Immunohistochemical detection of p140trkA and p75LNGFR neurotrophin receptors in neuroblastoma

In neuroblastoma, high levels of mRNA for p14h ^trkA and p75 ^LNGFR neurotrophin receptors are predictive of favorable outcome. Their evaluation by Northern blot, however, requires substantial amounts of tissue and this prevents their routine evaluation as well as the possibility for multicenter studies to be easily carried out. In an attempt to overcome these limitations, the feasibility and reliability of determining both neurotrophin receptors on cryostat sections by immunohistochemistry were assessed, and these findings were compared to those obtained from Northern blot analysis. Primary tumor samples from 28 untreated patients at all stages were evaluated by using H10 anti-p140 ^trkA and ME20.4 anti-p75 ^LNGFR mAbs. Although weak, positiveimmunostaining was found in 9 of 28 tumors for p140 ^trkA and in 5 of 28 tumors for p75 ^LNGFR . As compared to Northern blot, the concordance rate was 79% (22 of 28 cases) for p140 ^trkA (p < 0.05) and 71% (20 of 28 cases) for p75 ^LNGFR (p < 0.05). No case negative for Northern blot was found to be positive with immunohistochemistry. Since only high mRNA levels for both receptors have been shown to be clinically relevant, their immunohistochemical detection, although less sensitive than Northern blot, can be just as sufficient and reliable as a prognostic tool, and possibly with a better cost-benefit ratio.     

Cytogenetic analysis of human oocytes parthenogenetically activated by puromycin

Purpose

Treatment of aged human oocytes by puromycin allows a high rate of parthenogenetic activation and development until the first cleavage division. This technique was used for the study of the chromosome complement of oocytes which remained unfertilized after in vitro fertilization. Three hundred four unfertilized oocytes were treated with 10 Μg/ml puromycin for 6–8 hr and further cultured for 12–15 hr.

Results

Activation occurred in 90.5% of the oocytes. Heterozygous diploids with two pronuclei predominated (61%), which is in contrast to the mouse, where the majority of oocytes activated by puromycin are uniform haploids (89%).

Conclusions

Therefore we conclude that puromycin treatment induces retention of the second polar body in human oocytes, unlike in mouse oocytes treated in the same way. Chromosome analysis performed on 182 oocytes suggested a nondisjunction (ND) rate for the second meiotic division of 12.7%. This is a low figure considering the fact that puromycin itself has been reported to induce nondisjunction. For the first meiotic division a ND rate of only 5.6% was found. This rate is lower than the one found in metaphase II arrested oocytes and we believe that this difference is due to the technical differences between the study of meiotic and that of mitotic chromosomes.     

Adoptive Immunotherapy With Tumor-Infiltrating Lymphocytes and Subcutaneous Recombinant Interleukin-2 Plus Interferon Alfa-2a for Melanoma Patients With Nonresectable Distant Disease: A Phase I/II Pilot Trial

Background: On the basis of our previous experience, we designed this study to determine the activity and toxicity of outpatient treatment with autologous tumor-infiltrating lymphocytes (TIL) together with intermediate-dose recombinant interleukin-2 (rIL-2) and low-dose recombinant interferon alfa-2a (rIFN-2a), for patients with metastatic melanoma.Methods: Between April 1992 and October 1994, we processed 38 melanoma samples derived from 36 patients with metastases. Proliferative cultures of expanded lymphocytes (TIL) were infused only once into patients with metastatic melanoma. rIL-2 was administered subcutaneously for 1 month, starting on the day of TIL infusion, at an escalating dose of 6–18 × 10⁶ IU/m²/day for the first week and at the maximum-tolerated dose for the subsequent 3 weeks and then, after a 15-day interval, for 1 week/month for 3 months. rIFN-2a was administered subcutaneously at 3 × 10⁶ IU three times each week until progression.Results: Of 38 melanoma samples, 19 (50%) resulted in proliferative cultures and were infused. The median number of expanded lymphocytes was 18 × 10⁹ (range, 1–43 × 10⁹), and the median period of culture was 52 days (range, 45–60). rIL-2 was administered at doses ranging between 6 and 18 × 10⁶ IU/m²/day. Toxicity was mild or moderate, and no life-threatening side effects were encountered. Two of 19 treated patients experienced complete responses of their metastatic sites (soft tissue), 10 had stable disease, and 7 showed progressive disease. The response rate was 11% (95% confidence interval, 2–35%).Conclusions: Outpatient treatment with TIL plus rIL-2 and rIFN-2a is feasible, although, within the context of the small sample size, the activity of the combination was no different from the reported activity of any of the components used alone.     

Significance of wet autoclave pretreatment in immunohistochemistry

Until recently the only way to rescue masked epitopes in routinely processed surgical pathological material was enzymatic digestion. The use of heat for antigen retrieval, first by microwave irradiation, represents an important breakthrough in immunohistochemistry. With the acceptance of microwave oven pretreatment, various modified techniques and alternative heating methods have also been proposed. Wet autoclave pretreatment for tissue proteolysis is a highly reliable alternative to the microwave antigen retrieval technique. It provides uniform heating of the slides, hence an even enhancement of staining intensity in a variety of formalin-sensitive antigens, and it also offers consistent interlaboratory results. The method has been introduced in routine diagnostic immunohistochemistry for the detection of estrogen-and progesterone receptors, L26-, Ki-67- and bcl-2 antigens and variable types of cytokeratins (1/5/10/11, 8, 13, 19). Experimentally, wet autoclaving can be used very successfully for the immunophenotyping of p53 and mdm2 expression, for the detection of adhesion molecules (CD44, integrins) and some anti-inflammatory molecules (annexins), among others. It has produced a substantial improvement in the visualisation of silver-stained nucleolar organizer regionsassociated proteins (AgNORs) in routine paraffin sections and along with modified silver staining and standardized AgNOR parameters assessed by image analysis. Wet autoclaving-based AgNOR staining has been proposed by a European multicentric study group as the standardized method for AgNOR analysis in archival material.     

Smoking among participants in the childhood cancer survivors cohort: the Partnership for Health Study.

PURPOSE: This article describes baseline data collection and the intervention design of Partnership for Health, a smoking cessation intervention for smokers in the Childhood Cancer Survivors Study. The purpose of this article is to evaluate demographic, psychosocial, and cancer-related factors that are associated with smoking behavior and mediators of smoking cessation. PATIENTS AND METHODS: This study includes 796 smokers from the Childhood Cancer Survivors Study database who were diagnosed with cancer before the age of 21, had survived at least 5 years, and were at least 18 years of age at the time of the baseline survey. Correlates of smoking behaviors included smoking rate, number of recent quit attempts, and nicotine dependence; two key mediators of smoking cessation, readiness to quit smoking and self-efficacy, were also assessed. RESULTS: Participants smoked, on average, 14 cigarettes/day; 53.2% were nicotine dependent, and 58% had made at least one quit attempt in the past year. Smoking behaviors were primarily associated with demographic variables; mediators of cessation were primarily associated with age at cancer diagnosis and perceived vulnerability to smoking-related illnesses. Severity of psychologic symptoms was associated with increased smoking rate, high nicotine dependence, and low self-efficacy. Support for quitting was related to smoking rate, number of quit attempts, readiness to quit smoking, and self-efficacy. CONCLUSION: These findings indicate that many cancer survivors who smoke are receptive to smoking cessation interventions. Factors related to mediators of smoking cessation might be particularly good targets for intervention.     

Glutamatergic dysfunction in OCD.

The role of glutamatergic dysfunction in the pathophysiology of OCD has hardly been explored despite recent reports implicating glutamatergic dysfunction in OCD. We decided to investigate CSF glutamate levels in adult OCD probands compared to psychiatrically normal controls. In total, 21 consenting psychotropic drug-na?ve adult OCD patients, diagnosed using SCID-IV-CV, and 18 consenting psychiatrically normal controls with age within 10 years of age of the patients, who did not have any history of head injury or neurological illness, were included into the study. Aseptically collected and stored CSF samples obtained from the patients and control subjects were used for glutamate estimation, which was carried out by a modification of the procedure described by Lund (1986). CSF glutamate (micromol/l) level was found to be significantly higher [F(1,31)=6.846, p=0.014] in OCD patients (47.12+/-4.25) compared to control subjects (41.36+/-3.63) on analysis of covariance. There was no effect of gender, age, duration of illness, Y-BOCS score, or CGI-S score on CSF glutamate levels. Our study provides preliminary evidence implicating glutamatergic excess in the pathophysiology of OCD, which needs to be further explored by studies from other centers involving larger sample sets from different age groups.     

The effects of the ketogenic diet in refractory partial seizures with reference to tuberous sclerosis.

PURPOSE: Tuberous sclerosis complex (OMIM 191100) is a multiorgan disease commonly associated with epilepsy refractory to anticonvulsants. Individual reports indicate that seizures in children with tuberous sclerosis might benefit from a ketogenic diet. We studied the effects of the diet introduced at 3.5 years of age in three boys with tuberous sclerosis and refractory partial seizures. METHODS: On admission a classical LCT ketogenic diet was initiated and patients were followed for 12 months. Antiepileptic drugs were maintained unless adverse effects required reduction. RESULTS: Two patients became seizure-free within 2 months on the diet. In the third patient drop attacks decreased significantly. On follow-up the diet was well accepted and without adverse effects. CONCLUSION: The ketogenic diet should be considered as a treatment option for children with tuberous sclerosis and partial seizures refractory to anticonvulsants. Our data support the need for further studies in larger cohorts to confirm the effectiveness of the ketogenic diet in this entity.     

Randomized trial of amifostine in locally advanced non-small-cell lung cancer patients receiving chemotherapy and hyperfractionated radiation: radiation therapy oncology group trial 98-01.

PURPOSE: To test the ability of the cytoprotectant, amifostine, to reduce chemoradiotherapy-induced esophagitis and evaluate its influence on quality of life (QOL) and swallowing symptoms. PATIENTS AND METHODS: A total of 243 patients with stage II to IIIA/B non-small-cell lung cancer received induction paclitaxel 225 mg/m(2) intravenously (IV) days 1 and 22 and carboplatin area under the curve (AUC) days 1 and 22, followed by concurrent weekly paclitaxel (50 mg/m(2) IV) and carboplatin (AUC 2), and hyperfractionated radiation therapy (69.6 Gy at 1.2 Gy bid). Patients were randomly assigned at registration to amifostine (AM) 500 mg IV four times per week or no AM during chemoradiotherapy. Beyond standard toxicity end points, physician dysphagia logs (PDLs), daily patient swallowing diaries, and QOL (EORTC QLQ-C30/LC-13) were also collected. Swallowing AUC analyses were calculated from patient diaries and PDLs. RESULTS: A total of 120 patients were randomly assigned to receive AM, and 122, to receive no AM (one patient was ineligible); 72% received AM per protocol or with a minor deviation. AM was associated with higher rates of acute nausea (P = .03), vomiting (P = .007), cardiovascular toxicity (P = .0001), and infection or febrile neutropenia (P = .03). The rate of >/= grade 3 esophagitis was 30% with AM versus 34% without AM (P = .9). Patient diaries demonstrated lower swallowing dysfunction AUC with amifostine (z test P = .025). QOL was not significantly different between the two arms, except for pain, which showed more clinically meaningful improvement and less deterioration at 6 weeks follow-up (v pretreatment) in the AM arm (P = .003). The median survival rates for both arms were comparable (AM, 17.3 v no AM, 17.9 months; P = .87). CONCLUSION: AM did not significantly reduce esophagitis >/= grade 3 in patients receiving hyperfractionated radiation and chemotherapy. However, patient self-assessments suggested a possible advantage to AM that is being explored with modified dosing route strategies.