Epidemiological, clinical and viral determinants of the increased prevalence of high-risk human papillomavirus (HPV) infections in elderly women.

BACKGROUND: Population-based studies have reported a second peak of human papillomavirus (HPV) prevalence among women > 55 years, but reasons for this U-shaped HPV prevalence curve are poorly understood. OBJECTIVES: To analyse determinants of high-risk HPV (HR-HPV) infections among postmenopausal women. STUDY DESIGN AND METHODS: A cohort of 3,187 women was stratified into three age categories: i) youngest age group < 25 years (n = 1.103); ii) women between 26-55 years (n = 2.004), and iii) women > 55 years (n = 80), analysed for epidemiological, clinical and virological determinants of their HR-HPV infections. Real-time PCR was used for HPV genotyping, analysis of viral loads for HPV16, 18/45, 31, 33/52/58, 35 and 39, and load of integrated HPV16. RESULTS: Age-standardised prevalence of HR-HPV infections showed a second peak among women > 55 years, with a perfect U-shaped curve (R2 = 0.966). The factors explaining this increased HR-HPV prevalence among older women include: i) cohort effect, ii) higher viral loads for HR-HPV types with cubic model curve (R2 = 0.714) for HPV 16, iii) distinct shift (p = 0.0001) from multiple-type infections to single HR-HPV types, iv) transition from episomal to integrated HPV16 (p = 0.009), v) higher load of integrated HPV16 (p = 0.009), and, vi) higher proportion of incident infections, higher rate of viral persistence, and lower rate of HR-HPV clearance. CONCLUSIONS: These data suggest that in women who fail to eradicate their HR-HPV infection until menopause, selection of integrated viral clone has taken place, driving the process towards progressing disease. Consequent to this, most of the HR-HPV infections in women > 55 years were associated with high-grade CIN or invasive carcinoma.

Thymic hyperplasia and lung carcinomas in a line of mice transgenic for keratin 5-driven HPV16 E6/E7 oncogenes.

Human Papillomavirus type 16 (HPV-16) is the cause of both benign lesions and ano-genital cancers. In HPV-associated cancers the transforming properties of the expressed viral E6 and E7 proteins have been revealed by a number of different assays. We have generated transgenic mice expressing HPV-16 E6/E7 genes under the control of the murine keratin 5 gene promoter, which should confer cell-type specific expression in the basal cells of squamous stratified epithelia. Transgenic mice developed thymic hyperplasia and lung neoplasia with 100% frequency, the thymus showing a size increase at 2 months and reaching the maximum dimension at 6 months, when lung carcinomas appeared. After this time the size of hyperplastic thymi decreased, while malignant formations invaded the mediastinal area. Hepatic metastasis could be also observed in some of the animals at the autopsy and death invariably occurred around 10-11 months of age.

Clearance of high-risk human papillomavirus (HPV) DNA and PAP smear abnormalities in a cohort of women subjected to HPV screening in the New Independent States of the former Soviet Union (the NIS cohort study).

BACKGROUND: We analysed the temporal relationships of the clearance of human papillomavirus (HPV) DNA and cytological abnormalities in women participating in a screening study in three NIS countries. METHODS: The 274 patients included in this analysis were prospectively followed-up for 21.6 months (range: 0.5-42.9). All 274 women had abnormal PAP test (ASC-US or higher) and high-risk HPV-positive test (HCII) at baseline. Two groups were compared: 132 women who cleared both tests (Group 1), and 142 women who cleared either HPV or abnormal PAP test (Group 2). The first clearance during the follow-up, and the last visit clearance were modeled using life-table techniques, and the predictive factors were analysed using univariate (Kaplan-Meier) and multivariate (Cox) survival analysis. RESULTS: There was no difference in the mean clearance time for the abnormal PAP test (14.4 months; 0.7-40.5 and 12.6 months; 0.5-35.0) and high-risk HPV DNA (12.67 months; 0.6-33.5 and 10.8 months; 0.7-33.4) in Group 1 and Group 2 (Mann-Whitney: P = 0.107 and P = 0.082, respectively). Clearance times for HPV DNA and abnormal PAP test did not deviate from each other in either groups (Wilcoxon: P = 0.063 and P = 0.088). The monthly clearance rates for the abnormal PAP test are 1.32 and 1.38%, and those for the HPV DNA 1.62 and 1.61%, in Groups 1 and 2, respectively. Of the factors predicting the last visit clearance, the issues related to smoking are of particular interest. CONCLUSIONS: The clearance of high-risk HPV type and abnormal PAP test shows a close temporal relationship, the former preceding the latter, however, by an interval of 1.0-2.0 months.

Factors predicting persistence of high-risk human papillomavirus (HPV) infections in women prospectively followed-up in three New Independent States (NIS) of the former Soviet Union.

BACKGROUND: We completed an analysis of the factors predicting the persistence of high risk (HR) HPV infections in women participating in a multicenter screening trial in three NIS countries. METHODS: The 543 baseline HR HPV-positive women included in this analysis are derived from a sub-cohort of 887 women who were prospectively followed-up for a mean of 21.6 months (range: 0.5-42.9) as a part of a multi-center screening study in three NIS countries (the NIS cohort study; n = 3,187 women). Of these 543 women, 273 showed persistent HR-HPV in serial Hybrid Capture II (HCII) testing during the follow-up (Group 1), whereas 270 women cleared their infection (Group 2). These two groups were compared with their epidemiological, clinical, and virological data (HCII, PCR) to disclose the factors predicting persistent HR-HPV infection. RESULTS: Women with persistent HR-HPV infections were significantly younger (27.3 yrs) than those who cleared their infection (29.1 yrs) (p = 0.006), and their follow-up time was shorter; 14.1 and 21 months, respectively (p = 0.0001). Both variables were treated as confounders in the multivariate analyses. Of the 66 recorded epidemiological variables, only being a current smoker proved to be an independent predictor (OR 1.693; 95% CI 1.114-2.573; p=0.014). Baseline colposcopy, biopsy or Pap smear did not predict HPV persistence, whereas an incident or persistent abnormal Pap during the follow-up were independent predictors in a multivariate model (p = 0.005), together with the high viral load (HCII RLU/CO at 100 pg/ml cut-off), and HR HPV positive PCR test (p = 0.0001). When all significant variables were entered in the regression model, only the follow-up time (OR 0.950, 95% CI 0.924-0.976; p = 0.0001) and HR-HPV positive PCR (OR 4.169, 95% CI 1.741-9.987; p = 0.001), remained independent predictors. CONCLUSIONS: While several factors were related to HR-HPV persistence in univariate analysis and when adjusted for age and follow-up time as confounders, the only independent predictors in the multivariate regression model were follow-up time and HR-HPV positive PCR. Clearly more data are needed on type-specific persistence and HPV integration as its predictors.

Identification of Hürthle cell tumor by single-injection, double-phase scintigraphy with technetium-99m-sestamibi.

UNLABELLED: Early and late (double-phase) scintigraphy with 99mTc-MIBI was used in a comparative study of the scintigraphic aspects of Hürthle cell tumors and other thyroid tumors. METHODS: Single-injection, dual-phase (15-30 min and 3-4 hr) thyroid scintigraphy with 99mTc-sestamibi (MIBI) was performed on 41 patients who displayed a cold nodule on previous 99mTc scintigraphy. Visual scoring of nodular uptake was done to compare thyroidal and background tracer uptake. In addition, the nodular-to-thyroid (N/T) uptake ratio in the early and late images and the washout rate from the nodule (WON) and thyroidal tissue (WOT) were measured. Cytologic results were obtained for all patients; histopathologic results were obtained for the 20 patients who had surgery. RESULTS: In eight patients (Group A), the nodule displayed intense and persistent uptake of MIBI (N/T = 1.77 +/- 0.46 and 3.20 +/- 1.37; WON = 17.2% +/- 6.3%; WOT = 24.6% +/- 7.5%); histopathology revealed Hürthle cell tumors (two carcinomas and three adenomas) in five surgical patients. In 15 patients (Group B), the nodule displayed intense uptake in the early image with fading activity in the late image (N/T = 1.45 +/- 0.54 and 0.84 +/- 0.30; WON = 30.0% +/- 7.3%; WOT = 24.5% +/- 6.8%); histopathology revealed a colloid nodule (n = 1), papillary carcinoma (n = 4) and follicular carcinoma (n = 5) in 10 surgical patients. In the remaining 18 patients (Group C), the nodule was cold and late images were not acquired. Histopathology revealed colloid nodules (n = 2) and follicular adenoma (n = 3) in five surgical patients. CONCLUSION: Single-injection, dual-phase MIBI scintigraphy of the thyroid can identify Hürthle cell tumors because these tumors have intense, persistent tracer uptake in contrast to other thyroid tumors.

Hürthle cell tumor dwelling in hot thyroid nodules: preoperative detection with technetium-99m-MIBI dual-phase scintigraphy.

UNLABELLED: Single injection dual-phase scintigraphy (early and late acquisitions) with 99mTc-MIBI was used to differentiate benign and malignant hot thyroid nodules. METHODS: Thirteen euthyroid and two hyperthyroid patients displaying a hot thyroid nodule on the 99mTc scan due to an autonomously functioning thyroid nodule (AFTN) underwent early (15-30 min) and late (3-4 hr) thyroid scintigraphy after the administration of 740-1000 MBq 99mTc-MIBI. Visual scoring was done to assess nodular tracer uptake and retention. In addition, the nodular-to-thyroid (N/T) uptake ratio in the early and late image and the washout rates (WO) from the nodule and thyroidal tissue were measured. All patients underwent thyroid surgery. RESULTS: Histopathology revealed a Hürthle cell tumor in three nodules, a benign adenoma with oxyphilic metaplasia in two nodules and a benign adenoma without oxyphilic cells in the remaining 10 nodules. The Hürthle cell tumor nodules displayed intense and persistent uptake of 99mTc-MIBI (N/T was 2.81 +/- 0.52 and 5.53 +/- 1.06 in early and late images, respectively; WO from the nodule was 12.33 +/- 0.47, WO from the thyroidal tissue was 22.00 +/- 3.56). The benign nodules showed intense uptake in the early image and intense uptake to absent retention in the late image (N/T was 2.94 +/- 1.31 and 1.62 +/- 0.50 in the early and late images, respectively; WO from the nodule was 20.25 +/- 2.92, WO from the thyroidal tissue was 20.33 +/- 2.92). CONCLUSION: Single injection dual-phase 99mTc-MIBI scintigraphy of the thyroid with AFTN can identify nodules as a result of the activity of a Hürthle cell tumor, since these tumors cause intense and persistent tracer uptake in contrast with a benign AFTN.

Identification of urocortin mRNA and peptide in the human endometrium.

Urocortin is a 40-amino acid peptide belonging to the corticotropin-releasing factor (CRF) family. In human reproductive tissues, urocortin expression has been previously demonstrated in the ovary, in the placenta and fetal membranes and in pregnant uterine tissues, while no data are available on the expression of the peptide in the nonpregnant uterus. In this study, urocortin expression was evaluated by both immunohistochemistry and reverse transcription-polymerase chain reaction, in human uterine tissues and cells at different phases of the menstrual cycle. Urocortin was immunolocalized in endometrial epithelial and stromal cells, as well as in the myometrium, and in vascular smooth muscle cells. No differences between proliferative and secretory phase were observed. These results were confirmed by reverse transcription-polymerase chain reaction analysis of isolated endometrial epithelial and stromal cells, and myometrial specimens. These findings open new questions on the roles played by urocortin in the human uterus.

Cytokines in the viviparous reproduction of squamate reptiles: interleukin-1 alpha (IL-1 alpha) and IL-1 beta in placental structures of a skink.

Placental viviparity is known in many species of squamate reptiles. Among these, some scincids have developed an epithelio-chorial chorio-allantoic placenta which in the structure of its central ridged zone is similar to those of certain therian mammalian species. A broad range of immunoregulatory peptides, cytokines, has been identified at the maternofetal interface of several species of mammals, either with invasive or non-invasive types of placenta. Thus we began to study whether interleukin-1, which is considered to play a crucial role in mammalian pregnancy, might also be involved in the viviparity of reptilian species. Placentae of Chalcides chalcides L. were processed by immunohistochemistry and incubated in a culture medium for different times. A very strong immunoreactivity for interleukin-1 alpha (IL-1 alpha) and for interleukin-1 beta (IL-1 beta) was present in the chorial epiblast and in uterine epithelial cells, with varying degree and localization in different periods of pregnancy. IL-1 beta was also released into the medium at different amounts during incubation. In light of the mammalian data, our results suggest that the role of cytokines in pregnancy may represent a significant event in the evolution of placental viviparity.

11Beta-hydroxysteroid dehydrogenase expression in first trimester human trophoblasts.

This study evaluated the levels and the enzymatic characteristics of 11beta-hydroxysteroid dehydrogenase activity (11beta-HSD) of chorionic villi isolated from first trimester human placenta. The results demonstrated a predominant expression of the NAD-dependent dehydrogenase isoform (11beta-HSD2) over the NADP-dependent oxoreductase (11beta-HSD1). Thus, in tissue homogenates exogenous NAD increased the conversion of corticosterone to 11-dehydrocorticosterone of about 14-fold while NADP was ineffective. There was no conversion of 11-dehydrocorticosterone to corticosterone either with NADH or NADPH demonstrating the lack of reductase activity. In keeping with these results, RT-PCR analysis indicated a mRNA for 11beta-HSD2 in villous tissue while 11beta-HSD1 mRNA levels were undetectable. In addition, immunohistochemical staining localized the 11beta-HSD2 protein to syncytiotrophoblasts and cell columns of the chorionic villi. These results suggest roles for the trophoblast-associated 11beta-HSD2 oxidative activity in modulating the exposure of the embryo to active glucocorticoids in the early gestation and in regulating trophoblasts invasion of the uterine wall.

Identification of monoclonal B-cell populations in lymphoid infiltrates of lacrimal gland by rapid cycle polymerase chain-reaction.

Lymphoid hyperplasia of lacrimal gland may be difficult to be differentiated from lymphomas on the basis of morphology and immunohistochemistry. The results of this study indicate that polymerase chain reaction should be employed for confirming the diagnosis of lymphoma in cases with histological and immunophenotypical characteristics of lymphomas, and for detecting monoclonal lymphoid cells in an otherwise non-lymphomatous but dubious or borderline morphological context.

First trimester human trophoblast expresses both interferon-gamma and interferon-gamma-receptor.

Interferon-gamma (IFN-gamma) is a lymphokine, produced by activated T lymphocytes, which plays a key regulatory role in the host immunological responses. In addition, IFN-gamma is expressed by human and porcine trophoblast. As IFN-gamma exerts its biological functions through specific cell surface receptors and a great number of IFN-gamma receptors (IFN-gamma R) have been purified from human placenta, we have examined the relative distribution of IFN-gamma and IFN-gamma R in human placental tissues at different stages of pregnancy. By using immunohistochemical analysis and monoclonal antibodies, it was found that IFN-gamma expression is intense in the first trimester but almost imperceptible at term, whereas the expression of IFN-gamma R is present at both stages of pregnancy. For both lymphokine and receptor, the most intense expression was observed in villous syncytiotrophoblast and in extravillous interstitial trophoblast. From these results it appears that the expression of IFN-gamma R in trophoblast is related to the presence of the lymphokine in the early phase of gestation but not later. On this basis, it can be argued that IFN-gamma exerts its functional role via an autocrine and/or a paracrine loop mainly during the first trimester.

Progestin regulation of 11beta-hydroxysteroid dehydrogenase expression in T-47D human breast cancer cells.

This study examined the enzymatic characteristics and steroid regulation of the glucocorticoid-metabolizing enzyme 11beta-hydroxysteroid dehydrogenase (11beta-HSD) in the human breast cancer cell line T-47D. In cell homogenates, exogenous NAD significantly increased the conversion of corticosterone to 11-dehydrocorticosterone, while NADP was ineffective. There was no conversion of 11-dehydrocorticosterone to corticosterone either with NADH or NADPH demonstrating the lack of reductase activity. In keeping with these results, RT-PCR analysis indicated a mRNA for 11beta-HSD2 in T-47D cells, while 11beta-HSD1 mRNA levels were undetectable. In T-47D cells treated for 24 h with medroxyprogesterone acetate (MPA), 11beta-HSD catalytic activity was elevated 11-fold, while estrone (E(1)), estradiol (E(2)) and the synthetic glucocorticoid dexamethasone (DEX) were ineffective. The antiprogestin mifepristone (RU486) acted as a pure antagonist of the progestin-enhanced 11beta-HSD activity, but did not exert any agonistic effects of its own. In addition, RT-PCR analysis demonstrated that MPA was a potent inducer of 11beta-HSD2 gene expression, increasing the steady-state levels of 11beta-HSD2 mRNA. Taken together, these results demonstrate that 11beta-HSD2 is the 11beta-HSD isoform expressed by T-47D cells under steady-state conditions and suggest the existence of a previously undocumented mechanism of action of progestins in breast cancer cells.

Patients with different lung cancers show normal expression of fra(3)(p14.2) in aphidicolin-treated lymphocyte cultures.

Among common fragile sites, fra(3)(p14.2) is the most expressed either spontaneously or after treatment with aphidicolin (APC) in lymphocyte cultures. Because recurrent chromosomal abnormalities involving the short arm of chromosome 3 in tumor tissue are present in various malignancies, including lung cancer, the induction of fra(3)(p14.2) elicited by APC was investigated with the aim of detecting possible interindividual polymorphism in its expression that might be relevant to predisposition toward cancer-related events. Thirty-four patients affected with various lung cancers (14 squamous cell carcinomas, 13 adenocarcinomas, and seven small cell carcinomas) and 14 controls (patients undergoing routine routine follow-up after coronary by-pass) were included in this study. The frequency of fra(3)(p14.2) expression was not significantly different among the patients grouped either by disease or by sex and age. It was estimated that fra(3)(p14.2) accounts for about 20% of total breakage in APC-treated lymphocyte cultures from the general population.

Pathogenesis of posterior capsular opacification. Part II: Histopathological and in vitro culture findings.

The most interesting sources of information about the pathogenesis of posterior capsular opacification seem to be histopathological studies and in vitro tissue cultures. Since our surgical technique is extracapsular cataract extraction, the explants we used for tissue culture consisted of the anterior capsule epithelial sheet without the equatorial germinative zone. We successfully overcame several problems by using the autologous plasma clot culture method. This medium, considered the optimal one for this type of culture, allowed us to study the heterogeneous behavior of the epithelial cells in culture. Using the plasma clot culture method, we were able to demonstrate in vitro fibroblastic transformation of the epithelial cells. Histopathological findings of particular cases of posterior capsule opacification and immunohistochemistry of the human lens are also reported.

Histopathological study of iron deposit distribution in the rheumatoid synovium.

A qualitative and semi-quantitative evaluation of synovial iron deposits in 20 patients with rheumatoid arthritis (RA) and in 12 patients presenting with degenerative and traumatic joint disease was carried out. Ferric iron deposits, abundant and preferentially distributed in the superficial and deeper connective tissue layers in the RA patients, were more limited and prevalently sited in the synovial lining layer in the controls. These results further underline the increase in synovial iron stores found in active RA and the role played by iron deposits in sustaining inflammation, as has been reported in the literature.

Evaluation of HPV, CMV, HSV and EBV in esophageal squamous cell carcinomas from a high-incidence area of China.

BACKGROUND: Certain viruses, notably human papillomavirus (HPV), cytomegalovirus (CMV), herpes simplex virus (HSV) and Epstein-Barr virus (EBV), are known to produce tumors in animals and cell transformation in vitro and they have been implicated in the pathogenesis of human cancers. All these viruses are also known to infect the esophagus. This study was aimed to determine whether these viruses play any causal role in the etiology of esophageal squamous cell carcinoma. MATERIALS AND METHODS: A series of 103 esophageal squamous cell carcinomas derived from patients in the high-incidence area of northern China were analyzed by DNA in situ hybridization and polymerase chain reaction (PCR) for the presence of HPV DNA sequences and, using immunohistochemistry, for the demonstration of CMV, HSV and EBV infections. RESULTS: Six (5.8%) of the 103 tumors were found to contain HPV 16, 18 or 30 DNA sequences. HPV types 6, 11 and 53 were not detected in any of the cases. Amplified HPV DNA sequences were found in 17 out of 101 (16.8%) carcinoma specimens by PCR with L1 consensus primers. None of the 103 carcinomas tested was immunohistochemically positive for CMV, HSV or EBV. CONCLUSION: Our results confirmed the HPV involvement in esophageal carcinomas and provided further evidence to support a causal association of HPV infection with esophageal squamous cell carcinoma. However, the three herpesviruses, CMV, HSV and EBV, are highly unlikely to be involved in the pathogenesis of this malignancy in the high-incidence area of China.

Quantitative image analysis of oesophageal squamous cell carcinoma from the high-incidence area of China, with special reference to tumour progression and papillomavirus (HPV) involvement.

BACKGROUND: Despite much research effort, the major prognostic factor of oesophageal squamous cell cancer (ESCC) remains the pathological stage of the disease as defined by the TNM classification, whereas tumour grading is of limited value in this respect, mainly due to its low reproducibility. A better means for disease prognostication based on improved understanding of the pathogenetic mechanisms is urgently required. MATERIALS AND METHODS: Among the cohort of 700 ESCC patients from the high-incidence area of China, previously subjected to extensive testing for Human papillomavirus (HPV) involvement by in situ hybridization (ISH) and PCR, a group of 273 patients was randomly selected for analysis of the primary tumour, adjacent mucosa and regional lymph nodes, by histopathology and quantitative image analysis. All these and the HPV data were subjected to extensive univariate and multivariate analysis to disclose independent predictors of progressive disease. RESULTS: For the analyses, the tumours were graded into two categories: well-moderately and poorly-differentiated. HPV DNA was detected in 116 (18.9%) of the carcinomas by ISH and in 15.2% by PCR. In univariate analysis, lymph node status (considered as the surrogate marker of progressive disease) was significantly (p < 0.01) predicted by the following nuclear parameters: nuclear area, G0/G1 ratio, HPV DNA status, integrated optical density (IOD), mean optical density (MOD) and S-Phase. In multivariate (stepwise backward LR) analysis, 6 variables remained as independent predictors of disease progression (at p < 0.05 level), the three most significant ones being nuclear perimeter, nuclear roundness and equivalent diameter (p < 0.01). CONCLUSION: A series of quantitatively measured nuclear parameters seem to bear a close correlation with ESCC differentiation and progression in univariate analysis and some of these variables proved to be significant independent predictors of disease progression in multivariate modelling as well. These data clearly advocate the use of quantitative image analysis in searching for additional prognostic factors of ESCC.

Cytokeratin expression patterns as an indicator of tumour progression in oesophageal squamous cell carcinoma.

Recent studies point out that cytokeratins (CKs) are involved in dynamic cell remodeling during cancer progression and particularly, CK expression patterns have been associated with invasion and metastasis. In oesophageal squamous cell cancer (ESCC), lymph node (LNN) metastasis is an important step in disease progression, invariably associated with an ominous prognosis. To assess whether specific CK expression patterns could represent reliable markers of tumor progression, a series of 111 ESCCs (59 lymph node-positive, 52-negative) derived from the high- incidence area of Linxian (Northern China), were subjected to immunohistochemical (IHC) analysis with an extensive panel of CK antibodies. Statistically significant differences were observed for CK18 (p=0.01), CK19 (p=0.04) and PKK1 (p=0.02) expression between the LNN-negative and LNN-positive ESCCs. Furthermore, significant correlation between specific CK distribution pattern and progressive disease (i.e., LNN metastasis) was evidenced. The results suggest that CK8, CK18 and CK19 expression and distribution pattern could be of predictive value as a marker of disease progression as defined by the appearance of lymph node metastases in oesophageal squamous cell cancer.

Neuronal intranuclear inclusion disease: neuropathologic study of a case.

We report neuropathological findings in a 22-year-old man affected with neuronal intranuclear inclusion disease. The inclusions affected to different extents the various structures of the central nervous system, being more numerous in cerebral cortex, inferior olives, hypoglossal and oculomotor nuclei. They ultrastructurally differed from Marinesco bodies. In the neurons of the substantia nigra, we occasionally observed intranuclear inclusions resembling the so-called rodlets of Roncoroni. We did not observe inclusions in the extraneuronal tissues. There was no apparent correlation between frequency of the inclusions and neuronal loss. Intranuclear inclusions were found in many morphologically normal neurons. We suggest that the intranuclear inclusions are the marker of a distinctive disorder, even though their role in neuronal degeneration remains to be clarified.

Expression of phosphorylated and non-phosphorylated neurofilament subunits and cytokeratins in neuroendocrine lung tumors.

In this study antibodies specific for different intermediate-sized proteins (cytokeratins and neurofilaments) have been tested on a series of neuroendocrine (NE) lung tumors in order to evaluate their diagnostic validity. In particular we used a panel of polyclonal anti-neurofilament 200-kilodalton subunits whose reactivity against phospho-dependent epitopes was known. At least one NF subunit was constantly present and in all cases coexpression of cytokeratins and neurofilaments was confirmed. However, in cases of carcinoid tumor (CT) the results were homogeneous, while the cases of small cell lung carcinoma (SCLC) showed a much wider range of immunostaining. Our investigation confirms the hypothesis that the phosphorylation state is a significant determinant of immunohistochemical properties of neurofilaments. This might explain the large number of negative results obtained in previous investigations on NE tumors. The phosphorylation of neurofilaments may also be considered an indication of the degree of differentiation of the tumor.