Concurrent intrathecal and intravenous nivolumab in leptomeningeal disease: phase 1 trial interim results.

There is a critical need for effective treatments for leptomeningeal disease (LMD). Here, we report the interim analysis results of an ongoing single-arm, first-in-human phase 1/1b study of concurrent intrathecal (IT) and intravenous (IV) nivolumab in patients with melanoma and LMD. The primary endpoints are determination of safety and the recommended IT nivolumab dose. The secondary endpoint is overall survival (OS). Patients are treated with IT nivolumab alone in cycle 1 and IV nivolumab is included in subsequent cycles. We treated 25 patients with metastatic melanoma using 5, 10, 20 and 50 mg of IT nivolumab. There were no dose-limiting toxicities at any dose level. The recommended IT dose of nivolumab is 50 mg (with IV nivolumab 240 mg) every 2 weeks. Median OS was 4.9 months, with 44% and 26% OS rates at 26 and 52 weeks, respectively. These initial results suggest that concurrent IT and IV nivolumab is safe and feasible with potential efficacy in patients with melanoma LMD, including in patients who had previously received anti-PD1 therapy. Accrual to the study continues, including in patients with lung cancer. ClinicalTrials.gov registration: NCT03025256 .

Oncogenic Ras/Src cooperativity in pancreatic neoplasia.

Pancreas cancer is one of the most lethal malignancies and is characterized by activating mutations of Kras, present in 95% of patients. More than 60% of pancreatic cancers also display increased c-Src activity, which is associated with poor prognosis. Although loss of tumor suppressor function (for example, p16, p53, Smad4) combined with oncogenic Kras signaling has been shown to accelerate pancreatic duct carcinogenesis, it is unclear whether elevated Src activity contributes to Kras-dependent tumorigenesis or is simply a biomarker of disease progression. Here, we demonstrate that in the context of oncogenic Kras, activation of c-Src through deletion of C-terminal Src kinase (CSK) results in the development of invasive pancreatic ductal adenocarcinoma (PDA) by 5-8 weeks. In contrast, deletion of CSK alone fails to induce neoplasia, while oncogenic Kras expression yields PDA at low frequency after a latency of 12 months. Analysis of cell lines derived from Ras/Src-induced PDA's indicates that oncogenic Ras/Src cooperativity may lead to genomic instability, yet Ras/Src-driven tumor cells remain dependent on Src signaling and as such, Src inhibition suppresses growth of Ras/Src-driven tumors. These findings demonstrate that oncogenic Ras/Src cooperate to accelerate PDA onset and support further studies of Src-directed therapies in pancreatic cancer.

Identification of metastasis-associated genes by transcriptional profiling of a pair of metastatic versus non-metastatic human mammary carcinoma cell lines.

Cell lines 4A4 and 2C5 are the respective metastatic and non-metastatic variants of the human mammary carcinoma cell line MDA-MB-435 in the nude mouse system. We compared the transcriptional profile of approximately 5000 full-length genies using the Affymetrix HuGene FL Array technology. We have shown that the metastatic phenotype is mediated by different functional categories of genes, e.g. genes involved in immune response, genes responsible for tumor antigens, genes involved in migration and invasion, genes involved in mediating signal transduction, genes responsible for transcription factors, genes involved in phospholipid signaling, genes involved in modulation of extracellular matrix and cytoskeleton, genes with a cell-type specific mode of expression and genes which do not fit into the subclasses as defined above. Our results suggest an important role of Autocrine Motility Factor (AMF) as a mediator of metastasis in this system.

PTEN expression is maintained in sporadic colorectal tumours.

Loss of PTEN (phosphatase and tensin homologue deleted from chromosome 10) function has been implicated in the progression of several types of cancer. Allele loss close to the PTEN locus occurs in sporadic colon cancer and germline PTEN mutations cause Cowden disease, an inherited cancer syndrome characterized by an increased incidence of gastrointestinal tract lesions that can progress to colorectal carcinoma. However, although PTEN is a good candidate for involvement in the pathogenesis of sporadic colon cancer, previous analyses have not revealed a high frequency of somatic mutations in colorectal tumours. Alternative mechanisms which could lead to a loss of PTEN expression in colon cancer have not been investigated. This study monitored PTEN mRNA and protein levels in a panel of 50 tumour tissues obtained from 35 patients with sporadic colon cancer. RT-PCR and immunohistochemistry were used to evaluate the expression of mRNA and protein, respectively, in normal, adenoma and adenocarcinoma colorectal tissues as well as in metastatic lesions. To overcome the problem of heterogeneity and normal stromal cell contamination in homogenized tissue specimens, specific cell types were isolated by microdissection prior to PCR analysis. No loss of PTEN expression was evident in any of the colon tissues examined. PTEN protein was localized exclusively in the cytoplasm of normal and tumour cells and no correlation of immunostaining intensity and tumour stage or grade was revealed. As with previous deletion and mutation analyses, the present study suggests that loss of PTEN expression is not prevalent in sporadic colon cancer.

Noninvasive diagnosis of bladder carcinoma by enzyme-linked immunosorbent assay detection of CD44 isoforms in exfoliated urothelia.

The expression of variant isoforms of the adhesion molecule CD44 is correlated with the onset of neoplasia in many carcinomas. We have previously shown that noninvasive detection of bladder carcinoma is possible by analysis of anomalous CD44 expression in exfoliated urothelia. Although the sensitivity and specificity values obtained for the detection of bladder tumors using RT-PCR and Western blotting methods were superior to those obtained using urine cytology, the application of such techniques is inconvenient for routine diagnostic use. We now report the design and development of a sandwich-ELISA system for the reliable detection of CD44 protein extracted from sedimented urothelial cells in voided urine. Naturally micturated urine samples were obtained from 53 patients with newly diagnosed bladder cancer and from 65 subjects with no evidence of disease; patients with gross hematuria were excluded because of interference with the assay. To demonstrate the diagnostic potential of the system, a "gate" was imposed at N (max), i.e., the highest absorbance value obtained from a sample known to be tumor free. All values above this value were assumed to be indicative of the presence of a tumor. Using this parameter, 42 of 53 (81.1%) patients with histologically confirmed bladder tumors were correctly diagnosed. Correspondingly, under these conditions, the assay is 100% specific for tumor detection, with a sensitivity of 81.1%, which equates to a positive predictive value of 100% and a negative predictive value of 81.1%. A further 54 patients who had previously received treatment for bladder cancer but were currently clinically disease-free were also investigated. Of these, 47 of 54 (87%) were correctly diagnosed to be tumor-free, which in this group equates to a positive predictive value of 87% and a negative predictive value of 100%. The data presented demonstrate that the rapid and accurate detection of elevated levels of CD44 protein isoforms in exfoliated urothelial cells is applicable to the identification and monitoring of primary and recurrent bladder cancer.

Role of telomerase in cell senescence and oncogenesis.

The ends of linear chromosomes are capped by specialized nucleoprotein structures termed telomeres. Telomeres comprise tracts of noncoding hexanucleotide repeat sequences that, in combination with specific proteins, protect against degradation, rearrangement, and chromosomal fusion events. Due to the polarity of conventional DNA synthesis, a net loss of telomeric sequences occurs at each cell division. It has been proposed that this cumulative telomeric erosion is a limiting factor in replicative capacity and elicits a signal for the onset of cellular senescence. To proliferate beyond the senescent checkpoint, cells must restore telomere length. This can be achieved by telomerase, an enzyme with reverse-transcriptase activity. This enzyme is absent in differentiated somatic tissues, but telomerase reactivation has been detected in most tumors. Much investigative effort is focusing on telomere dynamics with a view to possible manipulation of cellular proliferative potential. In this article, we review the role of telomeres and telomerase in senescence and tumor progression, and we discuss the potential use of telomerase in diagnosis and treatment.

Telomerase activity in soft-tissue and bone sarcomas.

The telomerase enzyme is a reverse transcriptase capable of replacing the telomeric DNA sequences that are lost at each cell division. Telomerase activation permits extended cell proliferation beyond normal senescence checkpoints, and accordingly, telomerase activity has been detected in a wide range of malignant cells and tissues but is absent in terminally differentiated somatic cells. To date, the majority of cancer-related telomerase analyses have been performed on carcinomas that originate from epithelial cells, and few reports have included tumors originating from nonepithelial cells. In this study, we used the PCR-based telomeric repeat amplification protocol (TRAP) to assay telomerase activity in nuclear protein extracts obtained from a range of malignant and benign connective tissue lesions. In total, 62 histologically diagnosed specimens were analyzed including 37 sarcomas, 7 benign mesenchymal tumors, 12 normal tissue samples, and 6 carcinoma metastases obtained from bone. Thirty (81%) of the 37 primary sarcoma samples contained telomerase activity, and four of the six carcinoma metastases were also positive. Conversely, telomerase activity was detectable in only one of seven benign lesions and in none of the 12 normal connective tissue controls. Tumors of connective tissue origin can sometimes be difficult to categorize and to evaluate microscopically with regard to clinical management. As is the case in carcinomas, the presence of telomerase activity appears to be indicative of malignancy in mesenchymal tumor biopsy material and therefore may be useful as an adjunct to the pathologist in the assessment of borderline cases.

Comparison of telomerase and CD44 expression as diagnostic tumor markers in lesions of the thyroid.

The analysis of the tissue expression patterns of both the telomerase enzyme and the adhesion molecule CD44 has highlighted these molecules as potential tumor markers. In this study, the expression of these markers was analyzed in frozen tissue samples of the same human thyroid lesions, and the data were compared to evaluate their application to tumor diagnosis. The study analyzed 12 malignant specimens, including 5 papillary, 3 follicular, 2 anaplastic, 1 medullary, and 1 low-grade Hurthle cell carcinoma and 17 specimens from benign lesions, including cases of adenoma, hyperplasia, and Graves' disease. Telomerase expression was analyzed by assay of enzyme activity using the telomeric repeat amplification protocol and by reverse transcription-PCR detection of human telomerase reverse transcriptase (hTERT) mRNA. Nine of 12 (75%) malignant samples and the two Graves' disease samples were evaluated as positive for telomerase activity by the telomeric repeat amplification protocol assay. The presence of hTERT mRNA was detected in 8 (67%) of 12 malignant tissues and in 5 (29%) of 17 benign thyroid tissue samples. The expression of CD44 transcripts containing variant exons 7, 8, and 11 was evaluated by reverse transcription-PCR/Southern blot analysis. Of the 12 malignant samples, 9 (75%) included transcripts containing exon 7, 10 (83%) included transcripts containing exon 11, and 11 (92%) included transcripts containing exon 8. However, these CD44 exons were also present in transcripts in a high proportion of benign samples. Five (28%), 10 (59%), and 6 (35%) benign samples contained CD44 transcripts, including variant exons 7, 8, and 11, respectively. The measurement of telomerase activity proved to be the most specific for the detection of thyroid carcinoma in frozen tissue samples as a single analyte, but diagnostic accuracy was increased by the combination of telomerase and CD44 analyses.

CD44 expression in cervical intraepithelial neoplasia (CIN) and carcinoma.

BACKGROUND: The expression of the CD44 gene is markedly changed in many neoplastic tissues. The identification of tumor-specific CD44 expression patterns may aid tumor diagnosis. METHODS AND RESULTS: The transcription and translation of the CD44 gene were analyzed by reverse transcription polymerase chain reaction (RT-PCR), in situ hybridization and by immunohistochemistry. Samples were obtained from 24 normal and 24 neoplastic or malignant human cervical tissues. Southern blot hybridization analysis of RT-PCR products revealed an increase in the size and number of CD44 standard and variant transcripts in malignant cervical tissues compared with corresponding normal tissues. Misprocessing of mRNA was indicated in cervical carcinoma cells by the retention of intronic sequences. Multiple CD44 mRNA and protein isoforms were present throughout carcinoma tissues, whereas localization was restricted to the basal epithelium in normal cervical tissue. Analysis of desquamated cervical cells from cases of cervical intraepithelial neoplasia stages I-III showed progressively deranged patterns of CD44 expression, with more alterations being detected in the more advanced stages. CONCLUSIONS: Marked alterations in CD44 expression occur in cervical tissues during progression to malignancy. CD44 expression analysis could aid the early diagnosis of cervical malignancy using minimally invasive methods.

CD44 cell adhesion molecules.

The CD44 proteins form a ubiquitously expressed family of cell surface adhesion molecules involved in cell-cell and cell-matrix interactions. The multiple protein isoforms are encoded by a single gene by alternative splicing and are further modified by a range of post-translational modifications. CD44 proteins are single chain molecules comprising an N-terminal extracellular domain, a membrane proximal region, a transmembrane domain, and a cytoplasmic tail. The CD44 gene has only been detected in higher organisms and the amino acid sequence of most of the molecule is highly conserved between mammalian species. The principal ligand of CD44 is hyaluronic acid, an integral component of the extracellular matrix. Other CD44 ligands include osteopontin, serglycin, collagens, fibronectin, and laminin. The major physiological role of CD44 is to maintain organ and tissue structure via cell-cell and cell-matrix adhesion, but certain variant isoforms can also mediate lymphocyte activation and homing, and the presentation of chemical factors and hormones. Increased interest has been directed at the characterisation of this molecule since it was observed that expression of multiple CD44 isoforms is greatly upregulated in neoplasia. CD44, particularly its variants, may be useful as a diagnostic or prognostic marker of malignancy and, in at least some human cancers, it may be a potential target for cancer therapy. This review describes the structure of the CD44 gene and discusses some of its roles in physiological and pathological processes.

Telomerase in cancer: clinical applications.

The biology of telomeres and telomerase has been the subject of intensive investigative effort since it became evident that they play a significant role in two important biological processes, the loss of cellular replicative capacity inherent to organismal ageing and the unrestricted cell proliferation characteristic of carcinogenesis. Telomere shortening in normal cells is a result of DNA replication events, and reduction beyond a critical length is a signal for cellular senescence. One of the cellular mechanisms used to overcome proliferative restriction is the activation of the enzyme telomerase, which replaces the loss of telomeric DNA that occurs at each cell division. Studies have demonstrated that tumours have shorter telomeres than normal tissue and that telomerase is activated in up to 90% of all human cancers while it is present only in a limited range of normal adult tissues. The role of telomerase in the extension of the cellular replicative lifespan has recently been shown by ectopic expression of the enzyme, being consistent with the oncogenesis model whereby the acquisition of an 'immortal' phenotype is a requirement for advanced tumour progression. In this article we review the present knowledge of telomeres and telomerase in cancer and discuss the potential use of this enzyme as a diagnostic and prognostic tumour marker and as a target for cancer therapy.

Multiple intron retention occurs in tumor cell CD44 mRNA processing.

Markedly increased overall levels of CD44 transcripts and proteins have been recognized in many tumors and the inappropriate expression and abnormal assembly of the CD44 variable exons has been linked to both tumor growth and metastatic potential. We have also previously observed the aberrant inclusion of intron 9 in CD44 mRNA transcripts in tumor tissues. In this study we assessed whether such retention is specific to certain introns or is a more general phenomenon affecting CD44 gene expression in tumor cells. Intron 18 was cloned and sequenced from genomic DNA and the novel sequences analyzed and used to create intron 18-specific probes. The newly characterized intron was found to have consensus 5' splice site and branchpoint sequences but a suboptimal 3' splice site. The status of CD44 intron 18 retention or excision was assessed in a colon tumor cell line (HT29) and in tissue from 20 colorectal tumors and matched normal mucosa. The intron was shown to be retained in transcripts from 15 of the 20 (75%) carcinomas but in only 3 of the 20 (15%) matched normal samples. These results compare with 80% retention of CD44 intron 9 in colonic carcinoma tissue mRNA and confirm that multiple abnormalities of CD44 mRNA processing occur in tumor cells.

Telomerase activity in lesions of the thyroid: application to diagnosis of clinical samples including fine-needle aspirates.

The telomerase enzyme is capable of replacing telomeric DNA sequences that are lost at each cell division. It has been suggested that the function of this enzyme is necessary for cells to become immortal, and in concordance with this hypothesis, telomerase activity has been detected in malignant tumor cells, whereas the enzyme is inactive in normal somatic cells. The measurement of this activity in human tissue samples may have diagnostic value, and in this study, we examined whether such a measurement may be useful for the detection of malignant cells within the thyroid. Telomerase activity was assayed using the telomeric repeat amplification protocol and related to the histological diagnosis of thyroid biopsy tissue samples and of cells obtained from the thyroid by fine-needle aspiration (FNA). Extracts from 9 of 11 (82%) carcinoma biopsy tissue samples contained telomerase activity, whereas enzyme activity was detected in only 2 of 14 (14%) benign tissue sample extracts. These two positive cases were subsequently diagnosed as Graves' disease with severe lymphocytic infiltration. Five of six (83.3%) histologically confirmed carcinoma FNA samples were identified by using the telomeric repeat amplification protocol assay, and two samples considered to be suspicious by FNA cytology were also positive. Conversely, only 4 of 48 (8.3%) benign FNA samples had telomerase. These promising data indicate that this sensitive assay could become a useful adjunct to microscopic cytopathology in the detection of cancer cells in small tissue biopsies and in fine-needle aspirates of the thyroid.

Differential gene expression in mammary carcinoma cell lines: identification of DRIM, a new gene down-regulated in metastasis.

Differential display technique was applied to a pair of cell lines derived from human breast carcinoma cell line MDA-MB 435 with metastatic and non-metastatic properties in the nude mouse system, with the objective to isolate genes involved in metastasis. DRIM (Down-Regulated In Metastasis) was the only gene found to be differentially expressed in this system. DRIM encodes a protein comprising 2785 amino acids with significant homology to a protein in yeast and C. elegans. The protein contains a conserved positively charged tail and several HEAT repeats, designated after four functionally characterized proteins in which the repeat was detected. Most of the hydrophobic regions of DRIM can be assigned to HEAT repeats. Expression of DRIM at the RNA level was investigated in several normal tissues and tumor cell lines.

Clinical implications of anomalous CD44 gene expression in neoplasia.

An intensive search continues for reliable markers that would be clinically useful in the diagnosis of small tumors and in the evaluation of their predicted clinical outcome. One potential marker, extensively studied in human samples is the cell surface adhesion molecule CD44. The single CD44 gene codes for a large family of cell surface proteins by alternative splicing and severe abnormalities have been observed in the patterns of its expression in many types of human tumors using both protein and RNA-based analyses. These abnormalities are manifested by markedly increased levels of unusual transcripts and proteins in tumor cells compared to the corresponding normal tissues. Aberrant processing of immature CD44 transcripts has also been observed in tumor cells and this can lead to the inappropriate retention of introns and to the use of cryptic splice sites in the mRNA. Inappropriate expression patterns of the alternatively spliced exons have also been linked both to tumor progression and to metastatic potential. The clinical relevance of all these observations is demonstrated by the frequent detection of these abnormalities in samples from malignant tumors of many different organs and by their presence in pre-invasive and high risk pre-cancerous lesions. This article reviews the current information regarding the expression of the CD44 gene in tumor cells and its potential use as a marker in clinical evaluation. The overall conclusion is that with the use of the latest assay techniques and perhaps in combination with other molecular markers, analysis of CD44 expression can provide new and powerful assays for the detection of neoplastic disease.

CD44 variant isoform expression and breast cancer prognosis.

We examined the expression of CD44 isoforms in samples of breast cancer tissues from 95 patients by reverse transcription-polymerase chain reaction and immunohistochemistry, and tried to correlate the results with survival period. At the RNA level, expression of exon v2 was observed in 33 (35%) and that of v6 in 69 (73%) of the 95 specimens. Patients with CD44v2 mRNA expression had significantly shorter survival times than those with v2-negative tumors (P = 0.05), but there was only a weak correlation, if any, between v6 mRNA expression and overall survival (P = 0.06). Tumor tissue from 22 (23%) and 72 (76%) patients showed positive immunoreactivity with monoclonal antibody (mAb) M23.6.1. (CD44v2) and mAb 2F10 (CD44v6), respectively. Immunohistochemical evidence of CD44v2 peptide expression correlated with overall survival (P = 0.02), but there was no such association with CD44v6 expression in these tumors (P = 0.67). There were significant correlations between v2 immunoreactivity and higher histological grade and lower levels of estrogen and progesterone receptor. There was no significant correlation between v6 immunoreactivity and such clinicopathological characteristics. Although the expression of v2 was significantly associated with reduced overall survival, it was not an independent prognostic factor because it also correlated with progesterone receptor status. These findings suggest that v2 isoform expression might have more value than v6 expression for clinical use.

Disorderly CD44 gene expression in human cancer cells can be modulated by growth conditions.

Disorderly CD44 gene expression is a well-documented characteristic feature of tumour cells from cancers arising in many different organs of the human body. Molecular pathological studies have established that the pattern of the abnormal expression can differ according to the origin and the stage of the tumour. In this investigation it has been demonstrated that in some but not all tumour cell lines, which are undeniably and irreversibly malignant when inoculated in vivo, CD44 gene expression can still be modulated. In two cell lines, the pattern of CD44v expression was found to be affected by cell-to-cell and cell-to-substrate attachment. Expression was up-regulated by cell-substrate interactions, but only until cell-to-cell contact caused subsequent down-regulation of CD44v transcription. This information provides new opportunities for detailed investigation of the mechanisms of abnormal CD44 gene regulation in cancer and for exploring stage-related changes in the expression of this complex gene.

Telomerase activity and its inhibition in benign and malignant breast lesions.

Many types of human tumours and immortal cell lines have been demonstrated to exhibit telomerase activity with the recently formulated telomeric repeat amplification protocol (TRAP assay). However, a small proportion of undoubted tumour samples give a negative result and it has been postulated that, on occasion, the assay can be blocked by inhibitory factors in the cell or tissue extracts. To resolve this issue, a modified TRAP assay has been used to re-examine 45 previously negative breast tissue specimens. Phenol--chloroform extraction of the sample after the telomerase extension reaction revealed the presence of polymerase chain reaction (PCR) inhibitory factors in tissue from 6 of 14 (43 per cent) breast biopsies of fibrocystic disease (FCD), 6 of 12 (50 per cent) fibroadenomas (FAs), none of five carcinomas in situ, and 1 of 13 (8 per cent) invasive carcinoma (CA) tissue specimens. These results demonstrated that the enzyme telomerase can be active in some benign lesions as well as in carcinomas of the breast. Specimens which still remained negative for telomerase in the above experiment were next assayed for the presence of biologically relevant inhibitors of the enzyme by mixing the extracts with confirmed positive samples. Extracts from 12 of 17 carcinoma specimens (all of five carcinomas in situ and 7 of 12 invasive carcinomas showed dose-dependent inhibitory activity against telomere extension, whereas no inhibition was observed in those of three of eight FCD and 2 of seven FAs. These results indicate that telomerase activity may be regulated by a balance between inhibitory factors and an activated enzyme.