Dynamics of clinical biomarkers as predictors of immunotherapy benefit in metastatic melanoma patients

Purpose Baseline LDH, derived neutrophil–lymphocyte ratio (dNLR) and immune-related adverse events (irAEs) are associated with outcomes of patients with metastatic melanoma (MM). We hypothesized whether dynamic shifts in LDH, dNLR and incidence of irAEs may impact the prognosis of MM patients treated with anti-CTLA4 or anti-PD1 as single agents. Methods Retrospective analysis of medical charts from MM patients with prospective monitoring of dNLR, LDH values and irAE incidence. Primary endpoint was overall survival (OS).Results Patients switching from either high dNLR (≥2.5) to low dNLR (HR: 0.14; 0.03–0.74; p = 0.02) or high LDH (≥1.5 × ULN) to low LDH levels (HR: 0.08; 0.01–0.68; p = 0.02) had significantly better OS than those with high dNLR or LDH scores at the end of cycle 2. Longer OS was also observed in patients developing irAEs ≥ grade 2 as compared to no irAEs (HR: 0.2; 0.05–0.89; p = 0.03).Conclusions We found that major shifts in dNLR and LDH measures from baseline to cycle 2 measures and shifts from baseline to cycle 2 are significantly associated with OS in MM patients receiving single agent anti-PD1 therapy. Laboratory changes and clinical variables may help optimize prognostic estimates. (AU)

Dynamics of clinical biomarkers as predictors of immunotherapy benefit in metastatic melanoma patients.

PURPOSE: Baseline LDH, derived neutrophil-lymphocyte ratio (dNLR) and immune-related adverse events (irAEs) are associated with outcomes of patients with metastatic melanoma (MM). We hypothesized whether dynamic shifts in LDH, dNLR and incidence of irAEs may impact the prognosis of MM patients treated with anti-CTLA4 or anti-PD1 as single agents. METHODS: Retrospective analysis of medical charts from MM patients with prospective monitoring of dNLR, LDH values and irAE incidence. Primary endpoint was overall survival (OS). RESULTS: Patients switching from either high dNLR (≥2.5) to low dNLR (HR: 0.14; 0.03-0.74; p = 0.02) or high LDH (≥1.5 × ULN) to low LDH levels (HR: 0.08; 0.01-0.68; p = 0.02) had significantly better OS than those with high dNLR or LDH scores at the end of cycle 2. Longer OS was also observed in patients developing irAEs ≥ grade 2 as compared to no irAEs (HR: 0.2; 0.05-0.89; p = 0.03). CONCLUSIONS: We found that major shifts in dNLR and LDH measures from baseline to cycle 2 measures and shifts from baseline to cycle 2 are significantly associated with OS in MM patients receiving single agent anti-PD1 therapy. Laboratory changes and clinical variables may help optimize prognostic estimates.

Early evolutionary divergence between papillary and anaplastic thyroid cancers.

Background: Papillary thyroid cancer (PTC) is the most common thyroid carcinoma and exhibits an almost uniformly good prognosis, while anaplastic thyroid cancer (ATC) is less frequent and is one of the most aggressive cancers usually resistant to conventional treatment. Current hypothesis posits that ATC derives from PTC through the progressive acquisition of a discrete number of genomic alterations and implies that the mutational landscape of ATC resembles that of PTC. However, the clinical behaviour of ATC and PTC is radically different. We decided to address the disconnection between the clinical behaviour of ATC and PTC and the proposed model of the progressive development of ATC from PTC. Patients and methods: We carried out exome sequencing of DNA from 14 ATC specimens including three cases of concomitant ATC and PTC as well as their corresponding normal DNA from 14 patients. The sequencing results were validated using droplet digital PCR. We carried out immunohistochemistry and immunofluorescence studies of the concomitant ATC and PTC cases. In addition, we integrated our sequencing results with the existing TCGA data. Results: Most of the somatic mutations identified in the ATC component differed from the ones in PTC in the cases of concomitant ATC and PTC. The trunks of the phylogenetic trees representing the somatic mutations were short with long branches. In one case of concomitant PTC and ATC specimens, we observed an infiltration of PTC cells within the ATC component. Moreover, we integrated our results with data obtained from TCGA and observed that the most frequent mutations found in ATC presented high cancer cell fraction values and were significantly different from the PTC ones. Conclusion: ATC diverge from PTC early in tumour development and both tumour types evolve independently. Our work allows the understanding of the relationship between ATC and PTC facilitating the clinical management of these malignancies.

ALDH1A3 is epigenetically regulated during melanocyte transformation and is a target for melanoma treatment.

Despite the promising targeted and immune-based interventions in melanoma treatment, long-lasting responses are limited. Melanoma cells present an aberrant redox state that leads to the production of toxic aldehydes that must be converted into less reactive molecules. Targeting the detoxification machinery constitutes a novel therapeutic avenue for melanoma. Here, using 56 cell lines representing nine different tumor types, we demonstrate that melanoma cells exhibit a strong correlation between reactive oxygen species amounts and aldehyde dehydrogenase 1 (ALDH1) activity. We found that ALDH1A3 is upregulated by epigenetic mechanisms in melanoma cells compared with normal melanocytes. Furthermore, it is highly expressed in a large percentage of human nevi and melanomas during melanocyte transformation, which is consistent with the data from the TCGA, CCLE and protein atlas databases. Melanoma treatment with the novel irreversible isoform-specific ALDH1 inhibitor [4-dimethylamino-4-methyl-pent-2-ynthioic acid-S methylester] di-methyl-ampal-thio-ester (DIMATE) or depletion of ALDH1A1 and/or ALDH1A3, promoted the accumulation of apoptogenic aldehydes leading to apoptosis and tumor growth inhibition in immunocompetent, immunosuppressed and patient-derived xenograft mouse models. Interestingly, DIMATE also targeted the slow cycling label-retaining tumor cell population containing the tumorigenic and chemoresistant cells. Our findings suggest that aldehyde detoxification is relevant metabolic mechanism in melanoma cells, which can be used as a novel approach for melanoma treatment.

Identity between the PCPH proto-oncogene and the CD39L4 (ENTPD5) ectonucleoside triphosphate diphosphohydrolase gene.

PCPH was initially defined as a proto-oncogene on the basis of its frequent detection as an activated oncogene in tumorigenic Syrian hamster embryo fibroblast cell lines converted to the neoplastic state by a single treatment with the carcinogen 3-methylcholanthrene (MC). Further studies identified the translation product of the PCPH gene as a ribonucleotide-binding protein with special affinity for ribonucleoside diphosphates. Later, we showed that the PCPH protein was homologous to the product of the yeast GDA1 gene and demonstrated that it had intrinsic guanosine diphosphatase activity, although it did not complement the disrupted phenotype when expressed in gda1 null Saccharomyces cerevisiae strains. These results indicated that the primary function of PCPH was unlikely to be related to the ribonucleotide recycling function that its yeast counterpart performs in the Golgi during the process of protein glycosylation. However, taken together, our data strongly suggested that the normal cellular function of PCPH was related to ribonucleotide metabolism. We now report that PCPH is structurally and functionally identical to the mammalian ectonucleoside triphosphate diphosphohydrolase CD39L4 (ENTPD5), recently described as a member of the lymphoid activation antigen () CD39 protein family. These results may help to establish the normal cellular function of the PCPH proto-oncogene product and its role in neoplastic development during carcinogenesis.

Neonatal sunburn and melanoma in mice.

Retrospective epidemiological data have indicated that cutaneous malignant melanoma may arise as a consequence of intense, intermittent exposure of the skin to ultraviolet radiation, particularly in children, rather than from the cumulative lifetime exposure that is associated with other forms of skin cancer. Here we use a genetically engineered mouse model to show that a single dose of burning ultraviolet radiation to neonates, but not adults, is necessary and sufficient to induce tumours with high penetrance which are reminiscent of human melanoma. Our results provide experimental support for epidemiological evidence that childhood sunburn poses a significant risk of developing this potentially fatal disease.

Expression of the protein product of the PCPH proto-oncogene in human tumor cell lines.

Expression of the Protein Product of the PCPH Proto-oncogene in Human Tumor Cell Lines. Exposure of Syrian hamster embryo fibroblasts to chemical carcinogens resulted in the oncogenic activation of the PCPH proto-oncogene by induction of a single base-pair deletion that generated a truncated PCPH oncoprotein (mutated PCPH). Recently, we isolated and characterized the cDNA for the human PCPH proto-oncogene and determined that in humans PCPH is a single-copy gene located in chromosome 14 (14q24.3). Pilot mRNA expression studies indicated that PCPH was expressed in the majority of normal organs tested, particularly in liver and kidney, but it appeared to be expressed either at low levels or not at all in tumor cells or cell lines derived from the high-expressing tissues. We have generated an antiserum against bacterial recombinant Syrian hamster PCPH. This antiserum recognizes both the normal and truncated, oncogenic Syrian hamster PCPH proteins and cross-reacts with the yeast, mouse, rat and human homologue proteins. Using this antibody, we have performed a study of PCPH expression in a larger sample of human neoplastic cell lines, including some derived from breast, nervous system, colon, lung and pancreas tumors. Results confirmed the frequent lack of PCPH expression in malignant cells and identified several immunoreactive forms of PCPH being differentially expressed in cells of diverse tissue origins.

Both normal and transforming PCPH proteins have guanosine diphosphatase activity but only the oncoprotein cooperates with Ras in activating extracellular signal-regulated kinase ERK1.

Previous reports from our laboratory described the activation of the PCPH gene into the PCPH oncogene (mt-PCPH, reported previously as Cph) by a single point mutational deletion. As a consequence, the mt-PCPH oncoprotein is a truncated form of the normal PCPH protein. Although both proteins have ribonucleotide diphosphate-binding activity, only mt-PCPH acted synergistically with a human H-Ras oncoprotein to transform murine NIH3T3 fibroblasts. We report here the expression of the PCPH and mt-PCPH proteins in Escherichia coli and the finding that the purified bacterial recombinant proteins have intrinsic guanosine diphosphatase (GDPase) activity. However, expression of the Syrian hamster PCPH and mt-PCPH proteins in haploid yeast strains engineered to be GDPase deficient by targeted disruption of the single GDA1 allele did not complement their glycosylation-disabled phenotype, suggesting the existence of significant functional differences between the mammalian and yeast enzymes. Results from transient cotransfections into NIH3T3, COS-7, or 293T cells indicated that, in mammalian cells, both PCPH and mt-PCPH cause an overall down-regulation of the stimulatory effect of epidermal growth factor or the activated ras or raf oncogenes on the Ras/mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) signaling pathway. However, despite this overall negative regulatory role on Ras signaling, mt-PCPH, but not PCPH, cooperated with the Ras oncoprotein to produce a prolonged stimulation of the phosphorylation of ERK1 but had no effect on the phosphorylation levels of ERK2. These results represent a clear difference between the mechanisms of action of PCPH and mt-PCPH and suggest that the ability to cause a sustained activation of ERK1 may be an important determinant of the transforming activity of mt-PCPH.

The human PCPH proto-oncogene: cDNA identification, primary structure, chromosomal mapping, and expression in normal and tumor cells.

We identified a human cDNA encoding a 47-kDa protein that shares 78% and 87% identity with the products of the Syrian hamster and mouse PCPH proto-oncogenes respectively. The human homolog was localized by radiation-hybrid mapping to chromosome band 14q24.3, a region syntenic to the Pcph location on mouse chromosome 12. Northern analyses revealed that PCPH mRNA was widely distributed in normal human adult tissues, but its expression varied significantly among human tumor cells and cell lines of several tissue types, regardless of the level of expression in the corresponding normal tissues. The highest levels of PCPH mRNA and protein were detected in kidney and liver. However, PCPH was not expressed in the majority of human neoplasms tested, including kidney tumors. These data provide suggestive evidence for a possible association of the lack of PCPH expression to the neoplastic phenotype of human tumor cells. Our results should prove instrumental in designing studies to define the cellular function of the human PCPH proto-oncogene.

Retinoic acid stimulates HIV-1 transcription in human neuroblastoma SH-SY5Y cells.

Although the brain is an important target for the human immunodeficiency virus type 1 (HIV) and viral infection causes neuronal degeneration and dementia, the mechanisms responsible for HIV transcription in neuronal cells are largely unknown. We show here that retinoic acid (RA) stimulates HIV transcription in human neuronal SH-SY5Y cells. The steroid receptor coactivator 1 (SRC-1) enhances the transcriptional response to RA, and the viral protein Tat cooperates with RA and SRC-1 to induce a strong transactivation. These results suggest that retinoid receptors could play an important role as activators of viral gene expression in the human brain.

cDNA isolation, expression, and chromosomal localization of the mouse pcph proto-oncogene.

We report here the isolation and characterization of a cDNA from mouse thymus encoding the murine homolog of the protein product of the Syrian hamster Pcph proto-oncogene. The single open reading frame identified in the cDNA sequence encoded a protein predicted to have 428 amino acids, which shared 93.7% amino acid identity with the Syrian hamster Pcph within the first 412 residues but had a shorter, highly dissimilar C-terminus. Northern and western analyses revealed that Pcph mRNA and protein were widely distributed in mouse embryo and adult tissues, with the highest expression in adults detected in kidney and liver. The mouse Pcph proto-oncogene was mapped by linkage analysis to within 3.3+/-2.3 cM of Pkch-rs1 on chromosome 12. These data should prove valuable in designing studies to define the cellular function of the Pcph proto-oncogene.

Activation of the HIV-1 long terminal repeat by nerve growth factor.

The brain is an important target for the human immunodeficiency virus type 1 (HIV-1). We show here that nerve growth factor (NGF), which induces neuronal differentiation and survival, causes a strong activation of the HIV-1 long terminal repeat by a Ras/Raf-dependent mechanism in PC12 cells. Mutation of the kappaB sequences contained whithin the long terminal repeat reduces NGF-mediated stimulation. NGF does not activate NF-kappaB in PC12 cells, but rather increases binding of other nuclear factors to the kappaB sequences. Furthermore, a nuclear receptor response element contributes to the stimulatory effect of NGF. The retinoids receptors have been identified as components of the nuclear binding to the nuclear receptor response element in NGF-treated PC12 cells. These results reveal the importance of neurotrophins and nuclear receptor signaling pathways as specific activators of HIV-1 gene expression in neural cells.

Influence of Ras and retinoic acid on nerve growth factor induction of transin gene expression in PC12 cells.

Nerve growth factor (NGF)- and ras-induced neuronal differentiation of PC12 cells is accompanied by expression of transin, a secreted metalloproteinase. Retinoic acid (RA) is known to exert important effects on neural cell proliferation and differentiation. In this study we have analysed different PC12 sublines which express either activated Ras or dominant negative p21N17 Ras, to evaluate the influence of retinoic acid (RA) on the response of the transin gene to NGF and Ras. There was a good correlation between neurite extension and induction of transin mRNA levels in the different subclones. NGF did not induce transin mRNA in cells which do not differentiate in response to this neurotrophin. In addition, incubation with RA did not detectably increase basal transin mRNA levels, but caused a significant increase in the transin response to NGF or Ras in cells in which these factors induce a neuronal morphology. Sequences contained within 750 base pairs of the 5' flanking region of the transin gene confer responsiveness to NGF and Ras, but do not mediate the stimulatory effect of RA. In addition, expression of oncogenic Raf increases transin promoter activity in PC12 cells, but a dominant-negative Raf mutant was unable to block NGF-induced transin activity suggesting the existence of a bifurcation downstream of ras in the signaling mechanism leading to transin expression by NGF.

Morphometric and immunocytochemical study of the fetal, infant, and adult human vas deferens.

The human vas deferens (VD) is often considered simply as a conduit to transfer mature sperm from the epididymis to the ejaculatory duct. The cells that make up the epithelium of the VD, however, exhibit many characteristics of cells found in more complex epithelia, which are involved in absorption and/or secretion. In the present investigation, morphometry was utilized to characterize in detail the changes incurred by the human VD during its development, growth, and aging and to determine if these changes correlate with testicular maturation. In addition, the specific types of keratins present in the epithelial cells were defined, as well as desmin distribution in the muscular layers, during the various phases of the development, growth, and involution of the human VD. Results of the morphometric study are consistent with the interpretation that the development, growth, and aging of the VD are delayed, but parallel to, the identical phases exhibited by the human testis. Further, a differential expression of distinct keratin types was observed in the VD during the various phases examined in this study. Taken together, these two correlations may suggest that the VD is unlikely to function solely as a conduit for sperm. The rationale for this interpretation is as follows: 1) the complex developmental and maturational changes measured in the present investigation in the human VD are common to other absorptive and/or secretory epithelia; and 2) these changes parallel developmental changes observed in other androgen-dependent epithelia of the male reproductive tract, which also function to contribute components to seminal fluid as well as to provide a conduit for sperm.