Targeted mutagenesis of the Vibrio fischeri flavin reductase FRase I to improve activation of the anticancer prodrug CB1954.

Phase I/II cancer gene therapy trials of the Escherichia coli nitroreductase NfsB in partnership with the prodrug CB1954 [5-(aziridin-1-yl)-2,4-dinitrobenzamide] have indicated that CB1954 toxicity is dose-limiting at concentrations far below the enzyme K(M). Here we report that the flavin reductase FRase I from Vibrio fischeri is also a CB1954 nitroreductase, which has a substantially lower apparent K(M) than E. coli NfsB. To enhance the activity of FRase I with CB1954 we used targeted mutagenesis and an E. coli SOS reporter strain to engineer single- and multi-residue variants that possess a substantially reduced apparent K(M) and an increased k(cat)/K(M) relative to the wild type enzyme. In a bacteria-delivered model for enzyme prodrug therapy, the engineered FRase I variants were able to kill human colon carcinoma (HCT-116) cells at significantly lower CB1954 concentrations than wild type FRase I or E. coli NfsB.

Lagtimes in diagnosis and treatment of colorectal cancer: determinants and association with cancer stage and survival.

BACKGROUND: Lagtimes to diagnostic colonoscopy have been used as practice performance measures. AIM: To evaluate the duration, determinants and outcomes of lagtimes between referral for endoscopic evaluation and colorectal cancer (CRC) diagnosis. METHODS: We examined the medical records of 289 patients with CRC and evaluated lagtimes, their potential determinants and their association with CRC stage at diagnosis as well as overall survival. RESULTS: Median lag between referral and CRC diagnosis was 41 days (41.5% > 60 days, 30.1% > 90 days). The only significant predictor of lagtime was the initiating event for referral: abnormal symptom, laboratory test or imaging study was associated with shortest and presence of family history was associated with longest lagtimes respectively. Longer lagtimes were associated with lower mortality risk, but this was completely explained by earlier CRC stage. An analysis restricted to 100 patients referred for abnormal CRC screening tests found no association between duration of lag and CRC stage or mortality. CONCLUSIONS: There seems to be no meaningful association between mortality in patients with CRC and lagtimes between referral for colonoscopy and CRC diagnosis for periods up to 2-3 months. On the contrary, longer lagtimes were inversely associated with CRC stage at the time of diagnosis.

Gynecomastia: cytologic features and diagnostic pitfalls in fine needle aspirates.

OBJECTIVE: To illustrate some of the uncommon cytologic findings of gynecomastia, such as apocrine metaplasia, cellular atypia and foamy macrophages, that can be misinterpreted as evidence of malignancy. STUDY DESIGN: The clinical data and fine needle aspiration (FNA) cytologic material from 100 men with the diagnosis of gynecomastia were retrospectively reviewed. The excisional biopsy slides were available for 16 cases. For comparison, FNA smears from five men with breast lesions other than gynecomastia were studied. RESULTS: The patients ranged in age from 23 to 91 years. Cytologic findings were as follows: cohesive sheets of cells containing 20-1,000 cells (98%); scattered, single, bipolar cells (78%); spindle cells (68%); ductal epithelial atypia (26%); apocrine metaplasia (8%); and foamy histiocytes (12%). In nine cases the atypia was marked, and in two of them the possibility of malignancy could not be ruled out. Surgical follow-up on 16 patients, including the cases with marked atypia, showed gynecomastia. In one case, gynecomastia was associated with intraductal papilloma. No correlation between the underlying etiology and atypical cytologic features of gynecomastia was identified. CONCLUSION: Apocrine metaplasia and epithelial atypia are common findings in gynecomastia. Attention to the cell patterns, the presence of sheets of ductal cells and absence of atypical single cells will point to the correct diagnosis.

Assessment of liver histology in chronic alcoholics with and without hepatitis C virus infection.

Patients with alcoholic liver disease have a high prevalence of hepatitis C virus (HCV) infection. The histological appearances of the liver in patients with alcoholic liver disease and HCV infection are well described. However, liver histology in individuals with dual pathology, both chronic alcohol abuse and HCV infection, is less well understood. The purpose of the present study was to examine this issue and to determine if there is any correlation between specific histological features and the serum biochemical abnormalities seen in these patients. Eighty-six chronic alcoholics, 65 with HCV infection and 21 uninfected subjects, were included in the study. All patients had history of heavy alcohol abuse (consuming 80 g or more of ethanol a day for at least 10 years). The following data were collected on each patient: demographic information (age, gender, race), the amount and duration of alcohol intake, biochemical results, and liver biopsy abnormalities including the histological activity index (HAI) score. HCV-infected alcoholics were younger (P = 0.05) and were more often African American than Caucasian (P < 0.01). Alcohol consumption was significantly greater in uninfected alcoholics compared to those with HCV infection (P < 0.05). Liver histology in subjects with HCV infection showed higher HAI scores for intralobular necrosis (P = 0.008) and periportal inflammation (P = 0.004). Features of "chronic hepatitis" and focal lymphoid aggregates were more frequent in HCV-infected alcoholics (P = 0.001 for each). By contrast, cirrhosis was present in a higher proportion of uninfected alcoholics compared to those with HCV infection (P = 0.05). Histological findings of hepatic fibrosis and total HAI score showed a significant correlation with serum albumin and platelet count in HCV-infected alcoholics. Chronic alcoholics with HCV infection have specific histological appearances that can usually help distinguish these patients from uninfected alcoholics. Correlation analysis indicates that of the various laboratory tests, serum albumin and platelet counts are the best predictors of the severity of liver damage at histology. In chronic alcoholics, the development of cirrhosis is related more to the amount of alcohol consumed than to the presence of HCV infection.

Relationship between Epstein-Barr virus and lymphoepithelioma-like carcinoma of the lung: a clinicopathologic study of 6 cases and review of the literature.

Lymphoepithelioma-like carcinoma (LELC) is a rare form of lung cancer, usually encountered in Chinese patients. Similar to nasopharyngeal carcinoma, LELC of the lung is strongly associated with Epstein-Barr virus (EBV) infection in Asian patients, but there is controversy over whether an association exists in patients from Western countries. To determine whether such a relationship exists, we retrospectively studied 6 cases of primary LELC of the lung, all of which were in Western patients. There were 4 men and 2 women, ranging in age from 49 to 75 years. The tumors ranged from 1 to 4.5 cm in diameter. Four patients had stage I disease, 1 had stage IIb disease, and 1 had stage IIIa disease. All patients are alive without evidence of disease with a follow-up of 18 to 30 months. Formalin-fixed, paraffin-embedded tissue was stained with hematoxylin-eosin for routine evaluation and immunostained for keratin and leukocyte common antigen (LCA). LCA staining was performed to exclude large-cell lymphoma. Immunoperoxidase staining (1:500 clone CS1-4; Dako, Carpinteria, CA) and in situ hybridization were performed to detect EBV. Tumors consisted of solid nests of undifferentiated tumor cells in a syncytial arrangement surrounded by heavy lymphoplasmacytic infiltrate. Tumor cells stained positively for keratin but negative for LCA. All 6 cases were negative for EBV, suggesting no association between EBV and LELC in the Western population.

Modifications in the B10 and B26-30 regions of the B chain of human insulin alter affinity for the human IGF-I receptor more than for the insulin receptor.

Inversion of the natural sequence of the B chain of human insulin (HI) from ProB28LysB29 to LysB28ProB29 generates an insulin analogue with reduced tendency to self-associate. Since this substitution increases the homology of insulin to insulin-like growth factor-I (IGF-I), we have examined the affinity of a series of insulin analogues with the general modified structure XaaB28ProB29 HI for binding to both human placental insulin and IGF-I receptors. The XaaB28ProB29 HI series is approximately equipotent to HI in binding to the insulin receptor with the exception of when Xaa = Phe, Trp, Leu, Ile, and Gly (40-60% relative to HI). Substitution with basic residues in the B28 position increased the relative affinity to the IGF-I receptor approximately 1.5-2-fold (ArgB28ProB29 > OrnB28ProB29 = LysB28ProB29). Substitution with acidic residues reduced relative affinity for the IGF-I receptor approximately 2-fold (CyaB28ProB29 = GluB28ProB29 > AspB28ProB29). Combination of AspB10 substitution in conjunction with a modification in the B28-29 position (e.g. AspB10LysB28ProB29 HI) showed an additional 2-fold selective increase in affinity for the IGF-I receptor, suggesting that these two effects are additive. Addition of Arg residues at B31-32, on the backbone of either HI or AspB10 HI, increased affinity for the IGF-I receptor 10 and 28 fold, respectively, compared to HI, confirming the significance of enhanced positive charge at the C-terminal end of the insulin B-chain in increasing selectivity for the IGF-I receptor. This relative increase in IGF-I receptor affinity correlated largely, but not completely, with enhanced growth promoting activity in human mammary epithelial cells. In the case of LysB28ProB29 HI, growth activity correlated with dissociation kinetics from the insulin receptor which were shown to be identical with those of human insulin.

Needle biopsy for suspicious lesions of the head of the pancreas: pitfalls and implications for therapy.

Controversy continues to exist concerning the optimal diagnostic approach to a pancreatic head lesion suspected of being a neoplasm. The objective of this study was to evaluate the impact of needle biopsy in suspicious pancreatic head neoplasia and its effect on therapy and outcome. Seventy-three patients with symptoms or signs of periampullary neoplasia and a pancreatic head lesion identified on CT scan were reviewed retrospectively. Forty patients with potentially resectable lesions underwent intraoperative transduodenal core needle biopsy of the head of the pancreas. Thirty-three patients underwent CT-guided percutaneous fine-needle aspiration. The sensitivity and specificity of core needle biopsy were 76% and 100%, respectively. One death was directly related to the procedure and therapy was adversely affected in one patient with a false negative result. The sensitivity and specificity of percutaneous fine-needle aspiration were 85% and 92%, respectively, and were not significantly different from the core needle biopsy results (P >0.3). Three false negative fine-needle aspiration biopsies occurred in patients with potentially resectable lesions and a low clinical suspicion for malignancy. In patients with a mass in the head of the pancreas on CT scan, fine-needle aspiration biopsy offers results similar to those of intraoperative transduodenal core needle biopsy. In patients estimated to have resectable disease, a pancreaticoduodenectomy should be performed without a biopsy. For patients with unresectable disease, cytologic examination of fine-needle aspirate should be performed. If this examination is positive, it offers the advantage of facilitating the construction of a rational plan for palliation.

Intracranial metastases as the first manifestation of prostate cancer.

We report on 2 patients with neurologic symptoms secondary to intracranial metastases from carcinoma of the prostate. In one of these patients, the intracranial lesion was the only clinically detectable metastasis. We have found only three other such cases reported in the English-language literature.

Increased natural killer cells in fluids. A new, sensitive means of detecting carcinoma.

OBJECTIVE: Natural killer (NK) cells are cells of undefined lineage that are capable of lysing certain tumor cell lines in vitro. Determination of NK cell percent (NK%) in effusions by flow cytometry could aid in the detection of malignancies. STUDY DESIGN: Over a six-month period at the Houston Veterans Affairs Medical Center, fresh effusions were routinely processed for cytology, and a portion was submitted for lymphocyte immunophenotyping using the FACScan and a panel including CD16/CD56 for NK cells. Seventy fluids (42 pleural, 28 peritoneal) from 62 men were examined. RESULTS: NK cell percents were markedly increased in 15 cases (29-68%, mean 45.5) and low in 55 (2-20%, mean 8). Fourteen of the 15 cases with increased NK% were positive for carcinoma (93%), while 54/55 cases with low ones were negative for carcinoma (98%). Mesotheliomas, lymphomas and leukemias had low NK%. CONCLUSION: Using the Mann-Whitney U test, an increase in NK% predicts metastatic carcinoma with a P level of < .00001.

p53 gene product in pleural effusions. Practical use in distinguishing benign from malignant cells.

OBJECTIVE: To determine the utility of positive p53 immunostaining as an adjunct in the diagnosis of malignancy in pleural effusions, we reviewed 103 effusions representing the typical range of diagnoses encountered in the evaluation of pleural fluid cytology. STUDY DESIGN: Immunohistochemistry was performed on paraffin-embedded cell blocks using a monoclonal antibody to the p53 suppressor gene product clone BP53-12 and a standard avidin-biotin complex technique with a citrate buffer antigen retrieval solution. RESULTS: Forty-one of 75 effusions with an unequivocal cytologic diagnosis of malignancy were immunopositive for p53 protein (55%). One of nine effusions cytologically interpreted as showing reactive mesothelial cells showed immunopositivity; that case was subsequently diagnosed as a mesothelioma on pleural biopsy. Nineteen cases were interpreted as suspicious for malignancy. Of these, 16 were negative, and 3 were positive for p53 protein. Of the three positive cases, two showed the presence of non-small cell and poorly differentiated large cell carcinoma. CONCLUSION: These findings suggest that p53 protein immunostaining is relatively sensitive and highly specific in differentiating benign mesothelial cells from malignant cells in pleural effusions. While negative p53 protein immunostaining does not exclude malignancy, positive staining in reactive or suspicious cells warrants further diagnostic evaluation of the patient.

Neuroendocrine neoplasms of the larynx.

Neuroendocrine neoplasms of the larynx are a rare group of tumors that include carcinoid tumor, atypical carcinoid tumor, and small cell carcinoma. These neoplasms pose interesting diagnostic, prognostic, and therapeutic dilemmas, and they are, as a whole, aggressive tumors with a tendency for local and distant spread. The authors of this study examined six new cases of laryngeal neuroendocrine neoplasms. One case manifested itself as a primary atypical carcinoid tumor and caused a "carcinoid syndrome." The remaining five cases were small cell carcinomas of the larynx. Histologic, immunocytochemical, DNA flow cytometric, and p53 studies were performed on all cases. The expression of neuron-specific enolase and chromogranin were the most useful markers in this group of tumors. Overexpression of p53 protein was present in the majority of cases, including the atypical carcinoid tumor. The implications of these studies for diagnosis, classification, and treatment are discussed.

Case report: vitamin D-mediated hypercalcemia in fungal infections.

Hypercalcemia has been well described in a variety of neoplastic and granulomatous diseases. One mechanism for this hypercalcemia is via the excess production of 1,25-dihydroxyvitamin D from extra-renal sources. The authors describe an AIDS patient infected with Cryptococcus neoformans who had suggestive evidence of vitamin D-mediated hypercalcemia. He had an elevated serum 1,25-dihydroxyvitamin D value, a normal 25-hydroxyvitamin D value, and low values for parathyroid hormone and parathyroid hormone-related peptide. Most previously reported cases of hypercalcemia associated with fungal infections did not include sufficient evidence to implicate a role for excess 1,25-dihydroxyvitamin D production, except for two case reports involving patients with hypercalcemia with infections due to Pneumocystis carinii and Candida albicans. The authors' patient's hypercalcemia resolved during treatment of his underlying infection. Patients with hypercalcemia or in whom hypercalcemia develops during a disseminated fungal infection should have vitamin D metabolites measured as part of their work-up.

Leu-7 immunoreactivity in cytologic specimens of thyroid lesions, with emphasis on follicular neoplasms.

Anti-Leu-7 (HNK-1, CD 57) antibody, a marker for natural killer lymphocytes, was employed by Ghali et al. (Hum Pathol 1992;23: 21-25) to study surgically resected formalin-fixed, paraffin-embedded thyroid lesions. They demonstrated strong immunoreactivity of this antibody with thyroid carcinomas, both follicular and papillary, and only occasional weak immunoreactivity with colloid goiters and follicular adenomas. We studied cytologic specimens (primarily fine-needle aspiration biopsy specimens) from 44 thyroid lesions, including 10 follicular carcinomas, 14 follicular adenomas, seven adenomatous nodules, six papillary carcinomas, and seven cases of Hashimoto's thyroiditis. All follicular carcinomas exhibited immunoreactivity to anti-Leu-7 antibody, usually of a moderate to strong degree (9/10); however, six of 14 follicular adenomas yielded similar results. The patterns of immunoreactivity in the other lesions were similar to those previously described (Ghali et al., Hum Pathol 1992;23:21-25). It does not appear that anti-Leu-7 antibody can be used as a specific marker of malignancy in the cytologic assessment of follicular neoplasms of the thyroid.

A trinucleotide repeat-associated increase in the level of Alu RNA-binding protein occurred during the same period as the major Alu amplification that accompanied anthropoid evolution.

Nearly 1 million Alu elements in human DNA were inserted by an RNA-mediated retroposition-amplification process that clearly decelerated about 30 million years ago. Since then, Alu sequences have proliferated at a lower rate, including within the human genome, in which Alu mobility continues to generate genetic variability. Initially derived from 7SL RNA of the signal recognition particle (SRP), Alu became a dominant retroposon while retaining secondary structures found in 7SL RNA. We previously identified a human Alu RNA-binding protein as a homolog of the 14-kDa Alu-specific protein of SRP and have shown that its expression is associated with accumulation of 3'-processed Alu RNA. Here, we show that in early anthropoids, the gene encoding SRP14 Alu RNA-binding protein was duplicated and that SRP14-homologous sequences currently reside on different human chromosomes. In anthropoids, the active SRP14 gene acquired a GCA trinucleotide repeat in its 3'-coding region that produces SRP14 polypeptides with extended C-terminal tails. A C-->G substitution in this region converted the mouse sequence CCA GCA to GCA GCA in prosimians, which presumably predisposed this locus to GCA expansion in anthropoids and provides a model for other triplet expansions. Moreover, the presence of the trinucleotide repeat in SRP14 DNA and the corresponding C-terminal tail in SRP14 are associated with a significant increase in SRP14 polypeptide and Alu RNA-binding activity. These genetic events occurred during the period in which an acceleration in Alu retroposition was followed by a sharp deceleration, suggesting that Alu repeats coevolved with C-terminal variants of SRP14 in higher primates.

Utilization of fine-needle aspiration biopsy in the diagnosis of metastatic tumors to the pancreas.

There is relatively little information concerning the use of fine-needle aspiration biopsy (FNAB) to diagnose a mass in the pancreas that is secondary to metastatic tumor. This study reviews the incidence and types of neoplasms which metastasize to the pancreas and assesses the contribution FNAB can make in their diagnosis. Of 117 radiologically guided FNABs of the pancreas, 11% (n = 13) showed metastatic malignancy. Nine patients had a previous history of malignancy while four patients presented with a pancreatic mass and were subsequently found to have wide-spread malignant disease. The majority of metastatic lesions were epithelial (77%, n = 10). Patient outcomes were generally poor (mean survival 2.8 mo). Metastases to the pancreas occur from a variety of primary sites and should be considered in patients with a pancreatic mass and a history of prior malignancy. FNAB is useful in diagnosing these metastases and this is clinically important because of their poor prognosis.

Keratoacanthoma: when to observe and when to operate and the importance of accurate diagnosis.

Although keratoacanthoma regresses spontaneously, treating physicians seldom allow these lesions to progress through their natural course. We describe two cases of skin lesions, the first being a keratoacanthoma, which enlarged rapidly and then involuted with minimal scarring. The second lesion was initially misdiagnosed and failed to respond to medical management. An accurate diagnosis of squamous cell carcinoma was not made until the lesion had become deeply invasive. These two lesions have helped clarify our thoughts about the observation of keratoacanthomas, the need for frequent patient follow-up when a lesion is not surgically ablated, and the importance of providing the pathologist with an adequate biopsy specimen for diagnostic purposes.