Impact of Prosigna test on adjuvant treatment decision in lymph node-negative early breast cancer-a prospective national multicentre study (EMIT-1).

BACKGROUND: EMIT-1 is a national, observational, single-arm trial designed to assess the value of the Prosigna, Prediction Analysis of Microarray using the 50 gene classifier (PAM50)/Risk of Recurrence (ROR), test as a routine diagnostic tool, examining its impact on adjuvant treatment decisions, clinical outcomes, side-effects and cost-effectiveness. Here we present the impact on treatment decisions. PATIENTS AND METHODS: Patients with hormone receptor-positive, human epidermal growth factor receptor 2-negative pT1-pT2 lymph node-negative early breast cancer (EBC) were included. The Prosigna test and standard histopathology assessments were carried out. Clinicians' treatment decisions were recorded before (pre-Prosigna) and after (post-Prosigna) the Prosigna test results were disclosed. RESULTS: Of 2217 patients included, 2178 had conclusive Prosigna results. The pre-Prosigna treatment decisions were: no systemic treatment (NT) in 27% of patients, endocrine treatment alone (ET) in 38% and chemotherapy (CT) followed by ET (CT + ET) in 35%. Post-Prosigna treatment decisions were 25% NT, 51% ET and 24% CT + ET, respectively. Adjuvant treatment changed in 28% of patients, including 21% change in CT use. Among patients assigned to CT + ET pre-Prosigna, 45% were de-escalated to ET post-Prosigna. Of patients assigned to ET, 12% were escalated to CT + ET and 8% were de-escalated to NT; of those assigned to NT, 18% were escalated to ET/CT + ET. CT was more frequently recommended for patients aged ≤50 years. In the subgroup with pT1c-pT2 G2 and intermediate Ki67 (0.5-1.5× local laboratory median Ki67 score), the pre-Prosigna CT treatment decision varied widely across hospitals (3%-51%). Post-Prosigna, the variability of CT use was markedly reduced (8%-24%). The correlation between Ki67 and ROR score within this subgroup was poor (r = 0.25-0.39). The median ROR score increased by increasing histological grade, but the ROR score ranges were wide (for G1 0-79, G2 0-90, G3 16-94). CONCLUSION: The Prosigna test result changed adjuvant treatment decisions in all EBC clinical risk groups, markedly decreased the CT use for patients categorized as higher clinical risk pre-Prosigna and reduced treatment decision discrepancies between hospitals.

[Salivary gland drainage into the thyroglossal duct]. / Spyttkjerteldrenasje til ductus thyreoglossus.

BACKGROUND: Failure in regression of the thyroglossal duct is one of the most common reasons for midline swellings in the neck. Several authors have described recurrent thyroglossal duct remnants with persisting draining sinuses. However, few have described accessory salivary glands that drain into the thyroglossal duct. MATERIAL AND METHODS: In this article we report two such cases with midline salivary glands in the floor of the mouth. RESULTS: These two patients were subsequently successfully treated with radical tissue resection in the area between the hyoid bone and foramen cecum. INTERPRETATION: Preoperative fistulography or sinography was useful to demonstrate the ductal ramification of the salivary glands, and use of methylene blue during surgery proved of significant value for the result.

Differential expression of bcl-2 homologs in human CD34(+) hematopoietic progenitor cells induced to differentiate into erythroid or granulocytic cells.

The Bcl-2 family of proteins has been shown to play a central role in the regulation of apoptosis. We have examined the expression of several Bcl-2 homologs upon stimulation of CD34(+) human hematopoietic progenitor cells. CD34(+) cells were induced to differentiate into predominantly erythroid cells in the presence of erythropoietin (Epo) and stem cell factor (SCF), while the addition of G-CSF and SCF led to differentiation predominantly into granulocytic cells, as demonstrated by immunophenotyping and morphological examination of cultured cells. In Epo- and SCF-stimulated cells, we found a marked increase in the level of Bcl-x(L) protein expression and downregulation of Bax expression, apparent from day 4 and more pronounced on days 8 and 21. In contrast, Bcl-x(L) protein expression was downregulated in G-CSF- and SCF-stimulated cells compared with cells cultured in medium alone, whereas there was no sign of change in the level of Bax. Mcl-1 expression showed a biphasic expression pattern in both early erythropoiesis and early granulopoiesis, but with an inverse regulation. Thus, Mcl-1 levels initially decreased in granulocytic progenitor cells and increased in erythroid progenitor cells. Finally, Bcl-2 expression was significantly downregulated in both Epo and SCF and G-CSF- and SCF-stimulated cells. The role of the distinct upregulation of Bcl-x(L) in early erythroid differentiation was further examined by use of specific ribozymes against Bcl-x(L). Addition of Bcl-x(L) ribozymes promoted a clear increase in cell death of Epo- and SCF-stimulated cells, while erythroid differentiation was not affected. In conclusion, we found a distinct regulation of several Bcl-2 family members in CD34(+) cells dependent on the cytokine stimulation given. The use of Bcl-x(L)-specific ribozymes suggested that Bcl-x(L) is important for survival but not for differentiation of erythroid progenitor cells.

[Will the revolutionary knowledge of apoptosis play an important role in medicine?]. / Vil kunnskapsrevolusjonen om apoptose få medisinsk betydning?

Apoptosis is a phenomenon of fundamental importance in embryonal development and the homeostatic regulation of mature tissue. We review the present knowledge on apoptosis and the evidence that apoptosis may play a role in the pathogenesis of human disease. As examples we discuss its role in cancer, ischemic diseases, heart failure and neurodegenerative diseases.

Retinoic acid induces apoptosis of human CD34+ hematopoietic progenitor cells: involvement of retinoic acid receptors and retinoid X receptors depends on lineage commitment of the hematopoietic progenitor cells.

Retinoids are bifunctional regulators of growth and differentiation of hematopoietic cells. In this study we explored the effects of retinoic acid (RA) on apoptosis of human CD34+ hematopoietic progenitor cells isolated from normal bone marrow. RA (100 nM) induced an increase in the percentage of dead cells from 24% to 44% at day 6 (p < 0.05, n = 6) as compared to control cells cultured in medium alone. The effect was dose dependent and appeared relatively late. Significant differences were observed from day 4 onward. Apoptosis, or programmed cell death, was demonstrated as the mode of cell death by using the TUNEL assay, which detects single strand nicks in DNA, or by the Nicoletti technique demonstrating a subdiploid population by DNA staining. RA previously was found to inhibit granulocyte colony-stimulating factor--and not granulocyte-macrophage colony-stimulating factor--stimulated proliferation of CD34+ cells. However, we found that RA opposed anti-apoptotic effects of G-CSF and GM-CSF on CD34+ cells (G-CSF: 8% dead cells at day 6; G-CSF + RA: 20%; GM-CSF: 12%; GM-CSF + RA: 27%). Moreover, RA induced apoptosis of CD34+ cells and CD34+CD71+ cells stimulated with erythropoietin. To explore the receptor signaling pathways involved in RA-induced apoptosis, we used selective ligands for retinoic acid receptors (RARs; RO13-7410) and retinoid X receptors (RXRs; RO 25-6603). We found that RARs were involved in RA-mediated apoptosis of myeloid progenitor cells, whereas RARs as well as RXRs were involved in RA-mediated apoptosis of erythroid progenitor cells.

[Apoptosis--new possibilities in the treatment of cancer]. / Apoptose--nye muligheter i behandlingen av kreft.

Apoptosis (also called programmed cell death) is an active, regulated form of cell death that differs in a fundamental way from necrosis, the death type occurring in various diseases. Recent insight into apoptosis is changing important concepts in oncology. It has become clear that cancer cells die to a large extent by apoptosis, not necrosis, and that resistance to treatment may arise because of resistance to apoptosis. This leads to new strategies for cancer treatment: the possibility of induction of cell death in tumours, based on the triggering of molecules involved in the signalling of apoptosis, and managing treatment resistance by devising therapy targeted towards anti-apoptotic molecules within the cell. This article presents an overview of the intracellular mechanisms of apoptosis, and explains the possibilities for future therapy. A summary of the studies of treatment carried out to date is also given.

Multiplication and death-type of leukemia cell lines exposed to very long-chain polyunsaturated fatty acids.

Polyunsaturated fatty acids (PUFA) may reduce cell multiplication in cultures of normal, as well as transformed, white blood cells. We assessed the sensitivity of 14 different leukemia cell lines to PUFA by measuring cell number after 3 days of incubation. Ten of the examined cell lines were sensitive to 30, 60 and/or 120 microM of arachidonic, eicosapentaenoic and docosahexaenoic acid, whereas four cell lines were resistant. The sensitivity to PUFA was not associated with any particular cell lineage, clinical origin or specific mRNA pattern of bcl-2 and c-myc. Effects on cell viability were assessed by studying cell membrane integrity, DNA fragmentation and cell morphology. The sensitive cell lines Raji and Ramos died by necrosis and apoptosis, respectively, during incubation with eicosapentaenoic acid, whereas the viability of the resistant U-698 cell line was unaffected. The effects of EPA on Raji cells, was counteracted by vitamin E, indicating that lipid peroxidation was involved. However, apoptosis induced by eicosapentaenoic acid in Ramos cells, was unaffected by vitamin E, as well as eicosanoid synthesis inhibitors. In conclusion, our results indicate that a majority of leukemia cell lines are sensitive to PUFA. This sensitivity may be caused by induction of apoptosis or necrosis by very long-chain polyunsaturated fatty acids.

Ambulatory liver biopsy: complications and evolution in 264 cases.

AIM: To determine the safety and acceptance of outpatient liver biopsies. PATIENTS AND METHODS: Data from all liver biopsies were collected in a prospective way over a period of 18 months. Information was gathered on complications, evolution of patients outside the hospital and opinion relating to the test. All patients were required to previously present: platelet count > 60.000/mm3 prothrombin time within 4 seconds of control and absence of ascites or encephalopathy. Criteria for outpatient liver biopsy also included cooperative patient, a partner or friend who stayed with the patient during 12-24 hours following the test and easy access to the hospital. Out of a total of 378 biopsies, 264 (70%) were ambulatory. RESULTS: Five of the 264 outpatients were hospitalized (1.9%), due to a subcapsular hematoma in one case, persistent pain in 3 cases and sever hypotension in the other; all of them evolved favorably in the first 24 hours. Among the inpatients, 2 had complications (1.7%): one subcapsular hematoma resolved without treatment and one abdominal hemorrhage requiring transfusion. Of the ambulatory patients, 46 (18%) presented pain whilst at home, being more frequent in females than in males (30% vs 15%, p = 0.004) and in those who needed more than one attempt to obtain histological material compared with those of a single attempt (33% vs 17%; p = 0.008). Twenty four hours after the test, 83% of the patients had returned to their normal activities. Ninety five percent of the patients questioned considered that the test was not traumatic, and 88% stated a preference for liver biopsy as a day case procedure. CONCLUSIONS: Liver biopsy performed on an ambulatory basis is safe, well tolerated and acceptable by the majority of patients.

[Arterioportal fistula and hemobilia in a patient with hepatic transplant]. / Fístula arterioportal y hemobilia en un paciente con trasplante hepático.

The case of a 36-year old male liver transplant recipient hospitalized for upper digestive hemorrhage, jaundice and pain in the right hypochondrium is herein reported. Two hepatic biopsies had been performed 60 and 7 days prior to admission. Bleeding was observed to be from the biliary tract by endoscopy and an arterioportal fistula in the right hepatic lobe by echo-doppler and arteriography was seen. Treatment with selective embolization by arteriography was satisfactory with biliary tract drainage not being required. Doppler echography was used to control the evolution of the patient.

RAR-, not RXR, ligands inhibit cell activation and prevent apoptosis in B-lymphocytes.

We have previously shown that retinoids inhibit activation of human peripheral blood B-lymphocytes. In the present paper, we wished to explore the involvement of nuclear retinoid-specific receptors in this process by using ligands specific for the retinoic acid receptors (RARs) and retinoid X receptors (RXRs). We found that the RAR-specific ligand TTAB reduced anti-IgM-induced B-cell activation in a dose-dependent manner. Thus, at 100 nM of TTAB, DNA synthesis was reduced by approximately 60%. In contrast, the RXR-selective ligand SR11217 had no effect on DNA synthesis. Similar findings were obtained when the expression of the activation antigen CD71 (appears late in G1) was examined. The role of retinoids in apoptosis of resting peripheral blood B-lymphocytes was examined using the same receptor-selective ligands. Again, we found that the RAR-selective ligands were more potent effectors than were the RXR-selective ligands. In spite of the inhibitory effects of retinoids on B-cell proliferation, the same retinoids significantly promoted the survival of the cells. Thus, 10 nM TTAB significantly reduced spontaneous apoptosis of in vitro cultured B-cells at day 3 from 45% to 30%, as determined by vital dye staining and DNA end-labeling. Again, the RXR-specific ligand SR11217 had no effect. Interestingly, we found that CD40 ligand was able to potentiate the retinoid-mediated inhibition of apoptosis. By reverse transcriptase polymerase chain reaction (PCR), we found that peripheral blood B-lymphocytes expressed RARalpha, RARgamma, and RXRalpha, but not RARbeta, RXRbeta, or RXRgamma. Hence, the lack of effect of the RXR-specific ligand SR11217 on growth and apoptosis was not due to absence of RXRs. In conclusion, the ability of retinoids to inhibit growth and prevent apoptosis of normal human B-lymphocytes indicates a dual role of retinoids in this cell compartment, and it appears that both effects of retinoids are mediated via RARs and not RXRs.

Interleukin-13 in combination with CD40 ligand potently inhibits apoptosis in human B lymphocytes: upregulation of Bcl-xL and Mcl-1.

Interleukin-13 (IL-13) is a novel T-cell-derived cytokine with IL-4-like effects on many cell types. In human B lymphocytes, IL-13 induces activation, stimulates proliferation in combination with anti-IgM or anti-CD40 antibodies, and directs Ig isotype switching towards IgE and IgG4 isotypes. We show here that IL-13 also regulates human B-cell apoptosis. IL-13 reduced spontaneous apoptosis of peripheral blood B cells in vitro, as shown by measurement of DNA fragmentation using the TUNEL and Nicoletti assays. The inhibition of cell death by IL-13 alone was significant but modest, but was potently enhanced in combination with CD40 ligand (CD40L), a survival stimulus for B cells by itself. Interestingly, IL-13 increased the expression of CD40 on peripheral blood B cells, providing a possible mechanism for the observed synergy. IL-13 alone was a less potent inhibitor of apoptosis than IL-4. Moreover, there was no additive effect of combining IL-4 and IL-13 at supraoptimal concentrations, which is consistent with the notion that the IL-4 and IL-13 binding sites share a common signaling subunit. The combination of IL-13 with CD40L augmented the expression of the Bcl-2 homologues Bcl-xL and Mcl-1, suggesting this as a possible intracellular mechanism of induced survival. By contrast, levels of Bcl-2, and two other Bcl-2 family members, Bax and Bak, remained unaltered. Given the importance of the CD40-CD40L interaction in B-cell responses, these results suggest a significant role of IL-13 in the regulation of B-cell apoptosis.

Involvement of ICE (Caspase) family in gamma-radiation-induced apoptosis of normal B lymphocytes.

Normal lymphocytes are highly sensitive to the damaging effects of ionizing radiation, and undergo cell death by apoptosis. We have investigated the possible involvement of the Interleukin-1 beta-converting enzyme (ICE) (Caspase) protease family, which appears to play an important role as intracellular mediator of apoptosis. Resting B lymphocytes isolated from human peripheral blood were irradiated (6 Gy) and cultured for 24 h, resulting in 25 +/- 5.1% apoptotic cells, as measured by the TUNEL assay (mean +/- SD, n = 6). Addition of the ICE family inhibitor Z-VAD.fmk (50 microM) completely inhibited apoptosis (2.0 +/- 1.5% at 24 h). By using fluorogenic substrates containing the peptide recognition sequences DEVD and YVAD, the type of ICE family protease involved was examined more closely. A marked transient increase in DEVD-, and absent YVAD-cleavage activity indicated the involvement of a CPP32-like protease, not an ICE-like protease. Western blot analysis demonstrated that untreated B lymphocytes expressed the proform of the ICE family members CPP32 and ICH1L, but no detectable ICE. The induction of cell death by radiation was accompanied by the activation of CPP32 as shown by the cleavage of the proform to the active subunit p17, and the cleavage of poly(ADP-ribose) polymerase (PARP), one of the known substrates of CPP32. In contrast, no activation of ICH1L could be detected. These results indicate the involvement of CPP32 and possibly other CPP32-like proteases in radiation-induced apoptosis of resting B lymphocytes.

[Acute hepatitis associated with terbinafine]. / Hepatitis aguda asociada a terbinafina.

Oral terbinafine is a recently introduced antifungal drug with slight side effects that rarely includes liver involvement. A case of toxic hepatitis secondary to terbinafine administration in a patient with no previous history of liver disease and in whom other possible causes of liver damage and histologic study were performed is reported. A mixed lesion was presented with predominance of cholestasis. The initial worsening following discontinuation of the drug is of note as are the prolonged course of the enzymatic alterations which normalized at one year. The precise mechanism by which terbinafine produced liver damage is unknown and is probably due to an idiosyncratic type reaction.

Expression of the Bcl-2 homologue Mcl-1 correlates with survival of peripheral blood B lymphocytes.

Normal peripheral blood B lymphocytes undergo spontaneous apoptosis in vitro, and this process is regulated positively and negatively by several immunomodulatory stimuli. We have shown previously that Bcl-2 protein levels are unaltered by these factors, suggesting a Bcl-2-independent regulation of apoptosis in this system. Here, we have investigated the possibility that the three recently identified Bcl-2 homologues, Bax, Bcl-x, and Mcl-1, could be involved instead. Freshly isolated cells expressed both Bax and Mcl-1 protein, but only low levels of Bcl-xL and no detectable Bcl-xS, as determined by Western blot analysis. Upon culture of cells with apoptotic or survival stimuli, Bax and Bcl-xL protein levels remained relatively unchanged. By contrast, Mcl-1 levels decreased markedly in cells undergoing apoptosis in medium and, even more dramatically, after treatment with the apoptotic stimuli transforming growth factor beta 1 and forskolin. This decrease was rapid and preceded cell death. Furthermore, all the survival stimuli tested (interleukin 4, anti-IgM antibodies, and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate) prevented the decline in Mcl-1 levels. This striking correlation between cell survival and Mcl-1 expression in peripheral blood B cells suggests the possible involvement of Mcl-1, instead of Bcl-2, in the regulation of apoptosis in these cells. The present study is the first one linking this novel Bcl-2 homologue to the control of cell death in normal cells.

All-trans- and 9-cis-retinoic acid inhibit growth of normal human and murine B cell precursors.

In the present paper we demonstrate that physiologic levels (10 nM) of both all-trans- and 9-cis-retinoic acid (RA) are potent inhibitors of the growth of human as well as murine B cell precursors in vitro. Ten nanomolar concentrations of all-trans- and 9-cis-RA reduced the DNA synthesis ([3H]thymidine uptake) of human B cell precursors (CD19+ IgM-) stimulated with O-tetradecanoylphorbol-13-acetate and ionomycin by approximately 55% and 70%, respectively. Human B cell precursors stimulated with low m.w. B cell growth factor were also inhibited by RA. Ten nanomolar concentrations of either isoform of RA reduced DNA synthesis by approximately 50%. No effect of RA on differentiation to sIgM positive cells was noted. The potent growth-inhibiting effect of RA on human B cell precursors was confirmed in the murine cell system. B lymphopoiesis from murine hematopoietic precursors (Lin-B220(+)-containing cells) was induced by stimulation with IL-7. Concentrations of all-trans- and 9-cis-RA as low as 10 pM reduced the colony-forming ability of the IL-7-stimulated Lin-B220(+)-containing cells. Ten nanomolar concentrations of either isoform reduced colony formation by approximately 60%. RA was not toxic to the cells, as the inhibition of colony formation after 24 h was reversible at concentrations as high as 1 microM. The growth-inhibiting effect of RA was directly mediated, as revealed by single cell analysis of the Lin-B220(+)-containing cells. Thus, vitamin A appears to have an important role in regulation of B lymphopoiesis.

TGF-beta 1 and cyclic AMP promote apoptosis in resting human B lymphocytes.

TGF-beta and agents that elevate intracellular cAMP levels are potent inhibitors of B cell activation in vitro and have been shown to arrest stimulated B cells in the G1 phase of the cell cycle. We tested the effects of TGF-beta 1 and the cAMP-inducing agent, forskolin, on the viability of resting B cells from human peripheral blood, and found that both agents caused a significant, dose-dependent increase in cell death relative to spontaneous death in medium alone, as measured by vital dye staining with propidium iodide. Apoptosis was shown to be the overall mode of death by demonstrating DNA fragmentation using DNA nick end labeling and by verifying the characteristic morphologic changes. In contrast with TGF-beta 1 and forskolin, various B cell activation stimuli generally inhibited spontaneous apoptosis of resting cells. The most potent effects were observed with IL-4 and the phorbol ester, O-tetradecanoylphorbol-13-acetate (TPA), an activator of protein kinase C. IL-4 also partly inhibited TGF-beta 1 and forskolin-induced apoptosis. In contrast, TPA completely reversed cell death in forskolin-treated cultures, but had no effect on TGF-beta 1-induced apoptosis, indicating that TGF-beta 1 and forskolin promote apoptosis by different mechanisms. The relative protein expression of bcl-2, a proto-oncogene that inhibits apoptosis, was unaltered by the apoptotic as well as the survival stimuli tested, suggesting that apoptosis was regulated by a bcl-2-independent mechanism. We conclude that apoptosis is a regulated phenomenon in resting human B cells. Furthermore, TGF-beta and cAMP may inhibit B cell responses not only by blocking cell cycle progression in activated cells, but also by inducing apoptosis in resting cells.

Differential effects of interferon-gamma and low molecular weight BCGF on growth of human B lymphocytes; interferon-gamma prolongs the increased c-MYC mRNA levels after activation.

We have characterized the growth-stimulating effect of Interferon-gamma (IFN-gamma) on various parameters of B cell growth, and compared the effects with those of low molecular weight B cell growth factor (lmw BCGF). We have found that IFN-gamma did not affect early changes induced by anti-mu, like initial calcium-flux and rise in mRNA-and protein levels of the proto-oncogene c-myc measured at 3 h. On the other hand, IFN-gamma enhanced the effect of anti-mu on parameters measured later in the G1 phase of the cell cycle, such as expression of the transferrin receptor and general transcriptional activity, measured as an increase in 7-aminoactinomycin D binding. In particular, whereas the c-myc levels in anti-mu-treated cells peaked at 3 h and then gradually declined, IFN-gamma together with anti-mu maintained the c-myc levels at 24 h at approximately the same levels as seen at 3 h. Overall, lmw BCGF had a more potent effect on the parameters affected by IFN-gamma, correlating with stronger enhancement of DNA synthesis. However, in contrast to IFN-gamma lmw BCGF did not affect anti-mu-induced c-myc mRNA levels. Thus this study has revealed differences between two B cell growth factors in effects on B cell cycle parameters.

Downregulation of c-myc RNA is not a prerequisite for reduced cell proliferation, but is associated with G1 arrest in B-lymphoid cell lines.

We related the effects of c-myc expression on the ability of growth inhibitors to block the cells in the G0/G1 phase of the cell cycle. In two different B-cell lines, there was an association between the accumulation of cells in the middle to late G1 phase of the cell cycle and a rapid transient downregulation of c-myc mRNA levels. The phorbol ester TPA and the adenylate cyclase activator forskolin reduced the c-myc RNA, levels and after 3 days of treatment a proportion of the cells accumulated in G1. In contrast, neither interferon-gamma, tumor necrosis factor-alpha nor the monoclonal antibody 33-1 against DQ major histocompatibility antigens changed the cell-cycle distribution or regulated the c-myc RNA levels. Yet, all five growth inhibitors reduced the proliferation to approximately the same extent. The growth reduction was not accompanied by definite differentiation, as judged by the absence of the B-cell differentiation marker B1 (CD20).