Prognostic impact of baseline tumour immune infiltrate on disease-free survival in patients with completely resected, BRAFv600 mutation-positive melanoma receiving adjuvant vemurafenib.

BACKGROUND: We conducted a retrospective exploratory analysis to evaluate the effects of baseline tumour immune infiltrate on disease-free survival (DFS) outcomes in patients with fully resected stage IIC-IIIC melanoma receiving adjuvant vemurafenib monotherapy or placebo in the BRIM8 study. PATIENTS AND METHODS: BRIM8 was a phase III, international, double-blind, randomised, placebo-controlled study. Eligible patients with BRAFV600 mutation-positive, completely resected melanoma were randomly assigned to oral vemurafenib (960 mg twice daily) or matching placebo for 52 weeks. The primary end point was DFS. The association of CD8+ T-cell infiltration and programmed death ligand 1 (PD-L1) expression with DFS, as measured by immunohistochemistry, was explored retrospectively. RESULTS: Four hundred ninety-eight patients were randomly assigned to receive adjuvant vemurafenib (n = 250) or placebo (n = 248); tumour samples were available for biomarker analysis for approximately 60% of patients. In the pooled biomarker population, placebo-treated patients with <1% CD8+ T cells in the tumour centre had shorter median DFS than those with ≥1% CD8+ T cells (7.7 versus 47.8 months). DFS benefit from vemurafenib versus placebo was greater in patients with <1% CD8+ T cells [hazard ratio (HR) 0.56; 95% confidence interval (CI) 0.34-0.92) than in patients with ≥1% CD8+ T cells (HR 0.77; 95% CI 0.48-1.22). Likewise, median DFS was shorter among placebo-treated patients with <5% versus ≥5% PD-L1+ immune cells (IC) in the tumour (7.2 versus 47.8 months). A greater DFS benefit with vemurafenib versus placebo was observed in patients with <5% PD-L1+IC (HR 0.36; 95% CI 0.24-0.56) than in patients with ≥5% PD-L1+IC (HR 0.99; 95% CI 0.58-1.69). CONCLUSIONS: The presence of CD8+ T cells and PD-L1+IC are favourable prognostic factors for DFS. Treatment with adjuvant vemurafenib may overcome the poor DFS prognosis associated with low CD8+ T-cell count or PD-L1 expression. CLINICALTRIALS. GOV IDENTIFIER: NCT01667419.

HCV viraemia associates with NK cell activation and dysfunction in antiretroviral therapy-treated HIV/HCV-co-infected subjects.

The impact of hepatitis C virus (HCV) RNA levels on immune status in chronically HCV mono-infected when compared to HIV/HCV co-infected on antiretroviral therapy (ART) remains poorly understood. A total of 78 African American subjects HCV viraemic/naïve to HCV treatment (33 HCV genotype 1 mono-infected, 45 ART-treated HIV/HCV genotype 1 co-infected) were studied. Clinical and liver enzyme measurements were performed. Whole blood was analysed for immune subset changes by flow cytometry. Peripheral blood mononuclear cells (PBMC) were used for same-day constitutive and in vitro Interferon (IFN)-α-induced signal transducer and activator of transcription (STAT) phosphorylation, K562 target cell lysis and K562 target cell recognition-mediated IFN-γ production. Statistical analysis was performed using R (2.5.1) or JMP Pro 11. While both groups did not differ in the level of liver enzymes, HIV/HCV had higher T-cell activation/exhaustion, and constitutive STAT-1 phosphorylation compared to HCV. In contrast, CD4+ FoxP3+ CD25+ frequency, IFN-αR expression on NK cells, as well as constitutive and IFN-α-induced direct cytotoxicity were lower in HIV/HCV. Linear regression models further supported these results. Finally, increase in HCV viral load and CD4+ T-cell count had an opposite effect between the two groups on NK cell activity and T-cell activation, respectively. HCV viral load in ART-treated HIV/HCV co-infection was associated with greater immune activation/exhaustion and NK dysfunction than HCV viral load alone in HCV mono-infection. The more pronounced immune modulation noted in ART-treated HIV-co-infected/untreated HCV viraemic subjects may impact HCV disease progression and/or response to immunotherapy.

Monokines regulate glycosylation of acute-phase proteins.

The acute-phase response to inflammatory stimuli, characterized by increased synthesis of acute-phase proteins (APP), is often accompanied by changes in the glycosylation patterns of some of these proteins. While expression of APP genes in hepatocytes is regulated by monokines, mechanisms governing changes in glycosylation are not known. Exposure of human hepatoma cell line Hep 3B to conditioned medium from LPS-activated human monocytes and to medium from the keratocarcinoma cell line COLO-16 led to increased synthesis of alpha 1 proteinase-inhibitor and ceruloplasmin and to alterations of their glycosylation patterns similar to those seen in human serum in various inflammatory states. IL-1, tumor necrosis factor, and hepatocyte stimulating factor I increased synthesis of ceruloplasmin without alterations in the pattern of its glycosylation. These findings demonstrate that altered glycosylation seen in plasma in some inflammatory states can be explained by the effects of monokines on glycosylation in hepatocytes and that gene expression and glycosylation of some APP during the acute-phase response may be regulated by different mechanisms.

The importance of ceruloplasmin oxidase activity in patients with chronic lower limb atherosclerotic ischemia.

AIM: Inflammatory reactions accompanying ischemia increase the cytokine synthesis what leads to the increase in serum ceruloplasmin (Cp) concentration and activity. The aim of the study was to evaluate the association between serum Cp oxidase concentration and activity and the grade of lower extremity ischemia. Moreover, the correlation of Cp concentration and activity with the levels of interleukin 6 (IL-6), C-reacting protein (CRP), and a-1-acid glycoprotein (AGP) in serum was studied. METHODS: Two groups of patients were examined: 15 patients with moderate (MI) and 32 patients with critical ischemia (CI) of the lower extremities. Cp oxidase activity was measured spectrophotometrically, after incubation with o-dianisidine. The concentration of IL-6 was measured with the ELISA method, and Cp, CRP and AGP concentration by rocket immunoelectrophoresis. RESULTS: Significant increase in Cp oxidase concentration and activity was observed in patients with critical limb ischemia (median: 164.8 U/L), especially in patients with necrotic changes (median: 216.6 U/L). Cp oxidase activity was dependent on its concentration in patients with critical limb ischemia with necrotic changes (r=0.56; P<0.01). In patients with critical limb ischemia, the increase in Cp concentration and activity correlated significantly with CRP concentration (r=0.46; P=0.0007) (r=0.62; P=0.0001), respectively. CONCLUSION: Concentration and the oxidase activity of Cp depend of the degree of lower extremity ischemia and correlates with the major markers of inflammation, such as CRP. Critical limb ischemia induces the inflammatory reaction triggering the increase in IL-6 and of acute phase protein production. These processes lead to the increase in Cp oxidase activity dependent of Cp and CRP concentration.

A common variant of CDKN2A (p16) predisposes to breast cancer.

BACKGROUND: A common missense variant of the CDKN2A gene (A148T) predisposes to malignant melanoma in Poland. An association between malignant melanoma and breast cancer has been reported in several families with CDKN2A mutations, OBJECTIVE: To determine whether this variant also predisposes to breast cancer. METHODS: Genotyping was undertaken in 4209 cases of breast cancer, unselected for family history, from 18 hospitals throughout Poland and in 3000 controls. RESULTS: The odds ratio (OR) associated with the CDKN2A allele for women diagnosed with breast cancer before the age of 50 was 1.5 (p = 0.002) and after age 50 it was 1.3 (p = 0.2). The effect was particularly strong for patients diagnosed at or before the age of 30 (OR = 3.8; p = 0.0002). CONCLUSIONS: CDKN2A appears to be a low penetrance breast cancer susceptibility gene in Poland. The association should be confirmed in other populations.

Comparative studies of cervical spine anterior stabilization systems--Finite element analysis.

BACKGROUND: The object of the study was to assess the impact of one-level stabilization of the cervical spine for both anterior static and dynamic plates. Segments C2-C6 of the cervical spine, were investigated, from which was determined the stress and strain fields in the region of implantation and adjacent motion segments. The purpose was the comparison of changes that affect the individual stabilizers. METHODS: For testing we used finite element analysis. The cervical spine model takes into account local spondylodesis. The study includes both an intact anatomical model and a model with implant stabilization. FINDINGS: The analysis covered the model loaded with a moment of force for 1 Nm in the sagittal plane during movement. We compared both the modeled response of the whole fragment C2-C6 and the response of individual motion segments. The largest limitation of range of motion occurred after implantation with static plates. The study also showed that the introduction of the one-level stabilization resulted in an increase in stress in intervertebral disc endplates of adjacent segments. INTERPRETATION: The results indicate that the increase in stress caused by stiffening may result in disorders in remodeling of bone structures. The use of dynamic plates showed improved continuity strains in the tested spine, thereby causing remodeling most similar to the physiological state and reducing the stresses in adjacent segments.

Interleukin-6 and oncostatin M-induced growth inhibition of human A375 melanoma cells is STAT-dependent and involves upregulation of the cyclin-dependent kinase inhibitor p27/Kip1.

Interleukin-6 (IL-6)-type cytokines lead to growth arrest of human A375 melanoma cells. The present study demonstrates that this effect depends on the activation of STAT transcription factors. We observed a correlation between the extent of growth inhibition exerted by IL-6, IL-6 plus soluble IL-6 receptor or oncostatin M (OSM) and the intensities of STAT3 and STAT1 signals. A truncated chimeric receptor retaining only the membrane-proximal region of gp130, the common signal transducer of IL-6-type cytokines, did neither activate STATs nor mediate growth arrest of stable transfectants. These functions were restored by the addition of short STAT recruitment modules comprising critical tyrosine residues from gp130 (Y767, Y814). A receptor carrying tyrosine module Y759 of gp130 effectively mediated activation of the phosphatase SHP-2 but did not alter cell growth. Overexpression of dominant negative forms of STAT3 but not STAT1 abrogated the inhibitory effect of OSM and IL-6 in A375 cells. In addition, we have identified the cyclin-dependent kinase inhibitor p27/Kipl as a novel target to be regulated by IL-6-type cytokines. Stimulation-dependent upregulation of p27 mRNA occurred STAT3-dependently. Also p27 protein accumulated which coincided with the disappearance of hyperphosphorylated retinoblastoma protein in three human melanoma cell lines sensitive to IL-6-type cytokines.

The designer cytokine hyper-IL-6 mediates growth inhibition and GM-CSF-dependent rejection of B16 melanoma cells.

The low immunogenic B16 melanoma cell line was transfected with a mammalian expression vector containing the complementary DNA for a sIL-6R/IL-6 fusion protein, termed Hyper-IL-6 (H-IL-6), which was shown to have biological activities at 100-1000-fold lower concentrations than IL-6 in combination with sIL-6R. The secreted p84 glycoprotein was detected in the supernatant of transfected cells and was fully active on BAF3/gp130 cells, which respond to IL-6/sIL-6R but not to IL-6 alone. Administration of recombinant H-IL-6 to C57BL/6 mice resulted in a prolonged acute phase protein gene expression indicating long systemic persistence of the fusion protein. Transfected B16 cells (B16/H-IL6 cells) showed morphological alterations in combination with a dramatic growth inhibition in vitro. Subcutaneous injection in C57BL/6 mice resulted in an almost complete rejection of B16/H-IL6 cells. This effect was partially abolished in FVB/BL/6 mice transgenic for a GM-CSF receptor antagonist, indicating a GM-CSF-dependent rejection of H-IL-6 transfected B16 cells. These results demonstrate that the anti-tumor effect of cytokines like IL-6 which are secreted by transfected melanoma cells at least in part depends on GM-CSF activity.

Novel BRCA1 mutations and more frequent intron-20 alteration found among 236 women from Western Poland.

Three different novel BRCA1 mutations, five independent cases of the same 12 bp insertion-duplication in intron-20 and two novel rare BRCA1 sequence variants were identified among 122 Polish women with positive, in most cases moderate family history of breast and/or ovarian cancer, 80 controls and 34 unselected breast cancer tissue specimens. All mutations and variants were germline. The 4153 delA frameshift mutation, the Tyr105Cys missense mutation and two cases of the alteration in intron-20 were found in the group of healthy women with positive family history. Two other cases of the intronic insertion were found in unselected controls. Their carriers had no family history of breast or ovarian cancer but other cancers occurred in their families. The 1782 Trp/STOP nonsense mutation and one case of the insertion in intron-20 were first found in tissue specimens of breast cancer patient and breast/ovarian cancer patient, respectively. Their carriers also had no family history of breast or ovarian cancer. The distribution of the insertion in intron-20 in analysed groups and results of RT-PCR experiments suggest a less prominent role for this variant considered earlier a splicing mutation. This study shows also, that more population-oriented research is needed, involving women with less profound or even no family history of breast and ovarian cancer, to better understand the role and significance of different BRCA1 variants and mutations.

Acute phase proteins and transformed cells.

Acute phase proteins (APP) are plasma proteins whose concentration and glycosylation alters in response to tissue injury, inflammation, or tumor growth. Significant interspecies and sex differences in APP response exist. APP are produced mainly by hepatocytes, and their synthesis and glycosylation are controlled by a network consisting of cytokines, their soluble receptors, and glucocorticoids. The major cytokines involved in these processes belong to a group of interleukin-6-type cytokines that act through the hematopoietin receptor complex on hepatocytes and JAK-STAT signal transduction pathway. Transformed cells (hepatoma) display significant differences in synthesis of APP, cytokine responsiveness, expression of cytokine-receptor subunits and signal-transduction machinery. The most striking variability relates to the glycosylation alterations induced by cytokines. However, transformed cells (hepatoma) form a basic model for studying and understanding mechanisms controlling the synthesis and glycosylation of APP. Furthermore, APP may be secreted by transformed (tumor) cells of various origins and may display a growth factor-like function in certain cancer types.

Potentiatied antitumor effectiveness of combined chemo-immunotherapy with interleukin-12 and 5-fluorouracil of L1210 leukemia in vivo.

In this study we investigated the efficacy of a combination of IL-12 and 5-FU, a chemotherapeutic exerting several immunomodulatory effects, in murine L1210 leukemia. Mice inoculated with 1 x 10(5) leukemia cells were treated with a single dose of 5-FU (50 mg/kg) and seven daily doses of IL-12 (100 ng/dose), and were observed for survival. Treatment with IL-12 or 5-FU given alone produced moderate anti-leukemic effects. However, combination of both drugs resulted in a significant prolongation of mouse survival time. Importantly, there were 70% of long-term (>60 days) survivors among mice treated with both agents simultaneously. Moreover, we observed 100% of long-term survivors when mice were treated with a minimally increased dose of IL-12 (170 ng) in combination with 5-FU (50 mg/kg). The antileukemic effects were completely abrogated in scid/scid mice and in mice depleted of peritoneal macrophages and significantly decreased after administration of anti-CD3+, anti-CD4+ or anti-CD8+ monoclonal antibodies. Administration of anti-NK1.1 antibodies did not decrease the antileukemic effects indicating that NK cells are not important effectors of this treatment regimen. Collectively, these results indicate that the combination of IL-12 and 5-FU is inducing strong antileukemic responses that are dependent on the presence and activity of macrophages and T lymphocytes and warrant further studies of combined chemo-immunotherapy with IL-12.

BRCA2 germline mutations in male breast cancer patients in the Polish population.

Breast cancer is a rare disease in men. Germ-line mutations in BRCA2 and androgen receptor (AR) genes are thought to be responsible for a proportion of male breast cancer cases. The present study was performed on a series of 37 consenting patients not selected for family history of breast/ovarian cancer. The entire coding region of the BRCA2 gene and two exons of the AR gene were analyzed for germ-line mutations to evaluate the association between BRCA2 and AR genes and male breast cancer in Poland. We identified four frameshift mutations (11%) in exons 10, 11, 17 and 18, two of them were novel: 6495del3insC and 8457insA. Three missense unclassified variants (8%) of the BRCA2 gene were also identified. The frequencies of missense alterations were examined in a set of 200 chromosomes. No alteration of the AR gene was found. We did not observe much difference in clinicopathological features between carriers and non-carriers of BRCA2 mutations. Five of 37 patients (14%) had a family history of breast cancer, in one first- or second-degree relative, among the latter was one mutation carrier. The results of this study suggest that germ-line BRCA2 mutations account for rather small proportion of male breast cancer in Poland.

Leukemia inhibitory factor, interferon gamma and dexamethasone regulate N-glycosylation of alpha 1-protease inhibitor in human hepatoma cells.

Hepatocytes respond to inflammatory stimuli by changing the synthesis and N-glycosylation of acute phase plasma proteins (APP). So far, interleukin (IL) 6, transforming growth factor beta (TGF beta), tumor necrosis factor alpha (TNF) and IL-1 have been found to control N-glycosylation patterns of APP. Cytokines either increased (type I) or decreased (type II) the ratio of bi-relative to more branched N-glycans on APP. In this study, we describe the effect of leukemia inhibitory factor (LIF), interferon gamma (INF gamma) and dexamethasone (dex) on production of alpha 1-protease inhibitor (PI) and alpha 1-antichymotrypsin (ACT) and on glycosylation of PI in the human hepatoma cell line HepG2. Cytokines and dex were used separately and in various combinations including also IL-6 and TGF beta. Production of the antiproteases was quantitated by immunoelectrophoresis of the proteins accumulated in the culture medium. Glycosylation pattern of PI was assessed by crossed immunoaffinity electrophoresis (CIAE) with Concanavalin A (Con A) as a ligand. The production of ACT and PI was increased by LIF, decreased by INF gamma and unaffected by dex. LIF and INF gamma each like IL-6, decreased PI-Con A reactivity while dex like TGF beta enhanced PI-Con A reactivity. Combination of dex with LIF yielded additive effects while combination of dex with either INF gamma, L-6 or TGF beta acted synergistically on PI-Con A reactivity. Combinations of multiple cytokines and dex produced additive, inhibitory or synergistic effects. The type of glycosylation profile of PI secreted by HepG2 cells depended on the composition and amounts of interacting cytokines and dex.

Genetically modified tumour vaccines--where we are today.

Tumour vaccines are based on weakly immunogenic specific tumour antigens admixed with adjutants in order to elicit, restore or augment antitumour immune responses against residual or metastatic tumour cells. Cellular cytotoxicity is considered to play a major role in eliminating tumour cells. Activation of cellular toxicity requires at least three synergistic signals: presentation of specific tumour antigen, constimulatory signal (B7 molecules) and propagation signal (cytokines). Recently several HLA-restricted specific tumour antigens recognized by cytotoxic T-cells have been characterized. Antibody defined antigens, heat shock proteins and viral antigens are also discussed. First generation vaccines made of whole cancer cells or tumour-cell lysates together with non-specific adjutants produced about 20% of clinical responses and are currently tested in prospective clinical trials. Novel second generation of tumour vaccines employ genetically modified tumour cells, antigen presenting cells (dendritic cells) or recombinant tumour antigens. Tumour cells are modified with genes encoding molecules providing signals for cytotoxic T-cells required for recognition and killing of cancer cells such as B7 constimulatory molecules, HLA proteins and genes of different cytokines. Dendritic cells are modified with genes of specific tumour antigens in order to activate both helper and cytotoxic T-cells. Novel vaccines produce specific immune responses and objective clinical responses with minimal toxicity in phase I/II trials. Advances in gene transfer technology, tumour immunology and better methods of monitoring specific antitumour immune responses allow the hope that tumour vaccines will be introduced into the clinic, at least in some malignancies resistant to systemic therapy so far such as melanoma and renal cell carcinoma.

Soluble human interleukin-6-receptor modulates interleukin-6-dependent N-glycosylation of alpha 1-protease inhibitor secreted by HepG2 cells.

Interleukin-6 (IL-6) induces changes in gene expression and the N-glycosylation pattern of acute-phase proteins in hepatocytes. IL-6 exerts its action via a cell surface receptor complex consisting of an 80 kDa IL-6 binding protein (gp80) and a 130 kDa glycoprotein (gp130) involved in signal transduction. A genetically engineered gp80-derived soluble human IL-6-receptor (shIL-6-R) significantly enhanced the IL-6 effect on N-glycosylation changes (revealed by reactivity with the lectin-concanavalin A) of a1-protease inhibitor (PI) secreted by human hepatoma cells (HepG2). Stable transfection of IL-6-cDNA into HepG2 cells (HepG2-IL-6) resulting in constitutive secretion of 2 micrograms of IL-6 per 10(6) cells in 24 h led to a down-regulation of surface-bound gp80 and subsequent homologous desensitization of HepG2-IL-6 cells towards IL-6. Soluble human IL-6-R functionally substituted membrane-bound gp80 resulting in a reconstitution of responsiveness of HepG2-IL-6 cells.

Antitumor effects of the combination therapy with TNF-alpha gene-modified tumor cells and interleukin 12 in a melanoma model in mice.

In the present study, TNF-alpha gene-transduced B78 melanoma cells (B78/TNF) were used as a vaccine and combined with interleukin (IL)-12 in the treatment of B78 melanoma-bearing mice. The combined administration of genetically modified melanoma cells and IL-12 induced specific protective antitumor immunity resulting in a decreased rate of the tumor take following a rechallenge with parental B78 cells. When used therapeutically, intratumoral injections of irradiated B78/TNF melanoma cells and IL-12 exerted strong antitumor effects and led to complete regression of established tumors in 50% of mice. Injections of irradiated B78/TNF cells alone did not influence tumor development and IL-12 itself significantly delayed tumor growth but without curative effect. FACS analysis of parental B78 melanoma cells and its B78/TNF genetically modified variant showed that a proportion of cells of both cell lines expressed 87-1 (CD80) costimulatory molecule and that the expression of this molecule was increased during incubation with IFN-gamma. Moreover, IFN-gamma markedly augmented expression of major histocompatibility class (MHC) class I and II molecules on B78/TNF cells that were primarily MHC class I and II negative with no substantial effect on MHC-negative parental B78 melanoma. IFN-gamma also synergized in cytostatic/cytotoxic effects with TNF-alpha against B78 melanoma in vitro. Lymphocyte depletion studies in vivo showed reduction of the antitumor response in mice treated with anti - NK monoclonal antibodies (mAbs) as well as in mice treated with anti-CD4+ anti-CD8 mAbs. The results suggest that, when used therapeutically, IL-12 and a vaccine containing TNF-alpha gene-transduced tumor cells may reciprocally augment their overall antitumor effectiveness by facilitating development of systemic antitumor immunity and by stimulating local effector mechanisms of the tumor destruction.

Genetically modified tumour vaccines (GMTV) in melanoma clinical trials.

Since melanoma is a model immunogenic malignancy incurable in the disseminated phase of its natural course different immunotherapeutic approaches are tested in clinical trials. A number of tumour vaccines genetically modified (GMTV), with various immunostimulatory factors, are tested in phase I/II clinical trials. These factors include cytokines, tumour antigens (TA), costimulatory molecules or HLA antigens. We have designed a novel, mixed auto/allogeneic cellular melanoma vaccine modified with the IL-6 and the sIL-6R genes. Preclinical studies in a mouse model demonstrated that the IL-6/sIL-6R based vaccine is able to elicit efficient anti-tumour responses, mediated by CD8+ and NK cells, which resulted in inhibition of the tumour growth, metastases formation and prolonged survival of the animals treated. Irradiation of vaccine cells does not only lead to their sterilisation but also causes increased secretion of exogenous IL-6 and sIL-6R. Since January 1996 we have vaccinated more than one hundred metastatic melanoma patients. Promising clinical results (22% CR+PR, 32% SD) and the evidence of immune responses in the vaccinated patients have prompted us to design a phase III clinical trial which is to be open in 2000.

New protein and PCR markers RAK for diagnosis, prognosis and surgery guidance for breast cancer.

Breast cancer antigens RAK-p120, -p42, -p25 were detected in 100% of breast cancer cases tested (71 cases). Only 10% of adjacent tissue cases tested positive for all three cancer antigens, and 17.5% of the cases tested positive for two antigens only. Eighty-five percent of histologically normal breast tissue samples, isolated either from breast cancer patients or patients with advanced fibrocystic disease, tested RAK-negative, with the exception of low expression of p25, observed in some patients. Polymerase chain reaction (PCR) with HIV-1 gp 41-derived primers revealed cancer-associated DNA fragments of similar size (140 bp) as in HIV-1 genome. Fifty-four percent of cancer adjacent tissues, and 50% of malignancy-free breast tissue samples, tested PCR-negative. It is suggested that genetic predisposition to cancer may be associated with the presence of RAK genes, while expression of RAK antigens marks an already ongoing process of malignant changes.

Microheterogeneity forms of alpha-fetoprotein present in amniotic fluid.

The microheterogeneity forms of human alpha-fetoprotein present in amniotic fluid were studied in affinity immunoelectrophoresis using two lectins: concanavalin A and Lens culinaris agglutinin. Ninety-seven samples of amniotic fluid collected throughout gestation were obtained from pregnancies in which there was no fetal malformation. Two samples of amniotic fluid from pregnancies complicated by anencephaly and one sample obtained from an empty gestational sac were also studied. The changes in relative concentrations of AFP microheterogeneity forms during gestation are described. It is suggested that concanavalin A is suitable for the demonstration of malformations of the central nervous system in the first and second trimester of pregnancy while in the third trimester it would be of no value. The results of this study suggest that Lens culinaris agglutinin might be suitable for this purpose in the third trimester of gestation, but this needs to be confirmed in a larger study. The optimal concentrations of both lectins were investigated. In addition, a simple method of rocket affinity electrophoresis is also described. The possible mechanisms responsible for the changes in relative concentrations of microheterogeneity forms of alpha-fetoprotein during gestation and in pathological conditions are discussed.

Diagnostic evaluation of cancer antigens RAK .1. Cervical and ovarian cancer.

Cancer antigens RAK-p120, p42, and p25, which exhibit biological, immunological and molecular similarity to the proteins expressed by Human Immunodeficiency Virus 1 (HIV-1), were found in 47 of 47 tested cases of serous adenocarcinoma of the ovary, and 45 of 45 tested cases of squamous carcinoma of the cervix. Normal ovary and cervix did not express antigens RAK. High molecular weight protein (RAK-p160) was detected in the blood of over 61% of ovarian and 72% of cervical cancer patients, and in 14.3% of healthy women with family history of breast and/or gynecological cancer. Antigens RAK might represent new diagnostic markers.