Tropism of human herpesvirus 8 for peripheral blood lymphocytes in patients with Castleman's disease

Multicentric Castleman's disease (MCD), also called multicentric angiofollicular lymphoid hyperplasia, is a systemic lymphoproliferative disorder causing fever, lymphadenopathy and splenomegaly. Recently, Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8 (KSHV/HHV-8) DNA sequences have been detected in cases of MCD. We examined HHV-8 DNA sequences in the peripheral blood mononuclear cells (PBMCs) of two HIV-negative patients with MCD and in PBMCs and the lymph node of a HIV-negative patient with localized Castleman's disease (LCD) by the polymerase chain reaction. The novel sequences were detected in all DNA samples. Furthermore, the sequences were detected in only the CD19+ B-lymphocyte fraction of the patient with LCD as previously reported. However, the sequences were detected in CD19+ B-lymphocyte and CD2+ T-lymphocyte fractions of two patients with MCD. These results suggest that HHV-8 has tropisms for both B lymphocytes and T lymphocytes in Castleman's disease.     

A case of concomitant tuberculosis and sarcoidosis with mycobacterial DNA present in the sarcoid lesion

A 35-year-old Chinese woman initially presented with histologically and bacteriologically confirmed tuberculous lymphadenitis. She was also found to have thrombocytopenia, elevated serum alkaline phosphatase, and bilateral lung infiltrates. After 15 months of antituberculosis treatment, despite resolution of the cervical lymphadenopathy, she started to experience dyspnea. Chest radiograph appearance, thrombocyte count, and liver biochemistry had all deteriorated as well. Histologic findings from tissues obtained via transbronchial biopsy and open lung biopsy were consistent with sarcoidosis but also showed the presence of mycobacterial DNA by the polymerase chain reaction. She subsequently achieved a very good response clinically, radiographically, hematologically, and biochemically with 1-year of corticosteroid treatment for her sarcoidosis, and she remained relapse-free afterwards. The concomitant presence of tuberculosis and sarcoidosis in this patient together with the presence of mycobacterial DNA in the sarcoid lesion reiterate the possibility that mycobacteria or some of its components may be capable of inducing the immune response and the pathologic changes of sarcoidosis.     

Cytokine patterns in tuberculous and sarcoid granulomas: correlations with histopathologic features of the granulomatous response

Cytokines play an important role in granuloma formation, but the extent that cytokine profiles are similar in different granulomatous diseases and whether differences in the histopathologic features of the granulomatous response results from differences in cytokine production have not been evaluated. To investigate these questions, we used RT-PCR to quantify the expression of mRNAs coding for 16 cytokines in granulomatous lymph nodes from patients with tuberculosis and sarcoidosis and from control tissues, and we sought correlations between the level of expression of these cytokines and the histopathologic features of the granulomas. Expression of mRNAs coding for a number of cytokines (IL-1beta, IFN-gamma, TNF-alpha, granulocyte-macrophage (GM)-CSF, IL-12 (p40), and lymphotoxin-beta) was increased in tuberculous and sarcoid granulomas compared with that of control tissues. All sarcoid granulomas were shown to express a Th1 pattern of cytokine mRNAs, while tuberculous lymph nodes expressed either a Th1 or a Th0 profile. GM-CSF and lymphotoxin-beta mRNAs were more abundant in sarcoid than in tuberculous granulomas, whereas IL-8 mRNA was strongly expressed only in tuberculous lymph nodes. Strong expression of GM-CSF, TNF-alpha, and IL-8 by granulomas was shown to be correlated, respectively, with the presence of florid granulomatous lesions, the absence of central necrosis, and the presence of neutrophil infiltration. These results demonstrate that the formation of tuberculous and sarcoid granulomas in humans is associated with the expression of characteristic cytokine profiles and indicate that the expression of certain cytokines is associated with the development of specific pathologic features in the resulting granulomas.     

Frequent detection of human herpesvirus-6 sequences by polymerase chain reaction in paraffin-embedded lymph nodes from patients with angioimmunoblastic lymphadenopathy and angioimmunoblastic lymphadenopathy-like lymphoma

In search of a possible involvement of viral agents in angioimmunoblastic lymphadenopathy with dysproteinaemia (AILD) and AILD-like lymphoma (AILD-L), we studied the presence of human herpesvirus-6 (HHV-6) in the lymph node biopsies of 12 cases by polymerase chain reaction (PCR). Given the rarity of this lymphoproliferative disorder, we investigated archival specimens, consisting of formalin-fixed and paraffin-embedded tissues, obtained from patients with a clinical and histologic diagnosis of AILD and AILD-L. HHV-6 sequences were detected in the lymph node biopsies of 7 out of the 12 AILD and AILD-L cases examined. HHV-6 sequences were identified also in the involved liver and spleen tissues of one patient and in the PBMCs of two patients, all carrying viral sequences in the affected lymph nodes. We also used PCR to characterize the HHV-6 genomes, showing that two different viral strains are represented in the pathologic tissues. This study provides evidence of the presence of HHV-6 specific sequences in an unexpectedly high proportion of our series of AILD and AILD-L cases, suggesting a possible involvement of this lymphotropic virus in the pathogenesis of at least some cases of the disease.     

Chromosome breakpoint distribution in nonmelanoma skin cancers

Human herpesvirus 8 (HHV-8) DNA sequences were examined in peripheral blood mononuclear cell (PBMC) DNA samples of 56 children, 15 healthy adults, and 10 renal transplant patients by the polymerase chain reaction (PCR). The PCR amplification was carried out using the published KS330(233) primer pairs to amplify HHV-8 DNA sequences. The PCR-amplified products were confirmed by Southern blot hybridization with radiolabeled 233 bp HHV-8 DNA fragment, which was cloned and sequenced from the PCR-amplified product of Kaposi's sarcoma tissue. Six PCR-amplified product of four children and two renal transplant patients were cloned and sequenced. HHV-8 DNA sequences were detected in 36 of 56 (64%) normal children, in 12 of 15 (80%) healthy adults, and in all 10 renal transplant patients by Southern blot hybridization of PCR-amplified products. Six PCR-amplified products were confirmed by sequencing. These results suggest that HHV-8 infection is prevalent in the Japanese population with infection occurring in early childhood.     

Failure to detect the presence of Chlamydia pneumoniae in sarcoid pathology specimens

The pathogenesis of sarcoidosis is not yet known. On the basis of seroepidemiological data, an association between Chlamydia pneumoniae infection and sarcoidosis has been suggested, but so far no study has addressed the direct detection of this agent in the affected tissues. The aim of the present study was to detect C. pneumoniae deoxyribonucleic acid (DNA) within sarcoid tissue specimens by means of a two-step polymerase chain reaction. Lung biopsy specimens of 33 patients with histologically confirmed pulmonary sarcoidosis and 21 control lung biopsies or pathology specimens of patients with pulmonary carcinoma or emphysema were retrospectively analysed. A nested polymerase chain reaction was applied using two sets of primers designed to detect a fragment of the 16 strand ribosomal ribonucleic acid (rRNA) gene of C. pneumoniae. The results of the study failed to demonstrate the presence of C. pneumoniae in biopsy specimens of sarcoid tissue and in the control lung biopsies or pathology specimens. Our results, therefore, tend to rule out the possibility of a direct involvement of Chlamydia pneumoniae in the pathogenesis of sarcoidosis.     

Human herpesvirus-8: detection of novel herpesvirus-like DNA sequences in Kaposi's sarcoma and other lesions

Kaposi's sarcoma (KS) is a malignancy suspected of having an infectious etiology. Unique viral DNA sequences were recognized in KS lesions, using a novel technique that identifies small differences between two complex genomes. The virus had homology with the herpesvirus family, especially Epstein Barr virus (EBV), yet it was distinct from the known herpesviridae, and was appropriately named human herpesvirus 8 (HHV-8) or Kaposi's sarcoma-associated herpesvirus (KSHV). HHV-8 DNA sequences were present in AIDS-associated KS, classic KS, African endemic KS, Mediterranean KS, iatrogenic KS, and KS in homosexual men without HIV infection. HHV-8 DNA sequences were also present in peripheral blood mononuclear cells (PBMC) of KS+ patients; body-cavity-based lymphomas in HIV positive patients without KS; and in tissue from a number of malignant and non-malignant lesions in patients without HIV infection. The role of HHV-8 in KS and other malignancies is not known. Viruses are notoriously trophic for lesional tissue. Therefore, in order to determine the role of HHV-8 in KS pathogenesis, HHV-8 needs to be isolated and shown to induce immortalization in a suitable system. Regardless of its role in KS, another human herpesvirus has been discovered, and the extent of its pathogenicity needs to be uncovered.     

Involvement of the IP-10 chemokine in sarcoid granulomatous reactions

The accumulation of T cells and monocytes at sites of ongoing inflammation represents the earliest step in the series of events that lead to granuloma formation in sarcoidosis. In this study, we evaluated the pulmonary production of IFN-inducible protein 10 (IP-10), a CXC chemokine that stimulates the directional migration of activated T cells. Striking levels of IP-10 were demonstrated in the bronchoalveolar lavage (BAL) fluid of 24 patients with pulmonary sarcoidosis and lymphocytic alveolitis, as compared with patients with inactive disease or control subjects. A positive correlation was demonstrated between IP-10 levels and the number of sarcoid CD45R0+/CD4+ cells in the BAL. Immunochemistry, performed with an anti-human IP-10 polyclonal Ab in lymph nodes displaying prominent sarcoid granulomas, showed that cells bearing IP-10 were mainly epithelioid cells and CD68+ macrophages located inside granulomatous areas. Macrophages recovered from the BAL of sarcoid patients stained positive for IP-10 protein. Furthermore, alveolar macrophages isolated from sarcoid patients with T cell alveolitis and cultured for 24 h in presence of IFN-gamma secreted definite levels of IP-10 capable of inducing T cell chemiotaxis. Interestingly, alveolar lymphocytes recovered from patients with active sarcoidosis were CD4+ T cells expressing Th1 cytokines (IL-2 and IFN-gamma) and high levels of CXCR3. Taken together, these data suggest the potential role of IP-10 in regulating the migration and activation of T cells toward sites of sarcoid inflammatory process and the consequent granuloma formation.     

Human Herpesvirus-6 (HHV-6) infection in multiple sclerosis: a preliminary report

We examined cerebral spinal fluid (CSF) from multiple sclerosis (MS) patients and patients with other neurological diseases (OND) for antibody specific for Human Herpesvirus-6 (HHV-6) and for HHV-6 DNA detectable by PCR. CSF from MS patients had a higher frequency of IgG antibody to HHV-6 late antigens (39.4%) compared with CSF from OND (7.4%). In contrast, the frequency of detectable IgG antibody in CSF from MS patients specific for Epstein-Barr Virus (EBV) (12.1%) and Human Cytomegalovirus (HCMV) (6.1%) was much lower. Two of 12 MS CSFs (16.7%) also contained HHV-6 DNA detected by PCR. None of four OND CSF were positive for HHV-6 DNA. Plasma from 16 patients with MS, eight with OND and 72 healthy donors were tested for antibodies by ELISA to HHV-6 early (p41/38) and late (gp110) proteins. Although no differences in anti-gp110 IgG antibody were detected between MS patients, patients with other neurological diseases, and normals, IgG antibody to early protein p41/38 was detected in > 68% of the plasma from MS patients, 12.5% from OND patients and 27.8% of the controls. IgM antibody to p41/38 was present in > 56% of MS patients, 12.5% of OND patients, and 19% of controls. These data suggest that more than half of the MS patients had active, ongoing HHV-6 infections. HHV-6 was also isolated from peripheral blood mononuclear cells (PBMC) from 3/5 MS patients who were in relapse or had progressive disease and was identified as HHV-6 Variant B. These preliminary results support the hypothesis that HHV-6 may be a co-factor in the pathogenesis of some cases of MS.     

A methodologic issue for ophthalmic telemedicine: image quality and its effect on diagnostic accuracy and confidence

Sarcoid reactions in lymph nodes with or without metastasis from a primary malignant neoplasm are well-known. However, it is extremely rare to find these reactions associated with cutaneous solid tumors; only one such case has appeared in the literature. Here we describe a case of an 83-year-old man with cutaneous squamous cell carcinoma accompanied by sarcoid reactions and metastatic foci in the regional lymph nodes. The possibilities of systemic sarcoidosis and tuberculosis were excluded after extensive examinations specific for these diseases. Some authors regard the sarcoid reaction to be a sign of a good prognosis on the basis of studies of a few patients with solid tumors. In our case, however, the patient died of pulmonary metastasis with pleuritis carcinomatosa shortly after surgery. Systematic analysis of a sufficient number of cases should be carried out to evaluate the clinical significance of this type of reaction.     

A case of sarcoid reaction associated with papillary thyroid carcinoma

Sarcoid reaction, a granulomatous lesion similar to those seen in sarcoidosis, has been reported to be associated with various disorders. Here we describe a 54-year-old woman, who was diagnosed with sarcoid reaction associated with papillary carcinoma of the thyroid. Her history included total thyroidectomy with radical neck dissection for a papillary carcinoma of the thyroid. She was found to have a right subclavian mass. Dissection of the mass was performed for the diagnosis of metastatic papillary carcinoma to the lymph node, but the pathological examination showed granuloma without caseation as well as metastasis to the lymph node. Polymerase chain reaction (PCR) of the specimen excluded a possibility of Mycobacterium infection. There was no supporting evidence for systemic sarcoidosis in this patient; the patient showed no skin, eye, or lung lesions, or bilateral hilar lymphadenopathy, and she did not show increase in serum gamma-globulin or in plasma angiotensin-converting enzyme (ACE) levels, or increased CD4/CD8 ratio of lymphocytes obtained from bronchoalveolar lavage. These findings suggest that the present case had sarcoid reaction associated with papillary carcinoma. Although sarcoid reaction has been reported to be associated with various malignancies, only five cases, to our knowledge, are reported in the literature, which were associated with papillary carcinoma.     

Absence of detectable human herpesvirus 8 in the semen of human immunodeficiency virus-infected men without Kaposi's sarcoma

The prevalence of human herpesvirus 8 (HHV-8)/Kaposi's sarcoma (KS)-associated herpesvirus was investigated in the semen of 99 human immunodeficiency virus (HIV)-infected men (median CD4 cell count, 357/mm3) by use of a polymerase chain reaction (PCR) assay capable of detecting <10 copies of HHV-8 DNA. Of the subjects, 95 (96%) self-identified as men who have sex with men (MSM), and 3 had a history of clinical KS. Seminal cell specimens were negative for HHV-8 in 98 subjects. None of the 26 without KS (27.1% of 96 tested) who were seropositive for HHV-8 by IFA for latency-associated nuclear antigens had HHV-8 detected in their semen. The only subject with any evidence for seminal HHV-8 DNA was seropositive for HHV-8 and had active KS. HHV-8 was detected in 10 (10.4%) of 96 peripheral blood mononuclear cell specimens. The prevalence of HHV-8 DNA by PCR in semen of HIV-infected MSM without KS is low.     

Human herpesvirus 8 DNA load in leukocytes of human immunodeficiency virus-infected subjects: correlation with the presence of Kaposi's sarcoma and response to anticytomegalovirus therapy

Specific human herpesvirus 8 (HHV-8) DNA sequences were found in leukocytes of 12 of 29 (41.4%) AIDS subjects with Kaposi's sarcoma (KS), whereas they were found in 4 of 43 (9.3%) AIDS subjects without KS (P = 0.003), although the peak HHV-8 DNA load in PCR-positive subjects with KS (mean, 425 copies per 0.2 microgram of DNA) did not significantly differ from the one found in PCR-positive patients without KS (mean, 218 copies). The use of intravenous ganciclovir or foscarnet therapy to treat cytomegalovirus disease did not affect the HHV-8 DNA load in seven patients for whom serial samples were analyzed.     

Pathological studies on sarcoidosis autopsy. II. Early change, mode of progression and death pattern

Three hundred and twenty autopsy cases of sarcoidosis in Japan were analyzed to determine the pathological changes in the early stage, the mode of progression in each organ and the changes in the final stage of the disease. The lung and the mediastinal lymph nodes were affected in most of the cases, while the lesions were limited to the lung and intrathoracic nodes in some of the cases. It was suspected that early changes developed in the lung and in the hilar, and then in the mediastinal lymph nodes. The progression of sarcoid granulomas in the lung was classified into three patterns: (i) probably of a disseminated hematogenous nature; (ii) of an interstitial lymphogenous nature; and (iii) of a local expansive nature. These three patterns were observed also in the heart. In the brain, perivascular granuloma formation was a prominent feature. In the other organs in which sarcoid lesions were not malignant nor disseminated and conglomerated, no interstitial patterns were observed. In chronic cases, repeated dissemination and particularly the interstitial spread of granulomatous changes led to a prominent interstitial fibrosis and dysfunction of the organs, finally resulting in death of the individual. In such long-standing cases, the mediastinal nodes deteriorated by hyalinous degeneration of the granulomas, and many active granulomas were formed in the intra-abdominal or body surface lymph nodes. These lymph nodes were likely to continue supplying sensitized lymphocytes to the whole body. A persistence of active change in the lymph nodes and the lymphogenous spread of granulomas in organs would appear to be key factors in the prognosis of sarcoidosis.     

Selection of T lymphocytes bearing limited TCR-Vbeta regions in the lung of hypersensitivity pneumonitis and sarcoidosis

Hypersensitivity pneumonitis (HP) and sarcoidosis are interstitial lung disorders (ILD) characterized by a lymphocytic alveolitis that, in the active phase of the disease, is sustained by different T-cell subsets, i.e., CD8+ cells in HP and CD4+ lymphocytes in sarcoid patients. To address the question of whether a bias in T-cell selection occurs in the lung of patients with HP and sarcoidosis, we analyzed the T-cell receptor beta chain variable region (TCR-Vbeta) repertoire by flow cytometry and polymerase chain reaction (PCR) analyses in blood and lung lymphocytes of 14 HP and 25 sarcoid patients. To verify whether these cells can be activated in vitro through the TCR, blood and lung lymphocytes were also assessed for their responsiveness to different superantigenic stimuli represented by staphylococcal enterotoxins, including SEA, SEB, SEC1, SEC2, SED, and SEE. Flow cytometry and PCR analyses demonstrated an overexpression of cells bearing Vbeta2, Vbeta3, Vbeta5, Vbeta6, and Vbeta8 gene segments in the lung of HP patients as compared with the peripheral blood. In sarcoid patients cells bearing Vbeta2, Vbeta5, and Vbeta6 gene segments in the lung of HP patients as compared with the peripheral blood. In sarcoid patients cells bearing Vbeta2, Vbeta5, and Vbeta6 gene segments were overrepresented in the lung rather than in the blood. Both in HP and sarcoid patients almost all T cells bearing the dominant Vbeta segment belonged to the T-cell subset that sustains the alveolitis, i.e., CD8 in HP patients and CD4 in sarcoid subjects. Follow-up studies demonstrated that the recovery of the alveolitis was characterized by the disappearance of cells bearing a limited T-cell repertoire. Interestingly, T-lymphocyte response to different superantigens demonstrated that the proliferation elicited by different staphylococcal toxins was more pronounced in the lung than in the blood. Taken together, our findings indicate a compartmentalization of cells bearing discrete Vbeta gene products in the pulmonary microenvironment and suggest that the expansion of specific Vbeta region subsets occurring in the lung might result from triggering by a specific antigen. In fact, the removal from exposure in HP patients or specific treatment in sarcoidosis resulted in the decrease of the overrepresented cell population accounting for the lymphocytic alveolitis.     

Human herpesvirus 8 infection occurs following adolescence in the United States

Most recent evidence suggests that human herpesvirus 8 (HHV-8) infection is restricted to persons with Kaposi's sarcoma (KS) or to persons who may subsequently develop KS. To accurately determine the prevalence of infection in the United States, children and adults with AIDS were examined for evidence of HHV-8 infection to see whether HHV-8 (like other herpesviruses) would be readily detected in immunosuppressed persons. By use of nested polymerase chain reaction, DNA specific for HHV-8, Epstein-Barr virus, and cytomegalovirus was detected in blood leukocytes from 0, 26 (51%), and 9 (18%), respectively, of 51 children. Similarly, HHV-8-specific antibodies were not detected in analyses of sera from the children. By contrast, HHV-8 DNA was detected in 9 (27%) of 33 adult AIDS patients without KS. These findings suggest that the pattern of transmission of HHV-8 in the United States differs from that of other herpesviruses in that primary infection occurs predominantly in adults.     

HHV-8 infection is specific for cell lines derived from primary effusion (body cavity-based) lymphomas

Human herpes virus 8 (HHV-8; or KSHV, Kaposi's sarcoma-associated herpes virus) is a gamma herpes virus with sequence homology to Epstein-Barr virus (EBV). It was first isolated from Kaposi's sarcoma tumor cells and subsequently from tumor cells and peripheral blood mononuclear cells from patients with primary effusion lymphomas (PEL; or body cavity-based lymphomas). PEL has been recognized as an individual nosologic entity based on its distinctive biological-pathological features and its consistent infection with HHV-8 (commonly, but not always co-infected with EBV), occurring predominantly in human immunodeficiency virus (HIV)-infected patients but occasionally also in HIV-negative cases. Whether HHV-8 sequences can be found also in non-hematopoietic tumor cells other than Kaposi's sarcoma and in malignant hematopoietic malignancies other than PEL, has been the focus of the present studies. We examined the presence of HHV-8 sequences by polymerase chain reaction (PCR) using (1) a panel of 133 human cell lines established from a large variety of solid tumors; (2) a spectrum of 114 hematopoietic cell lines derived from the different cell lineages including 50 B cell leukemia/lymphoma-derived cell lines and seven cell lines established from patients with PEL. Besides the seven PEL cell lines, 46 cell lines that were derived from malignant pleural effusion or ascitic fluid material (25 non-hematopoietic and 21 hematopoietic cell lines) were examined. Except for the seven PEL cell lines that were strongly HHV-8+ in the PCR, all solid tumor cell lines and all hematopoietic cell lines scored consistently negative for the presence of HHV-8 sequences. These results confirm the absolute specificity of HHV-8 infection (within the hematopoietic malignancies) for PEL. PEL cell lines represent useful tools for the analysis of the biology of this neoplasm and of the pathogenetic role of the virus in the disease development.     

Human herpesvirus 7 in patients with pityriasis rosea. Electron microscopy investigations and polymerase chain reaction in mononuclear cells, plasma and skin

BACKGROUND: Clinical evidence suggests a viral etiology for pityriasis rosea (PR). OBJECTIVE: To evaluate human herpesvirus (HHV)-6 and HHV-7 as candidates for the etiology of PR. METHODS: Blood and skin tissue from 12 patients with acute PR, and 12 patients with other dermatoses were studied, as well as blood samples from 25 healthy persons. Serum interferon (IFN)-alpha and IFN-gamma were analyzed by ELISA. Analysis of morphological changes in cocultured peripheral blood mononuclear cells (PBMC) and electron microscopy (EM) to identify viral particles were performed. Polymerase chain reaction (PCR) with specific primers for HHV-6 and HHV-7 DNA sequences was performed on the plasma and PBMC of patients and healthy controls and on the skin of patients with PR and other skin diseases. RESULTS: PR plasma contained detectable IFN-alpha and IFN-gamma, whereas plasma from controls did not. PBMC from PR patients showed ballooning cells and syncytia after 7 days in culture whereas PBMC from controls and recovered PR patients did not. This cytopathic effect was also documented in a PR patient who relapsed and in Sup-T1 cell cultures inoculated with the cell-free supernatant from centrifuged cultured PBMC; in this supernatant, herpesvirus, virions were detected by EM, PCR identified HHV-7 DNA in PBMC, plasma and skin from all patients with active PR and in the PBMC only of 5 patients tested 10-14 months later. Weaker signals of HHV-7 DNA were detected in PBMC of 11 controls, but not in their plasma. Skin was negative for HHV-7 in all control specimens. CONCLUSIONS: Although the detection of HHV-7 DNA in PBMC and tissues does not prove directly a causal role, HHV-7 DNA in cell-free plasma corresponds to active replication which supports a causal relationship. We propose that PR is a clinical presentation of HHV-7 reactivation.     

Mutations in exon 7 and 8 of p53 as poor prognostic factors in patients with non-small cell lung cancer

This study was performed to clarify the different effects of each mutant exon of p53 as indicators of a poor prognosis in patients with non-small cell lung cancer (NSCLC). Tumor tissues of 204 patients with NSCLC were analysed; 96 tumors were stage I, 22 stage II, and 86 stage III. DNA was extracted from frozen specimens and polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and direct sequencing were performed to investigate mutations of p53 from exon 5 to exon 8. Seventy-five patients with NSCLC (36.8%) had mutations in p53 which included 72 cases of missense mutations and three cases of non-missense mutations. The overall survival rate of patients with mutant p53 adenocarcinomas was strikingly worse than that of patients whose tumors had wild-type p53 (35.7% vs 53.8%; P=0.041), but no significant difference in survival was found in the patients with NSCLC and squamous cell carcinoma. Mutations in exon 5 of p53 occurred in 33 cases (16.2%), mutation in exon 6 was detected in only one case (0.5%), mutations in exon 7 in 20 cases (9.8%), and mutations in exon 8 in 18 cases (8.8%). The overall survival rate of patients with mutations in exon 7 was worse than that of patients with wild-type p53 in NSCLCs and adenocarcinomas (42.9% vs 56.0%; P=0.025 and 33.3% vs 53.8%; P=0.048, respectively), whereas the overall survival of patients with mutations in exon 5 was almost the same as that of patients with wild-type p53. In addition, the overall survival rate of patients with mutations in exon 8 was strikingly worse than that of patients with wild-type p53 in NSCLCs, adenocarcinomas and squamous cell carcinomas (22.9% vs 56.0%; P<0.001, 19.0% vs 53.8%; P=0.004 and 33.3% vs 62.5%; P=0.042, respectively). Multivariate analysis with the Cox regression model of patients with NSCLC, adenocarcinoma and squamous cell carcinoma indicated that mutations in exon 8 were best correlated with the overall survival rate, followed by lymph node status (P<0.001, P=0.015 and P=0.006, respectively), and mutations in exon 7 of NSCLC were also revealed to have good correlation, followed by lymph node status and mutations in exon 8 (P=0.031). Mutation of p53 was a poor prognostic factor for adenocarcinoma as described previously. Moreover, mutations in exon 8 were more useful indicators of prognosis not only for adenocarcinoma but also for NSCLC. Worse overall survival of the patients with mutations in exon 8 of p53 was suggested to be associated with codon 273 mutations as well as mutations between codon 280 and 285 included into the H2 alpha helix corresponding to residues 278-286. These results suggested that abnormal conformation of H2 alpha helix might play an important role not only in the loss of normal function but also in the acquisition of tumorigenesis. Investigation of mutations in exon 8, especially codon 273 mutation and mutant H2 alpha helix was considered to be a clinically useful approach for determining the prognosis of patients with NSCLC.