloid leukemia, and
the 8p11 syndrome that is associated with fibroblast growth factor receptor 1
(FGFR1) rearrangement. Hypereosinophilic syndrome
(HES) is a subcategory of idiopathic eosinophilia and
is characterized by an absolute eosinophil count of
> or =1.5 x 10(9)/l for at least 6 months as well as eosinophil-mediated
tissue damage. At present, a working diagnosis of primary eosinophilia
mandates a bone marrow examination, karyotype analysis,
and additional molecular studies in order to provide the patient with accurate
prognostic information as well as select appropriate therapy. For example, the
presence of either PDGFRA or PDGFRB mutations warrants the use of imatinib in clonal eosinophilia. In HES, prednisone, hydroxyurea,
and interferon-alpha constitute first-line therapy, whereas imatinib,
cladribine, and monoclonal antibodies to either
interleukin-5 (mepolizumab) or CD52 (alemtuzumab) are considered investigational. Allogeneic transplantation offers a viable treatment option
for drug-refractory cases.
Acta Haematol. 114(1):52-60., 2005
Isolated tuberculous
epididymitis: A review of forty cases
Viswaroop
BS, Kekre N, Gopalakrishnan
Ganesh
Background: Tuberculous epididymitis is one of the causes of chronic epididymal lesions. It is difficult to diagnose in the absence of renal involvement.
Aim: To profile isolated tuberculous epididymitis and to assess our approach in the evaluation of this group of patients.
Setting and Design: Retrospective study
done at
Methods and Materials: Between 1992 and 2002, 156 fine needle aspiration cytology specimens and 108 epididymal biopsies were carried out in 187 men for evaluation of chronic epididymal nodules. Isolated epididymal tuberculosis was defined as "tuberculous infection affecting the epididymis without evidence of renal involvement as documented by the absence of acid fast bacilli in the urine sample and on imaging". The age, laterality, mode of presentation and method of histological diagnosis were studied with the objective of profiling isolated tuberculous epididymitis.
Results: Fifty-four of the 187 men (median age 32 years; interquartile range: 21-37 years) had tuberculous epididymitis. Fourteen were excluded from the analysis (10 had associated urinary tract tuberculosis and 4 were lost to follow-up). None of the 40 men with isolated tuberculous epididymitis had urinary symptoms. Bilateral involvement was seen in five (12.5%) cases. The salient presenting features included painful swelling (16 subjects, 40%), scrotal sinus (4, 20%) and acute epididymitis (2, 10%). Past history or concomitant presence of tuberculosis was noted in three subjects each. Anti TB treatment resulted in a complete response in 10 and partial response in 18. Five subjects underwent epididymectomy. Tuberculous epididymitis was found incidentally in 5 (10%) cases on high orchiectomy specimen done for suspected testicular tumour.
Conclusions: Tuberculous epididymitis can be the sole presentation of genitourinary tuberculosis.
J Postgrad Med, 51:109-111,2005 July
Carcinoma cuniculatum of the esophagus
Giovanni
De Petris, Matthew Lewin,
Toru Shoji
Aims Extremely well-differentiated squamous cell carcinoma with the features of so-called
carcinoma cuniculatum (CC) is a rare neoplasm. Auithors describe the clinicopathologic
findings of the first 2 cases of CC of the esophagus.
Methods
and Results Two elderly men
presented with symptoms and clinical signs of esophageal malignancy. Repeated endoscopic biopsies of their esophageal tumors were
inconclusive. Resection revealed CC of the esophagogastric
junction in both cases. The tumors extended into the adventitia but no lymph
node metastases were present. In situ hybridization for human papillomavirus HPV subtypes was negative.
Conclusion Carcinoma cuniculatum
is reported for the first time in the esophagus. The diagnosis of this tumor
variant is difficult by means of cytological examination or by endoscopic biopsies alone. Carcinoma cuniculatum
in this location shows biologic features similar to verrucous
carcinoma (deep penetration, no lymph nodes metastases, and location at one end
of the esophagus). No evidence of human papillomavirus
could be demonstrated.
Annals of Diagnostic
Pathology, Volume 9 • Number 3, June 2005
Review Article
Endometrial hyperplasia/adenocarcinoma. A
conventional approach
Michael T. Mazur
Hyperplasia
of the endometrium is a process characterized by an
irregular, noninvasive proliferation of glands with a variable amount of stroma. Precise classification of endometrial hyperplasia
in biopsy material is important in order to identify those hyperplasias
that are likely to be precursors of endometrial adenocarcinoma.
The current World Health Organization (WHO) classification provides a scheme
that has become widely accepted, primarily dividing hyperplasias
in to those with and those without cytologic atypia while the degree of glandular crowding (simple vs.
complex) has secondary importance. A wide variety of other endometrial changes,
ranging from artifacts, metaplasias and polyps to
well-differentiated adenocarcinoma must be considered
in the differential diagnosis. Well-differentiated adenocarcinoma
is diagnosed when one of 3 essential criteria is found in biopsy specimens: (1)
a confluent gland pattern; (2) an extensive papillary pattern; or (3) a desmoplastic stromal response.
Using the WHO classification allows segregation of endometrial hyperplasia into
clinically meaningful categories. Strict morphologic criteria also enable
separation of hyperplasia from well-differentiated adenocarcinoma.
Annals of
Diagnostic Pathology, Volume 9 • Number3, June 2005
Hodgkin lymphoma involving Waldeyer ring: a clinicopathologic
study of 22 cases
Quinones-Avila Mdel P, Gonzalez-Longoria AA,
Admirand JH, Medeiros LJ.
Authors report 22 cases of Hodgkin lymphoma involving Waldeyer ring seen at their institution during a 31-year interval. There were 16 males (73%) and 6 females (27%) with a median age of 48 years (range, 5-81 years), and 15 (68%) patients had airway obstruction or tonsillar enlargement. For 19 patients, the clinical stage was as follows: I, 7 (32%); II, 11 (50%); and III, 1 (5%). The 3 patients (14%) whose disease was unstaged had concurrent or a history of non-Hodgkin lymphoma. Histologically, the neoplasms were classified as follows: lymphocyte-rich classical, 8 (36%); nodular sclerosis, 7 (32%); mixed cellularity, 4 (18%); unclassified, 2 (9%); and lymphocyte depletion, 1 (5%). Of 7 stage I cases, 4 (57%) were the lymphocyte-rich classical type. Reed-Sternberg and Hodgkin cells were positive for CD15 and CD30 in 20 cases assessed. Epstein-Barr virus latent membrane protein type 1 was positive in 12 (67%) of 18 cases assessed. Authors conclude that Hodgkin lymphoma rarely involves Waldeyer ring, with the lymphocyte-rich classical type being common at this location.
Am
J Clin Pathol. 123(5):651-6., 2005
Significance of
the Depth of Tumor Invasion and Lymph Node Metastasis in Superficially Invasive
(T1) Esophageal Adenocarcinoma
Liu, Lixia,
Hofstetter, Wayne L , Rashid, Asif
, Swisher, Stephen G et al
Superficially invasive esophageal adenocarcinomas are a heterogeneous group of tumors, including tumors invading into mucosa and submucosa. The prognostic significance of the depth of tumor invasion and lymph node status in this group of patients remain unclear. Authors evaluated 90 consecutive patients with resected T1 adenocarcinoma of esophagus or esophagogastric junction. The T1 tumors were classified into four groups based on the depth of invasion: T1a, invading into lamina propria; T1b, into muscularis mucosae; T1c, into superficial submucosa; and T1d, into deep submucosa. The depth of tumor invasion was compared with clinicopathologic features. The depth of tumor invasion was significantly associated with the presence of lymph node metastasis (36% in T1d, 8% in T1c, 12% in T1b, and 0% in T1a; P < 0.001) and with tumor size (76% > 1.2 cm in T1d, 75% in T1c, 35% in T1b, and 25% in T1a; P < 0.001). The 5-year recurrence-free and overall survivals were significantly better in patients with tumors confined to mucosa (100% and 91%, respectively) than invasive into submucosa (60% and 58%; P = 0.0005 and P = 0.02, respectively). Lymph node metastasis was associated with tumor recurrence (P = 0.01) but not overall survival. Lymphovascular invasion was associated with both tumor recurrence (P = 0.001) and overall survival (P < 0.001) and was an independent prognostic factor in multivariate analysis (P = 0.04). The study indicated evaluation of depth of tumor invasion, status of lymph nodes, and lymphovascular invasion is important in resected superficially invasive esophageal adenocarinoma and may provide supportive information for the decision about postoperative adjuvant therapy.
American Journal of Surgical Pathology,
29(8):1079-1085, August 2005.