Epigenetic mechanisms in alcohol- and adversity-induced developmental origins of neurobehavioral functioning.

The long-term effects of developmental alcohol and stress exposure are well documented in both humans and non-human animal models. Damage to the brain and attendant life-long impairments in cognition and increased risk for psychiatric disorders are debilitating consequences of developmental exposure to alcohol and/or psychological stress. Here we discuss evidence for a role of epigenetic mechanisms in mediating these consequences. While we highlight some of the common ways in which stress or alcohol impact the epigenome, we point out that little is understood of the epigenome's response to experiencing both stress and alcohol exposure, though stress is a contributing factor as to why women drink during pregnancy. Advancing our understanding of this relationship is of critical concern not just for the health and well-being of individuals directly exposed to these teratogens, but for generations to come.

Clonality Testing in Veterinary Medicine: A Review With Diagnostic Guidelines.

The accurate distinction of reactive and neoplastic lymphoid proliferations can present challenges. Given the different prognoses and treatment strategies, a correct diagnosis is crucial. Molecular clonality assays assess rearranged lymphocyte antigen receptor gene diversity and can help differentiate reactive from neoplastic lymphoid proliferations. Molecular clonality assays are commonly used to assess atypical, mixed, or mature lymphoid proliferations; small tissue fragments that lack architecture; and fluid samples. In addition, clonality testing can be utilized to track neoplastic clones over time or across anatomic sites. Molecular clonality assays are not stand-alone tests but useful adjuncts that follow clinical, morphologic, and immunophenotypic assessment. Even though clonality testing provides valuable information in a variety of situations, the complexities and pitfalls of this method, as well as its dependency on the experience of the interpreter, are often understated. In addition, a lack of standardized terminology, laboratory practices, and interpretational guidelines hinders the reproducibility of clonality testing across laboratories in veterinary medicine. The objectives of this review are twofold. First, the review is intended to familiarize the diagnostic pathologist or interested clinician with the concepts, potential pitfalls, and limitations of clonality testing. Second, the review strives to provide a basis for future harmonization of clonality testing in veterinary medicine by providing diagnostic guidelines.

Hepatosplenic and hepatocytotropic T-cell lymphoma: two distinct types of T-cell lymphoma in dogs.

The clinical, clinicopathologic, and pathological findings of 9 dogs with T-cell lymphoma that involved the liver in the absence of peripheral lymphadenopathy were assessed. Seven dogs had hepatosplenic T-cell lymphoma (HS-TCL). Dogs with HS-TCL presented with hepato- and/or splenomegaly, regenerative anemia, thrombocytopenia, and hypoproteinemia. The clinical course was rapidly progressive with all dogs but 1 dead within 24 days of initial presentation. Neoplastic lymphocytes were centered on hepatic and splenic sinusoids and had a CD3+ (5/7), TCRαß- (5/5), TCRγδ+ (3/5), CD11d+ (6/7), granzyme B+ (5/7) immunophenotype. Bone marrow and lungs were consistently but variably involved. These findings closely resemble the human disease and support the classification of HS-TCL as a distinct World Health Organization entity in dogs. The remaining 2 dogs markedly differed in the pattern of hepatic involvement by neoplastic lymphocytes, which were not confined to hepatic sinusoids but invaded hepatic cords. In addition, neoplastic cells had a CD11d- immunophenotype, and clinicopathologic data indicated marked cholestasis and mild to absent anemia. Based on the distinct tropism of neoplastic lymphocytes for hepatocytes, the name hepatocytotropic T-cell lymphoma (HC-TCL) is proposed. Given the histomorphologic, clinicopathologic, and immunophenotypic differences, HC-TCL likely represents a separate biological entity rather than a histomorphologic variant of HS-TCL.

Sensitivity and specificity of canine pancreas-specific lipase (cPL) and other markers for pancreatitis in 70 dogs with and without histopathologic evidence of pancreatitis.

BACKGROUND: Pancreatitis is a common disorder in dogs for which the antemortem diagnosis remains challenging. OBJECTIVES: To compare the sensitivity and specificity of serum markers for pancreatitis in dogs with histopathologic evidence of pancreatitis or lack thereof. ANIMALS: Seventy dogs necropsied for a variety of reasons in which the pancreas was removed within 4 hours of euthanasia and serological markers were evaluated within 24 hours of death. METHODS: Prospective study: Serum was analyzed for amylase and lipase activities, and concentrations of canine trypsin-like immunoreactivity (cTLI) and canine pancreas-specific lipase (cPL). Serial transverse sections of the pancreas were made every 2 cm throughout the entire pancreas and reviewed using a semiquantitative histopathologic grading scheme. RESULTS: The sensitivity for the Spec cPL (cutoff value 400 µg/L) was 21 and 71% in dogs with mild (n = 56) or moderate-severe pancreatitis (n = 7), and 43 and 71% (cutoff value 200 µg/L), respectively. The sensitivity for the cTLI, serum amylase, and lipase in dogs with mild or moderate-severe pancreatitis was 30 and 29%; 7 and 14%; and 54 and 71%, respectively. The specificity for the Spec cPL based on 7 normal pancreata was 100 and 86% (cutoff value 400 and 200 µg/L, respectively), whereas the specificity for the cTLI, serum amylase, and lipase activity was 100, 100, and 43%, respectively. CONCLUSION AND CLINICAL IMPORTANCE: The Spec cPL demonstrated the best overall performance characteristics (sensitivity and specificity) compared to other serum markers for diagnosing histopathologic lesions of pancreatitis in dogs.

Immunohistochemical detection of survivin in canine lymphoma.

Survivin is a member of the family of proteins known as 'inhibitors of apoptosis proteins'. Survivin has a role in cellular decisions concerning division and survival and is frequently expressed in neoplastic cells. The aim of the present study was to investigate immunohistochemically the expression of survivin in normal canine tissues and in canine lymphoma. A representative range of fetal and adult normal tissues as well as biopsy samples from dogs with lymphoma were assembled in tissue arrays. The lymphomas were classified according to the revised Kiel and to the Revised European American Lymphoma-World Health Organization (REAL-WHO) schemes. Polyclonal and monoclonal antisera cross-reactive with canine survivin identified cytoplasmic expression of the molecule in a broad range of normal canine cells. The same reagents demonstrated cytoplasmic labelling of more than 5% of cells in all 83 lymphoma samples tested with polyclonal antiserum and in 67 of 82 (82%) of samples tested with monoclonal antiserum. Survivin was expressed by a wide range of canine lymphoma subtypes, but the expression of this molecule in normal canine tissues must be considered if novel therapies targeting survivin are applied to the management of canine lymphoma.

A comprehensive test system to identify suitable antibodies against p53 for immunohistochemical analysis of canine tissues.

The tumour suppressor p53 is commonly detected in tissues of companion animals by means of antibodies raised against the human protein. The following three-step procedure was devised to test the suitability of such antibodies for immunohistochemistry on canine tissues. (1) Western blot and immunohistochemical analyses on bacterially expressed recombinant canine protein to assess human-to-canine cross-reactivity. (2) Immunohistochemistry of cultured, UVB-irradiated canine keratinocytes to evaluate suitability for detection of endogenous p53. (3) Immunohistochemistry on tissue arrays to further substantiate suitability of the antibodies on a panel of normal and neoplastic human and canine tissues. Five of six antibodies cross-reacted with recombinant canine p53. Three of these (PAb122, PAb240, CM-1) also immunolabelled stabilized wild type p53 in cell cultures and elicited a consistent, characteristic labelling pattern in a subset of tumours. However, two alternative batches of polyclonal antibody CM-1 failed to detect p53 in cell cultures, while showing a characteristic labelling pattern of a completely different subset of tumours and unspecific labelling of normal tissues. The test system described is well suited to the selection of antibodies for immunohistochemical p53 detection. The results emphasize the need to include appropriate controls, especially for polyclonal antibodies.

Genotyping of Turkish environmental Cryptococcus neoformans var. neoformans isolates by pulsed field gel electrophoresis and mating type.

A total of 26 environmental Cryptococcus neoformans var. neoformans strains isolated from 634 samples of pigeon droppings collected from 54 different provinces of Turkey in 1996 and 1997 were included in this study. The results of pulsed-field gel electrophoresis (PFGE) showed that the 26 strains could be separated into 24 different PFGE patterns. In a mating-type study, of 26 strains, 20 were MATalpha, four were MATa, one was MATa/alpha and one was non-typable by STE20 specific primers. By the polymerase chain reaction typing, all the isolates were serotype A. The extensive heterogeneity among these isolates suggests that a single clonal population may not be present in Turkey. Additionally, the presence of an AMATa/DMATalpha hybrid may indicate the existence of strains that are AMATa mating type in Turkish environment.

Molecular epidemiology of clinical Cryptococcus neoformans strains from India.

Little is known about the molecular epidemiology of the human pathogenic fungus Cryptococcus neoformans in India, a country now in the midst of an epidemic of AIDS-related cryptococcosis. We studied 57 clinical isolates from several regions in India, of which 51 were C. neoformans var. grubii, 1 was C. neoformans var. neoformans, and 5 were C. neoformans var. gattii. This strain set included 18 additional sequential isolates from 14 patients. Strains were characterized phenotypically by measuring the polysaccharide capsule and by determining the MICs of standard antifungals. Molecular typing was performed by a PCR-based method using the minisatellite-specific core sequence (M13), by electrophoretic karyotyping, by restriction fragment length polymorphisms with the C. neoformans transposon 1 (TCN-1), and by URA5 DNA sequence analysis. Overall, Indian isolates were less heterogeneous than isolates from other regions and included a subset that clustered into one group based on URA5 DNA sequence analysis. In summary, our results demonstrate (i) differences in genetic diversity of C. neoformans isolates from India compared to isolates from other regions in the world; (ii) that DNA typing with the TCN-1 probe can adequately distinguish C. neoformans var. grubii strains; (iii) that TCN-1 sequences are absent in many C. neoformans var. gattii strains, supporting previous studies indicating that these strains have a limited geographical dispersal; and (iv) that human cryptococcal infection can be associated with microevolution of the infecting strain and by simultaneous coinfection with two distinct C. neoformans strains.

Cloning and characterization of the lanosterol 14alpha-demethylase (ERG11) gene in Cryptococcus neoformans.

The ergosterol pathway in fungal pathogens is an attractive antimicrobial target because it is unique from the major sterol (cholesterol) producing pathway in humans. Lanosterol 14alpha-demethylase is the target for a major class of antifungals, the azoles. In this study we have isolated the gene for this enzyme from Cryptococcus neoformans. The gene, ERG11, was recovered using degenerate PCR with primers designed with a novel algorithm called CODEHOP. Sequence analysis of Erg11p identified a highly conserved region typical of the cytochrome P450 class of mono-oxygenases. The gene was present in single copy in the genome and mapped to one end of the largest chromosome. Comparison of the protein sequence to a number of major human fungal pathogen Erg11p homologs revealed that the C. neoformans protein was highly conserved, and most closely related to the Erg11p homologs from other basidiomycetes. Functional studies demonstrated that the gene could complement a Saccharomyces cerevisiae erg11 mutant, which confirmed the identity of the C. neoformans gene.

Molecular and genetic characterization of a serotype A MATa Cryptococcus neoformans isolate.

Cryptococcus neoformans comprises two varieties (neoformans and gattii) and four serotypes (A, B, C and D). Fertile isolates of both mating types have been identified in serotypes B, C and D; however, a fertile serotype A MATa strain has not been confirmed, although serotype A MATalpha strains will mate with serotype D MATa strains. Preliminary analysis of a recent Italian environmental isolate (IUM 96-2828) suggested that this strain was haploid, serotype A and MATa. In this study, IUM 96-2828 has been characterized in detail. A mating reaction between IUM 96-2828 and H99 (serotype A MATalpha) produced abundant spores with an equal distribution of MATa and MATalpha progeny, all of which were serotype A. Karyotypic analysis of F(1) spores revealed evidence of recombination, confirming that IUM 96-2828 was fertile. The MATa pheromone gene from IUM 96-2828 was sequenced and found to be most closely related to the serotype D MATa pheromone gene. Phylogenetic comparisons of other genes not linked to mating type also suggested IUM 96-2828 was most closely related to serotype A strains. Biochemical analysis showed that the carbon assimilation profiles of H99 and IUM 96-2828 were identical for 97 % (30/31) of the substrates while isozyme analysis showed 89 % (17/19) identity. Assays of major virulence factors found no difference between H99 and IUM 96-2828. Virulence studies using the mouse model demonstrated that IUM 96-2828 was virulent for mice, although it was less virulent than H99. These data strongly suggest that IUM 96-2828 is a true haploid serotype A MATa isolate that is fertile.

The influence of gender on survival and tumor recurrence following adjuvant therapy of completely resected stages II and IIIa non-small cell lung cancer.

This study evaluates the influence of gender on survival and tumor recurrence following adjuvant therapy of completely resected stages II and IIIa non-small cell lung cancer (NSCLC). The Eastern Cooperative Oncology Group conducted a randomized prospective trial of adjuvant therapy in patients with completely resected stages II and IIIa NSCLC. A laboratory correlative study assessed the prevalence and prognostic significance of p53 and K-ras mutations. Patients were randomized to receive either radiotherapy (RT) alone or four cycles of cisplatin and VP-16 administered concurrently with radiotherapy (CRT). Median survival was 35 months for the 285 men and 41 months for the 203 women enrolled in the study (P = 0.12). The relative risk (RR) of death for men vs women was 1.19 (95% confidence interval [CI], 0.95-1.49). Median survival of the 147 men and 95 women randomized to the RT arm was 39 months each (P = 0.35). Median survival of the 138 men and 108 women randomized to the CRT arm was 30 and 42 months, respectively (P = 0.18). Disease recurrence patterns were similar between the genders. Univariate and multivariate analyses demonstrated improved survival for women with tumors of non-squamous histology (P < 0.01). The distribution of p53 and K-ras mutations was similar between the genders and had no influence on survival. Gender does not influence survival following adjuvant RT or CRT administered to patients with completely resected stages II and IIIa NSCLC. However, women with non-squamous histology have increased survival when compared to men.

Lack of prognostic significance of p53 and K-ras mutations in primary resected non-small-cell lung cancer on E4592: a Laboratory Ancillary Study on an Eastern Cooperative Oncology Group Prospective Randomized Trial of Postoperative Adjuvant Therapy.

PURPOSE: To determine the prognostic and predictive significance of p53 and K-ras mutations in patients with completely resected non-small-cell lung cancer (NSCLC). PATIENTS AND METHODS: Patients were randomized preoperatively to receive adjuvant postoperative radiotherapy (Arm A) or radiotherapy plus concurrent chemotherapy (Arm B). p53 protein expression was studied by immunohistochemistry (IHC) and p53 mutations in exons 5 to 8 were evaluated by single-strand conformational analysis. K-ras mutations in codons 12, 13, and 61 were determined using engineered restriction fragment length polymorphisms. RESULTS: Four hundred eighty-eight patients were entered onto E3590; 197 tumors were assessable for analysis. Neither presence nor absence of p53 mutations, p53 protein expression, or K-ras mutations correlated with survival or progression-free survival. There was a trend toward improved survival for patients with wildtype K-ras (median, 42 months) compared with survival of patients with mutant K-ras who were randomized to chemotherapy plus radiotherapy (median, 25 months; P = .09). Multivariate analysis revealed only age and tumor stage to be significant prognostic factors, although there was a trend bordering on statistical significance for K-ras (P = .066). Analysis of survival difference by p53 by single-stranded conformational polymorphism and IHC, interaction of p53 and K-ras, interaction of p53 and treatment arm, nodal station, extent of surgery, weight loss, and histology did not reach statistical significance. CONCLUSION: p53 mutations and protein overexpression are not significant prognostic or predictive factors in resected stage II or IIIA NSCLC. K-ras mutations may be a weak prognostic marker. p53 or K-ras should not be routinely used in the clinical management of these patients.

A randomized trial of postoperative adjuvant therapy in patients with completely resected stage II or IIIA non-small-cell lung cancer. Eastern Cooperative Oncology Group.

BACKGROUND: We conducted a randomized trial to determine whether combination chemotherapy plus thoracic radiotherapy is superior to thoracic radiotherapy alone in prolonging survival and preventing local recurrence in patients with completely resected stage II or IIIa non-small-cell lung cancer. METHODS: After surgical staging and resection of the tumor (usually by lobectomy or pneumonectomy), the patients were randomly assigned to receive either four 28-day cycles of cisplatin (60 mg per square meter of body-surface area intravenously on day 1) and etoposide (120 mg per square meter intravenously on days 1, 2, and 3) administered concurrently with radiotherapy (a total of 50.4 Gy, given in 28 daily fractions) or radiotherapy alone (a total of 50.4 Gy, given in 28 daily fractions). RESULTS: Of the 488 patients who were enrolled in the study, 242 were assigned to receive radiotherapy alone and 246 were assigned to receive chemotherapy and radiotherapy. The median duration of follow-up was 44 months. Treatment-associated mortality was 1.2 percent in the group given radiotherapy alone and 1.6 percent in the group given chemotherapy and radiotherapy. The median survival was 39 months in the group given radiotherapy and 38 months in the group given chemotherapy and radiotherapy (P= 0.56 by the log-rank test). The relative likelihood of survival among patients assigned to receive chemotherapy and radiotherapy, as compared with those assigned to receive radiotherapy alone, was 0.93 (95 percent confidence interval, 0.74 to 1.18). Intrathoracic disease recurred within the radiation field in 30 of 234 patients (13 percent) in the group given radiotherapy and in 28 of 236 patients (12 percent) in the group given chemotherapy and radiotherapy (P=0.84); data on recurrence were not available for 18 patients. CONCLUSIONS: As compared with radiotherapy alone, adjuvant radiotherapy and chemotherapy with cisplatin and etoposide does not decrease the risk of intrathoracic recurrence or prolong survival in patients with completely resected stage II or IIIa non-small-cell lung cancer.

Mediastinal lymph node dissection improves survival in patients with stages II and IIIa non-small cell lung cancer. Eastern Cooperative Oncology Group.

BACKGROUND: Mediastinal lymph node dissection (MLND) is an integral part of surgery for non-small cell lung cancer (NSCLC). To compare the impact of systematic sampling (SS) and complete MLND on the identification of mediastinal lymph node metastases and patient survival, the Eastern Cooperative Oncology Group (ECOG) stratified patients by type of MLND before participation in ECOG 3590 (a randomized prospective trial of adjuvant therapy in patients with completely resected stages II and IIIa NSCLC). METHODS: Eligibility requirements for study entry included a thorough investigation of the mediastinal lymph nodes with either SS or complete MLND. The former was defined as removal of at least one lymph node at levels 4, 7, and 10 during a right thoracotomy and at levels 5 and/or 6 and 7 during a left thoracotomy, while the latter required complete removal of all lymph nodes at those levels. RESULTS: Three hundred seventy-three eligible patients were accrued to the study. Among the 187 patients who underwent SS, N1 disease was identified in 40% and N2 disease in 60%. This was not significantly different than the 41% of N1 disease and 59% of N2 disease found among the 186 patients who underwent complete MLND. Among the 222 patients with N2 metastases, multiple levels of N2 disease were documented in 30% of patients who underwent complete MLND and in 12% of patients who had SS (p = 0.001). Median survival was 57.5 months for those patients who had undergone complete MLND and 29.2 months for those patients who had SS (p = 0.004). However, the survival advantage was limited to patients with right lung tumors (66.4 months vs 24.5 months, p<0.001). CONCLUSIONS: In this nonrandomized comparison, SS was as efficacious as complete MLND in staging patients with NSCLC. However, complete MLND identified significantly more levels of N2 disease. Furthermore, complete MLND was associated with improved survival with right NSCLC when compared with SS.

Adjuvant therapy of resected non-small-cell lung cancer.

The administration of adjuvant therapy after complete resection of non-small-cell lung cancer is controversial. Radiation therapy and chemotherapy have been used individually and concomitantly in efforts to prevent local recurrence and improve survival. However, recent phase II and III trials and a meta-analysis have produced conflicting results. Postoperative adjuvant therapy remains a subject of active investigation.

Adjuvant therapy of non small-cell lung cancer.

Multiple randomized prospective trials have failed to show a definitive survival advantage for the administration of adjuvant therapy following complete resection of non small-cell lung cancer.

Thoracic surgical spectrum of HIV infection.

The thoracic surgeon is often called on to diagnose or treat a variety of disorders associated with human immunodeficiency virus (HIV) infection. Surgical mediastinal exploration through cervical and anterior approaches is a safe and valuable modality in appropriately selected patients with unexplained mediastinal lymphadenopathy. Open lung biopsy is used in a small subset of HIV-infected patients with undiagnosed diffuse or multifocal pulmonary disease, with an anticipated diagnostic yield of more than 70%. The biopsy can be performed either thoracoscopically or via thoracotomy, based on the expertise and discretion of the surgeon. Open lung biopsy should be used very selectively and in patients with bronchoscopically confirmed diagnoses who are failing optimal medical therapy, because the impact on outcome is minuscule and because open lung biopsy is best avoided altogether in patients with established respiratory failure. Patients with acquired immune deficiency syndrome (AIDS) have an increased incidence of pneumothorax, often associated with Pneumocystis carinii pneumonia. Depending on the clinical scenario, tube thoracostomy, pleurodesis, or pleurectomy may be used. Thoracic empyema in AIDS patients requires urgent intercostal drainage and close clinical surveillance to discern the need for decortication or rib resection and open drainage. A surgical approach to pyogenic lung abscess or invasive aspergillosis is occasionally useful. Although it is controversial whether the incidence of lung cancer is increased in patients with HIV infection, HIV-positive patients with early stage nonsmall-cell lung cancer who are otherwise surgical candidates should undergo resection, especially in the era of highly active antiretroviral therapy.

High dose chemoradiotherapy followed by esophagectomy for adenocarcinoma of the esophagus and gastroesophageal junction: results of a phase II study of the Eastern Cooperative Oncology Group.

BACKGROUND: To assess the toxicity, local response, and survival associated with multimodality therapy in a cooperative group setting, patients with biopsy-proven clinical Stage I or II adenocarcinoma of the esophagus (staged according to 1983 American Joint Committee on Cancer criteria) or gastroesophageal junction were treated with concomitant radiation and chemotherapy followed by esophagectomy. METHODS: Radiotherapy was administered in daily 2-gray (Gy) fractions 5 days a week until a total of 60 Gy was reached. 5-fluorouracil (5-FU) was infused continuously at a dose of 1000 mg/m2/day for 96 hours on Days 2-5 and 28-31. On Day 2, a 10 mg/m2 bolus of mitomycin was injected intravenously. Esophagectomy was performed 4-8 weeks following completion of the radiotherapy. RESULTS: During the 18-month study period (August 1991 through January 1993), 46 eligible patients were accrued from 21 institutions. Eight patients were Stage I and 38 Stage II. Eighty-seven percent of patients (40 of 46) received 6000 centigray (cGy), and all received >5000 cGy. Seventy-eight percent of patients (36 of 46) received >90% of the planned 5-FU dose. Follow-up ranged from 11 to 36 months (median, 22 months). There were eight treatment-related deaths; two were preoperative (from adult respiratory distress syndrome) and six were postoperative. Complete or partial response prior to esophagectomy was observed in 63% of cases, stable disease in 15%, and progression in 20%. Thirty-three patients underwent esophagectomy (transhiatal, n=14; Ivor Lewis, n=16; other, n=3). No tumor was found in the specimens resected from 8 of these 33 patients; this represented a pathologic complete response rate of 17% overall and 24% for those who underwent esophagectomy. Overall median survival was 16.6 months, 1-year survival 57%, and 2-year survival 27%. Survival was significantly worse for patients with circumferential cancers (median, 18.1 months vs. 8.3 months; P <0.05). CONCLUSION: High dose radiation therapy with concurrent 5-FU and mitomycin may be administered to patients with esophageal adenocarcinoma with acceptable morbidity. However, in a cooperative group setting, esophagogastrectomy following intensive chemoradiotherapy is associated with excessive morbidity and mortality. Circumferential tumor growth is a significant adverse prognostic factor.