Laparoscopic total fallopian tube removal at the time of bilateral salpingo-oophorectomy in BRCA2 positive women.

About 10% of all serous ovarian cancer has BRCA1 and/or BRCA2 mutations. Recent data showed that following the SEE FIM protocol it is possible to evidence more fimbriae cancers. Due to those studies, fallopian tube cancer in recent years has become the predominant site of cancer in BRCA1 and/or 2 mutation carriers. The pathological study of the fallopian tube is not complete during salpingo-oophorectomy because a small part (intramural site) is situated inside the uterus. In this case report we demonstrate how it is possible to remove the tubes entirely for pathological analysis without hysterectomy by laparoscopic surgery.

Prothymosin alpha 1 effects, in vitro, on the antitumor activity and cytokine production of blood monocytes from colorectal tumor patients.

Recent animal studies demonstrate that prothymosin alpha 1 (ProT alpha) enhances the antitumor response by stimulation of mononuclear phagocyte functions. The present study was aimed at characterizing the in vitro effects by ProT alpha on blood monocytes from human colon cancer patients. Purified peripheral blood monocytes were studied in terms of tumor cytostatic ability and cytokine production after incubation with ProT alpha or interferon (rIFN-gamma) and transforming growth factor-beta (TGF beta), used as reference substances. SW620 colon carcinoma cells were used as tumor target cells in growth inhibition experiments. The level of baseline growth inhibitory activity of unstimulated patient's monocytes was significantly lower than that of normal monocytes. The defective antitumor activity of patient monocytes was associated with a higher production of the inhibitory monokines prostaglandin E2 (PGE2) and TGF beta. The stimulation of monocytes by ProT alpha and/or rIFN-gamma elevated the average antitumor activity in all donor groups. The ProT alpha-induced increase was associated with a significantly higher monocytic secretion of IL-1 beta and TNF-alpha. Moreover, the concentrations of TGF beta and PGE2 in the culture supernatants decreased significantly, when patient's monocytes were treated with ProT alpha and/or rIFN-gamma. Additionally, ProT alpha enhanced the diminished antitumor activity of TGF beta-treated normal monocytes. These results suggest that ProT alpha selectively regulates distinct functions of blood monocytes, the effect of this cytokine varying with the parameter and donor population examined. These data provide a rational and biological endpoint for further studies with ProT alpha as an activator of mononuclear function in colon cancer.

Interleukin-2-activated killer cell activity in colorectal tumor patients: evaluation of in vitro effects by prothymosin alpha1.

The effects of prothymosin alpha1 (Pro alpha1) in combination with interleukin-2 (IL-2) on peripheral blood lymphocytes from 50 colorectal tumor patients at different stages were studied with respect to immunocytotoxicity, adhesion to cultured SW620 colon carcinoma cells, secretion of cytokines and expression of adhesion and surface marker molecules. On average, the patients showed lower natural killer (NK) cell activity than healthy donors, which was associated with a lower adhesion capacity to the tumor target cells. The NK cell activity of the patients was inversely related to the tumor stage. The generation of lymphokine(IL-2)-activated killer (LAK) cell activity was found to be comparable on lymphocytes from healthy individuals and patients and was not correlated to tumor stage. Pro alpha1 stimulated patients' LAK cell activity only, primarily at the early stage (Dukes A/B). The Pro alpha1 effect was associated with an increased adhesion of lymphocytes to tumor target cells and an increased secretion of the deficient IL-2-induced IFN gamma secretion. No significant effects on the low level of TNF alpha secretion was noted. By flow cytometry, Pro alpha1 in combination with IL-2 augmented the expression of the NK cell markers CD56, CD16/56, the subset CD3/16/56 and CD25 on lymphocytes of the patients. In contrast, Pro alpha1 was equally effective by increasing the expression of CD18 and CD11a on lymphocytes from the patients and from normal controls. In conclusion, Pro alpha1, in combination with IL-2, can partially normalize lymphocyte deficiencies of colon cancer patients in vitro. This potential might provide an experimental basis for applying Pro alpha1 or related thymic peptides in selected immunotherapies against colorectal tumors.

Preclinical studies with prothymosin alpha1 on mononuclear cells from tumor patients.

The immunomodulating potential of the thymic protein prothymosin alpha1 (Pro alpha1) on the lymphocyte and monocyte directed antitumor reactions of melanoma and colorectal tumor patients in comparison with healthy controls were studied in vitro. On average, tumor patients showed lower NK- and LAK-cell activities than healthy controls, being associated with a lower adhesion capacity to tumor target cells. The NK-cell activity of the tumor patients was inversely related to the tumor stage. Pro alpha1 stimulated the impaired patients LAK-cell activity only at an early stage of disease. The Pro alpha1 effects were associated with an increased adhesion of lymphocytes to tumor target cells and an increased secretion of deficient IFN-gamma and IL-2 secretion. By flow cytometry, Pro alpha1 in combination with IL-2 increased the NK-cell marker CD56, CD16/56 and CD25 as well as CD18/11a adhesion molecule expression. Monocytes from tumor patients showed deranged tumoristatic activities compared with healthy controls. Pro alpha1 elevated the mean of the antitumor activity, when applied alone or in combination with rIFN-gamma. In the presence of IFN-gamma, Pro alpha1 stimulated the adhesion of monocytes to cultured tumor cells, mainly by increasing CD54 expression. Pro alpha1 stimulated alone or in combination with IFN-gamma the TNF-alpha and IL-1beta secretion by monocytes and decreased the high PGE2 and TGF-beta level, especially in the patients groups. Perspectively, this may provide the basis for applying Pro alpha1 in selected antitumor immunotherapy protocols.

Prothymosin alpha 1 modulates lymphokine-activated killer cell activity and IL-2 production by peripheral blood lymphocytes from melanoma patients in vitro.

The effects of prothymosin alpha 1 (Pro alpha 1) on the natural killer (NK), lymphokine (IL-2)-activated killer (LAK) cell activity and the phytohaemagglutinin (PHA)-induced IL-2 secretion of peripheral blood T-lymphocytes (PBL) from 34 malignant melanoma patients of all clinical stages were studied in vitro. On average, melanoma patients showed lower NK and LAK cell activities than healthy donors. In particular, patients with metastases revealed an impaired NK cell activity. However, individuals showed a broad range of LAK cell sensitivity to Pro alpha 1 depending, among other factors, on the disease stage. LAK cell activities were not correlated to tumour stage. Patients' impaired LAK cell activity could be restored by Pro alpha 1. Only patients at stage II (regional metastases) responded to Pro alpha 1. The IL-2 secretion from PBL melanoma and healthy donors did not differ, Pro alpha 1 administration was without any significant effect. However, stage III (distant metastases) PBL expressed significantly lower IL-2 levels, compared to stage I (primary tumours). The highest IL-2 levels was found to be associated with tumour stage II. Pro alpha 1 enhanced the IL-2 secretion from stage I PBL. Therefore Pro alpha 1 administration abrogated the defective LAK cell activity and IL-2 secretion of PBL, mainly from patients at early melanoma stages.

The influence of the thymic preparation thymex-L on deficient antitumor-activity of monocytes from melanoma patients in-vitro.

Recently we demonstrated that, in vitro, prothymosin alpha 1 (ProT alpha), a polypeptide from calf thymus, was able to enhance the deranged tumoristatic activity of peripheral blood monocytes from melanoma patients. Now we report, that the thymic preparation Thymex-L significantly enhanced the level of depressed monocyte activity from 19% to 26%, whereas in normal donor groups no significant change of basal activity (35%) was seen. Although the improvement of median levels of killer cell activity was found to be independent from the disease stage, the Thymex-L effect was only statistically significant in stage I and melanoma patients after chemotherapy. In contrast to ProT alpha, Thymex-L did not further enhance monocyte-mediated cytotoxicity when it was applied in combination with rIFN-gamma. However, after stimulation with rIFN-gamma, the median level of TNF-alpha secretion by melanoma monocytes significantly increased (about 2-fold) when preincubated with Thymex-L. These results indicate that depressed monocyte functions in selected melanoma patients may be partially improved by Thymex-L.

Thymosin alpha 1 effects, in vitro, on lymphokine-activated killer cells from patients with primary immunodeficiencies: preliminary results.

In patients with primary immunodeficiencies the role of natural killer (NK)- and lymphokine (IL-2)-activated killer (LAK)-cells is not yet satisfactorily established. Using a clonogenic assay with K562 leukemia target cells, we studied their NK- and LAK-cell activity in vitro. Moreover, the effect of thymosin alpha 1 (T alpha 1) on LAK-cell activity was studied in 11 patients with different immunodeficiencies. The results were compared with data of healthy controls (n = 11) and cord blood samples (n = 6). Common variable immunodeficiency patients demonstrated a mean LAK-cell activity of about 65% of normal controls and cord blood samples. The moderately reduced LAK-cell activity was not affected by T alpha 1. In the immunodeficient other patients, low levels of LAK-cell activity with a mean value of 10% of normal controls were seen. The mean LAK-cell activity could be improved by T alpha 1: three patients showed an improvement of their LAK-cell activity up to 25-30% after T alpha 1 administration in vitro, but in one case T alpha 1 was without any effect. Analysis of the expression of the surface markers CD8, CD16, CD57 and CD8/CD57 revealed that only CD16 positive lymphocytes were significantly less in immunodeficient patients. We found a linear correlation between LAK-cell activity and CD8/CD57 double positive lymphocytes in all patients. Our results demonstrate that suppressed LAK-cell activity from immunodeficient patients can be individually improved by T alpha 1. Further in vivo studies should evaluate thymic peptide immunotherapy for individual immunodeficient patients.

Prothymosin alpha augments deficient antitumor activity of monocytes from melanoma patients in vitro.

Human peripheral blood monocytes derived from normal donors, melanoma patients (MP) before and after chemotherapy (MPa) were assayed for their capacity to inhibit SK-MEL-28 melanoma cell growth in vitro; in addition, growth modulating effects by prothymosin alpha 1 (ProTa) were studied. After preincubation with or without ProTa for 1 day, monocytes were cultured in the absence or presence of interferon-gamma (rIFN-gamma) for a further day, and after cocultivation with SK-MEL-28 melanoma cells for 3 days monocyte/macrophage-mediated tumoristatic activity was determined employing the microculture tetrazolium (MTT) assay. The level of baseline growth inhibitory activity of unstimulated MP and MPa monocytes was 22% and 15%, respectively, and was significantly lower (p < 0.001) than that of normal monocytes (35%). The stimulation of monocytes/macrophages by rIFN-gamma greatly elevated the mean of their antitumor activity up to 44%, 49% and 58% in the group of MP, MPa and normal donors, respectively. ProTa significantly increased the level of monocyte-mediated growth inhibition of MP and normal donors, when it was applied alone or in combination with rIFN-gamma. Monocytes of MP at early stages (I and II) of their disease, when incubated with rIFN-gamma, showed a higher increase in tumoristatic activity than at stage III and tended to be the most susceptible to preincubation with ProTa followed by rIFN-gamma activation. However, on average tumoristatic activity of MP/MPa monocytes was significantly lower compared with that of normal monocytes, when activated with rIFN-gamma or ProTa alone or combined. Moreover, no effects on TNF-alpha secretion of MP/MPa monocytes were found by ProTa and/or rIFN-gamma, whereas TNF-alpha levels from normal monocytes were significantly increased by the two stimuli. These results indicate that monocyte disorders in melanoma patients may be partially normalized by ProTa.

Bromelain proteinase-f9 augments human lymphocyte-mediated growth-inhibition of various tumor-cells in-vitro.

Bromelain, a crude extract from pineapple stem containing various thiol proteases, has previously been suggested for adjuvant therapy of malignant diseases. We hence tested in vitro whether a highly purified bromelain proteinase (F9) would affect the antitumor activity by human peripheral blood lymphocytes (PBL) against MCF-7 breast cancer, KB squamous carcinoma and SK-MEL-28 melanoma cells. The antiproliferative effects by pretreated PBL were determined using the microculture tetrazolium (MTT) assay. All three cell lines were susceptible to F9-treated PBL and KB cells were selected to examine the kinetics, the dose dependency and the specificity of the F9 effects on PBL. Maximal antitumor effects were obtained when PBL were incubated with 25 mug/ml of F9 for 3 days at which the proteolytical activity of the added F9 was 1.6 U/mg. The F9-induced PBL antitumor activity was dependent on the applied proteolytical activity and abolished when F9 was inactivated by iodoacetamide. In contrast to F9, trypsin or pronase were not able to induce PBL-mediated growth inhibition of KB target cells. In response to F9, the concentration of interleukin-2 (IL-2) and tumor necrosis factor-a increased 10 and 19 fold in the PBL supernatant, respectively. F9 was found to synergize LAK cell activity in addition to suboptimal concentrations (0.625-2.5 U/ml) of rIL-2. In contrast to rIL-2-activated PBL, no cytolytic effect by F9-activated PBL was measured in the BCECF release assay, suggesting that F9 acts by a mechanism different from that of IL-2. F9 was also found to be growth inhibitory in the MTT assay, when it was directly added to the tumor cells: The concentration, at 50% growth inhibition by F9, was in the range of 25-38 mug/ml at which the proteolytical activity of the added F9 was 2.5 U/mg. On the basis of the present study we suggest that F9 alone, or in combination with rIL-2, may be used as a potential biological response modifier in specific immunotherapy of distinct cancer diseases.

A semiautomatic microassay to measure human tumor clonogenic growth-invitro.

A semi-automatic micromethod was developed using 96-well microtiter plates in combination with a video monitoring system to assay human tumor clonogenic growth in vitro. The conditions to follow the growth of K562 human myeloid colonies were optimized and validated: About 250 cells in a 40 mul agar mixture are to be seeded per well. The colony number is proportional to the number of seeded cells. The cloning efficiency is about 75% and correlates well with the established glass capillary method. The system precision of the automatic colony counter, expressed by the coefficient of variation, is less than 2%. The mean of the intra- and inter assay coefficient of variation is low (8.5% and 7%, respectively). The microassay was applied to measure the lymphokine-activated killer (LAK)-activity of peripheral blood mononuclear cells (PBMC) from healthy donors against K562 target cells and to examine the effects of various thymic hormone preparations on the cytotoxicity of generated LAK cells. Thus, the new microassay provides a useful tool for measuring clonogenic tumor cell growth and its modulation by different agents on many samples in a short time.