Outcomes Analysis of Patients Receiving Local Ablative Therapy for Oligoprogressive Metastatic NSCLC Under First-Line Immunotherapy.

CONTEXT: Nonsmall Cell Lung Cancer (NSCLC) treatment relies on first-line immunotherapy as single agent or combined with chemotherapy. Oligoprogression may be observed in this setting. MATERIAL AND METHOD: We performed a European multicentric retrospective study on patients treated with first-line immunotherapy, who presented with oligoprogressive disease, treated with a local ablative treatment. RESULTS: A total of 61 patients were retrospectively included between 2018 and 2022. Twenty-four patients (39%) received immunotherapy as single agent, and 37 (61%) chemo-immunotherapy. First oligoprogression occurred more frequently in pre-existing metastatic sites (47% of patients). Median PFS1 (defined as time to first oligoprogression) was 11.5 months [IC95%: 10.0-12.3]. We observed that 37 patients (61%) progressed after first oligoprogression, and 20 (54%) from them presented second oligoprogression. Median OS for the whole cohort was 72.0 months [IC95%: 19.3-124.8], with positive correlation between OS and PFS1 (R=0.65, P < .0001). After loco-ablative treatment with radiotherapy, disease control rate was 89% with ablative radiotherapy: 88% with conventional radiotherapy, and 89% with stereotactic radiotherapy. CONCLUSION: Patients with oligoprogression under/after immunotherapy have better prognosis with a high risk of subsequent oligoprogression.

Cell cycle regulation of E2F site occupation in vivo.

DNA-binding E2F complexes have been identified throughout the mammalian cell cycle, including the transcriptionally inactive complexes with pocket proteins, which occur early in the prereplicative G1 phase of the cycle, and the transactivating free E2F, which increases in late G1. Here, a regulatory B-myb promoter site was shown to bind with high affinity to free E2F and to E2F-pocket protein complexes in an indistinguishable way in vitro. In contrast, in vivo footprinting with NIH 3T3 cells demonstrated E2F site occupation specifically in early G1, when the B-myb promoter is inactive. These observations indicate that a novel mechanism governs E2F-DNA interactions during the cell cycle and emphasize the relevance of E2F site-directed transcriptional repression.

Inhibition of Fos- and Ras-induced transformation by mutant Fos proteins with structural alterations in functionally different domains.

We show that transformation-defective Fos proteins lacking either a functional leucine zipper (mutants L345 and J/R510s) or the 110 amino-terminal amino acids (mutant BR800) inhibit the induction of morphological transformation by v-Fos. Both types of mutants specifically repress transformation without any significant effect on cell proliferation. In contrast, several transformation-defective Fos mutants with structural alterations in the acidic region or the right half of the adjacent basic DNA contact site do not show any inhibition of transformation. This result, taken together with the repression of transformation by the leucine zipper-deficient mutants L345 and J/R510s, indicates that the interaction of Fos with proteins other than Jun is necessary for transformation. The leucine zipper-deficient mutants also inhibit Fos-mediated activation of AP-1-dependent transcription. This suggests that their inhibitory effect on transformation may at least in part be the result of the squelching of proteins other than Jun family members that are required for Fos-mediated transactivation. All three mutants were also found to inhibit transformation by the point-mutated H-ras oncogene from EJ carcinoma cells and to trigger a partial reversion of the transformed phenotype of Ras-transformed fibroblasts. These findings support the conclusion that Ras-induced transformation involves signal transduction pathways inducing the c-fos gene.

Suppression of the growth factor-mediated induction of c-fos and down-modulation of AP-1-binding activity are not required for cellular senescence.

It has been suggested that the impaired response of the c-fos gene to serum growth factors and the concomitant loss of AP-1 activity may be a crucial step in the process of cellular senescence. In the present study, we provide evidence arguing against such a conclusion. Data obtained in five independent experiments showed that both c-fos RNA and protein expression were similar in 'young' and in senescent serum-stimulated WI-38 cells, suggesting that the previously reported suppression of c-fos induction is not an obligatory event in the process of cellular senescence. Likewise, expression of fra-1, c-jun and junB continued to be high in serum-stimulated senescent cells, while induction of fosB was reduced approximately fivefold. Among all genes tested fosB thus seems to be the most suitable marker for the detection of senescent cells. Stimulated senescent cells showed only a approximately twofold reduction of AP-1-binding activity, and senescent cells continuously exposed to serum exhibited normal AP-1-binding activity. These observations support the conclusion that a down-modulation of AP-1 is not crucial for human fibroblasts to enter the senescent state.

Inducible acceleration of G1 progression through tetracycline-regulated expression of human cyclin E.

Cyclin E is a cell cycle-regulated protein that activates the cdc2-related protein kinases cdk2 shortly before S-phase entry. In order to analyse the biological role of cyclin E, we have generated HeLa cells that allow the conditional expression of ectopic human cyclin E. In these cells, a cyclin E cDNA is under the control of a bacterial tetracycline repressor-VP16 activator hybrid protein. In the absence of tetracycline, the endogenous gene becomes activated and leads to the synthesis of elevated levels of cyclin E. Concomitant with this increase in cyclin E expression we show by a combined time-lapse video recording/5-bromo-deoxyuridine labelling procedure a significant acceleration of G1 transition by approximately 1.5 hours. This observation is consistent with the idea that cyclin E is a rate-limiting factor with respect to the control of G1-->S transition. The experimental system described here should also prove useful to address the function of cyclin E in further detail.

Oncogenic activity of cyclin D1 revealed through cooperation with Ha-ras: link between cell cycle control and malignant transformation.

Circumstantial evidence implicates the putative cell cycle regulator cyclin D1 in the process of malignant transformation. Overexpression of cyclin D1 is observed in mammary carcinomas as a result of gene amplification and in parathyroid adenomas and centrocytic B-cell lymphomas as a consequence of chromosomal rearrangements and juxtaposition of the cyclin D1 gene to strong transcriptional control elements. These findings suggest that deregulation of cyclin D1 expression may contribute to malignant transformation in these tumours. To date, however, an oncogenic potential of cyclin D1 has not been demonstrated and the mechanism of its oncogenic activation remains obscure although overexpression of the wild-type protein is likely. We report here that the overexpression of cyclin D1 induces transformation in primary rat embryo fibroblasts in cooperation with activated Ha-ras. Cyclin D1/Ha-ras transformed cells are immortalized, show anchorage independence and give rise to fibrosarcomas in nude mice. Our data directly demonstrate that cyclin D1 is a proto-oncogene that can be activated by transcriptional deregulation. Its previously demonstrated ability to interact with putative cell cycle regulators suggests that cyclin D1 defines a new class of proto-oncogenes.

A new model of cell cycle-regulated transcription: repression of the cyclin A promoter by CDF-1 and anti-repression by E2F.

Cell cycle regulation of the cyclin A gene is determined by a bipartite repressor binding site in the region of the basal promoter, termed CDE-CHR, which also controls the expression of cell cycle genes upregulated in S or G2 (such as cdc25C). The CDE-CHR in the cyclin A promoter is recognized by both E2F complexes and CDF-1, but the contribution of each of these factors in cell cycle regulation is unknown. In the present study, we have introduced mutations into the cyclin A promoter which lead to either a loss or enhancement of E2F binding, while having only marginal effects on the interaction with CDF-1. Unlike mutants deficient for CDF-1 binding, promoter variants lacking E2F binding showed an unchanged repression in G0, thus identifying CDF-1 as the principal repressor of the cyclin A gene. The same mutants did show, however, a delayed derepression while a mutation leading to increased E2F binding resulted in premature up-regulation. These findings clearly suggest that E2F contributes to the correct timing of cyclin A transcription, presumably by acting as an anti-repressor. In agreement with this conclusion, we find that the cyclin A promoter only poorly interacts with E2F-4, which is the major E2F family member in G0 cells, while a clear binding is seen with E2F-1 and -3, which are up-regulated in late G1.

Functional domains in cyclin D1: pRb-kinase activity is not essential for transformation.

Although cyclin D1 plays a major role during cell cycle progression and is involved in human tumourigenesis, its domain structure is still poorly understood. In the present study, we have generated a series of cyclin D1 N- and C-terminal deletion constructs. These mutants were used to define the domains required for transformation of rat embryonal fibroblasts (REF) in cooperation with activated Ha-ras and and to establish correlations with defined biochemical properties of cyclin D1. Protein binding and REF assays showed that the region of the cyclin box required for the interaction with CDK4 as well as C-terminal sequences determining protein stability were crucial for transformation. Surprisingly, however, the N-terminal deletion of 20 amino acids which impaired pRb kinase activity did not affect the transforming ability of cyclin D1. Likewise, no effect on transformation was observed with mutants defective in p21CIP interaction. These observations argue against a crucial role of pRb inactivation or p21CIP squelching in cyclin D1-mediated transformation.

Cell proliferation and cell cycle progression are not impaired in fibroblasts and ES cells lacking c-Fos.

The transcription factor AP-1 is thought to play an important role in the control of cell proliferation, but the function of individual Fos and Jun family members is a largely unresolved issue. To directly analyse the function of c-Fos in the control of cell proliferation we have used embryonic stem (ES) cells and fibroblasts lacking c-Fos due to a disruption of the c-fos gene by homologous recombination. Our results demonstrate that proliferation of normally cycling cells and reentry of quiescent cells into the cell cycle following serum stimulation are not c-Fos-dependent and occur with similar efficiency in c-fos-/- and control cells. We also show that there is no compensatory overexpression or activation of other known Fos or Jun family members. On the contrary, the c-fos-/- cells showed a reduced induction of fra-1 after serum stimulation which is in agreement with the previous identification of fra-1 as a c-Fos target gene. Comparison of the AP-1 binding and transactivation activities in c-fos-/- and +/+ fibroblasts by electrophoretic mobility antibody supershift and CAT assays suggests that c-Fos is not a major component of AP-1 complexes in these cells. It is therefore conceivable that the lack of any detectable effect on cell proliferation in c-fos-/- cells might be due to a functional redundancy among the different AP-1 family members.

Lack of effect of clonidine and pentoxifylline in short-term therapy of diabetic peripheral neuropathy.

The goal of this study was to confirm or rule out anecdotal reports of beneficial effects of clonidine and pentoxifylline in the treatment of painful diabetic peripheral neuropathy. Clonidine was administered to 16 subjects at two dosage levels (0.1 and 0.2 mg/day) and was compared to placebo in a crossover design, with each phase lasting 4 wk. Either pentoxifylline (400 mg 3 times/day) or placebo was given to 21 subjects in a 12-wk trial. Discomfort was characterized and rated with a subjective pain score (range 0-20). There was a significant decrease in pain score from baseline with both active drugs (P less than 0.05), but this was no better than the response to placebo (P less than 0.30 for clonidine and P less than 0.95 for pentoxifylline). This study does not demonstrate a short-term benefit of either clonidine or pentoxifylline in the treatment of peripheral neuropathy.

Dendritic cells derived from peripheral monocytes express endothelial markers and in the presence of angiogenic growth factors differentiate into endothelial-like cells.

CD14-positive monocytes obtained from human peripheral blood were cultured with GM-CSF and IL-4. During the early culture phase immature dendritic cells (DCs) developed which not only expressed CD1a, HLA-DR and CD86, but also expressed the endothelial cell markers von Willebrand factor (vWF), VE-cadherin and VEGF receptors Flt-1 and Flt-4. Further maturation of DCs was achieved by prolonged cultivation with TNFalpha. These cells showed typical DC morphology and like professional antigen-presenting cells (APCs) expressed CD83 and high levels of HLA-DR and CD86. However, if immature DCs were grown with VEGF, bFGF and IGF-1 on fibronectin/vitronectin-coated culture dishes, a marked change in morphology into caudated or oval cells occurred. In the presence of these angiogenic growth factors the cultured cells developed into endothelial-like cells (ELCs), characterized by increased expression of vWF, KDR and Flt-4 and a disappearance of CD1a and CD83. Addition of IL-4 and Oncostatin M also increased VE-cadherin expression, and the loosely adherent cells formed clusters, cobblestones and network-like structures. vWF- expressing ELCs mainly originated from CD1a-positive cells, and VEGF was responsible for the decrease in the expression of the DC markers CD1a and CD83. In mixed leukocyte cultures, mature DCs were more potent APCs than ELCs. Moreover, Ac-LDL uptake, and the formation of tubular structures on a plasma matrix was restricted to ELCs. These results suggest that in the presence of specific cytokines immature DCs have the potential to differentiate along different lineages, i.e. into a cell type resembling ELCs.

trans-repression of the mouse c-fos promoter: a novel mechanism of Fos-mediated trans-regulation.

Fos protein can trans-activate AP-1-dependent gene expression and trans-repress the c-fos promoter. Although we find that trans-repression is enhanced by coexpression of c-Jun, it does not require any of the AP-1 or ATF sites in the mouse c-fos promoter. A major target for repression is the serum response element (SRE). Fos mutants with an impaired leucine zipper are defective in trans-repression and transformation, suggesting that these functions involve the formation of Fos protein complexes. In contrast, mutations that abolish DNA binding of Fos enhance trans-repression but destroy the transforming potential of Fos. In addition, v-Fos protein efficiently transforms but is unable to trans-repress. These findings point to different mechanisms involved in trans-activation and trans-repression and suggest that trans-repression of the type described here is neither sufficient nor required for Fos-induced transformation.

Multiple regions of v-Fos protein involved in the activation of AP1-dependent transcription: is trans-activation crucial for transformation?

We show that trans-activation by v-Fos requires several functionally separable regions, including the leucine repeat, the basic DNA-binding region, a directly adjacent acidic cluster, and additional flanking sequences. Structural alterations in the flanking regions are in part responsible for the greater trans-activating potential of the fos gene product of the Finkel-Biskis-Reilly mouse osteosarcoma virus, FBR-MuSV. A point mutation in the acidic cluster, which is known to activate the immortalizing potential of Fos, leads to a significant increase in trans-activation. However, comparison of the trans-activating and transforming properties of mutant Fos proteins suggests that functions other than trans-activation are involved in the induction of transformation.

Alternative splicing of fosB transcripts results in differentially expressed mRNAs encoding functionally antagonistic proteins.

We show that serum-stimulated fibroblasts transiently express two different forms of fosB mRNA, which are generated by alternative splicing of the transcript from a single gene. In addition to the known long form (fosB-L), encoding a protein of 338 amino acids (FosB-L), a second shorter form (fosB-S) with a deletion of 140 bp was detected. This deletion creates a stop codon 3' to the leucine repeat, giving rise to a protein of 237 amino acids (FosB-S) lacking the carboxyl terminus of FosB-L. Only the long FosB form efficiently induces transformation in mouse and rat fibroblast cell lines and trans-represses the c-fos promoter. Both of these functions are suppressed by coexpressed FosB-S. Upon serum stimulation, maximum expression of the oncogenic fosB-L form precedes the expression of the antagonistic fosB-S form, indicating a new mechanisms regulating the action of members of the Fos family. However, FosB-L and FosB-S do not differ in all trans-regulatory properties: Trans-activation of a 5x TRE-CAT reporter construct in HeLa and NIH-3T3 cells was found with both FosB forms. These observations suggest a correlation between fosB-induced transformation and trans-repression, thus pointing to different mechanisms involved in transformation by fosB and c-fos/v-fos.

Multiple interdependent regulatory sites in the mouse c-fos promoter determine basal level transcription: cell type-specific effects.

Although the induction of the mouse c-fos promoter by growth factors and specific signal transduction pathways has been analyzed in some detail, the mechanisms involved in the control of basal level transcription remain largely elusive. In this study, we present evidence for the existence of at least 9 different elements, located between the putative TATA box and position -610, that figure in basal level transcription and represent protein binding sites in different cell types. A major regulatory site in F9END, NIH3T3 and HeLa cells is the CRE around position -60. Other sites, including the SRE, a NF1 site around position -165, a novel site downstream of the SRE and three new sites upstream of the SRE play different cell type-specific roles. In addition, we have identified two regions upstream of the SRE, which seem to have cell type-specific negative regulatory effects. We also find that the precise function of several of these sites depends on the presence or absence of other elements, indicating some form of interaction between different regulatory sites. Finally, we present evidence, that the block of c-fos transcription in F9EC cells is due to the lack of transregulatory proteins, which are induced during retinoic acid mediated differentiation.

A rapid micro method for counting cells "in situ" using a fluorogenic alkaline phosphatase enzyme assay.

A new method has been developed to count cells "in situ", based on a fluorogenic enzyme assay that measures the activity of alkaline phosphatase. Increasing cell number was shown to correlate closely with alkaline phosphatase activity and this relationship did not change with time in culture. The alkaline phosphatase assay (ALP assay) was able to estimate relative cell numbers over a range from about 10(4) to 5 X 10(5) for many cell types, including Hep-2, a derivative of HeLa, several human colorectal cell lines SW1222, SW837, LS174T and HT29, a normal human diploid cell strain MRC5 and a rodent line NIH-3T3. The ALP assay is rapid and efficient, making it a useful method for studying growth assays.