Phase I study of continuous and intermittent schedules of lapatinib in combination with vinorelbine in solid tumors.

BACKGROUND: Chemotherapy in combination with small-molecule epidermal growth factor receptor inhibitors has yielded inconsistent results. Based on preclinical models, we conducted a phase I trial of two schedules of lapatinib and vinorelbine. PATIENT AND METHODS: Patients had advanced solid tumors and up to two prior chemotherapeutic regimens. Patients were enrolled on two dose-escalating schedules of lapatinib, continuous (arm A) or intermittent (arm B), with vinorelbine on days 1, 8, and 15 of a 28-day cycle. Tumors from a subset of patients were evaluated for gene mutations and expression of targets of interest. RESULTS: Fifty-one patients were treated. The most common grade 3/4 toxic effects included leukopenia, neutropenia, and fatigue. Dose-limiting toxic effects were grade 3 infection, febrile neutropenia, and diarrhea (arm A) and bone pain and fatigue (arm B). The maximum tolerated dose was vinorelbine 20 mg/m(2) weekly and lapatinib 1500 mg daily (arm A) and vinorelbine 25 mg/m(2) weekly and lapatinib 1500 mg intermittently (arm B). One patient on each arm had a complete response; both had human epidermal growth factor receptor 2-positive breast cancer. In a subset of patients, lack of tumor PTEN expression correlated with a shorter time to progression. CONCLUSION: In an unselected population, two schedules of lapatinib and vinorelbine were feasible and well tolerated.

Consensus for HER2 alterations testing in non-small-cell lung cancer.

Human epidermal growth factor receptor 2 (HER2) is a transmembrane glycoprotein receptor with intracellular tyrosine kinase activity. Its alterations, including mutation, amplification and overexpression, could result in oncogenic potential and have been detected in many cancers such as non-small-cell lung cancer (NSCLC). Such alterations are, in general, considered markers of poor prognosis. Anti-HER2 antibody-drug conjugates, e.g. trastuzumab deruxtecan (T-DXd, DS-8201) and disitamab vedotin (RC48), were recently approved for HER2-positive breast and gastric cancers. Meanwhile, several HER2-targeted drugs, such as T-DXd, neratinib, afatinib, poziotinib and pyrotinib, have been evaluated in patients with advanced NSCLC, with several of them demonstrating clinical benefit. Therefore, identifying HER2 alterations is pivotal for NSCLC patients to benefit from these targeted therapies. Recent guidelines on HER2 testing were developed for breast and gastric cancer, however, and have not been fully established for NSCLC. The expert group here reached a consensus on HER2 alteration testing in NSCLC with the focus on clinicopathologic characteristics, therapies, detection methods and diagnostic criteria for HER2-altered NSCLC patients. We hope this consensus could improve the clinical management of NSCLC patients with HER2 alterations.

Bone turnover biomarkers identify unique prognostic risk groups in men with castration resistant prostate cancer and skeletal metastases: Results from SWOG S0421.

BACKGROUND: Skeletal metastases often occur in men with castration-resistant prostate cancer (CRPC) where bone biomarkers are prognostic for overall survival (OS). In those with highly elevated markers, there is preferential benefit from bone-targeted therapy. In the phase IIIS0421 docetaxel +/- atrasentan trial, clinical covariates and bone biomarkers were analyzed to identify CRPC subsets with differential outcomes. SUBJECTS AND METHODS: Markers of bone resorption [N-telopeptide-NTx; pyridinoline-PYD] and formation [C-terminal collagen propeptide-CICP; bone alkaline phosphatase-BAP] were measured in pre-treatment sera. Bone biomarkers and clinical covariates were included in a Cox model for OS; bone markers were added in a stepwise selection process. Receiver operating characteristic (ROC) curves were constructed for risk factor models +/- bone markers. Significant variables were allowed to compete in a classification and regression tree (CART) analysis. Hazard ratios(HR) were calculated by comparing OS in each of the terminal nodes to a reference group in a Cox model. RESULTS: 750 patients were included. Each bone marker significantly contributed to the risk factor-adjusted OS Cox model, with higher levels associated with worse OS. BAP (HR = 1.15, p = 0.008), CICP (HR = 1.27, p < 0.001), and PYD (HR = 1.21, p = 0.047) in combination were significantly associated with OS. Prognostic accuracy was improved by addition of bone markers to clinical covariates. CART analysis selected CICP, BAP, hemoglobin, and pain score for the final OS model, identifying five prognostic groups. CONCLUSIONS: Elevated serum bone biomarker levels are associated with worse OS in bone-metastatic CRPC. Bone biomarkers can identify unique prognostic subgroups. These results further define the role of bone biomarkers in the design of CRPC trials.

Origin and prognostic value of circulating KRAS mutations in lung cancer patients.

Because of the current controversy on the origin and clinical value of circulating KRAS codon 12 mutations in lung cancer, we screened 180 patients using a combined restriction fragment-length polymorphism and polymerase chain reaction (RFLP-PCR) assay. We detected KRAS mutations in 9% plasma samples and 0% matched lymphocytes. Plasma KRAS mutations correlated significantly with poor prognosis. We validated the positive results in a second laboratory by DNA sequencing and found matching codon 12 sequences in blood and tumor in 78% evaluable cases. These results support the notion that circulating KRAS mutations originate from tumors and are prognostically relevant in lung cancer.

Yttrium-90 chimeric L6 therapy of human breast cancer in nude mice and apoptosis-related messenger RNA expression.

Radioimmunotherapy (RIT) in breast cancer patients using I-131-chimeric L6 (ChL6) and in human breast cancer xenografts in nude mice using Y-90-1,4,7,10-tetraazacylododecant N,N',N",N"'-tetraacetic acid-peptide ChL6 (Y-90-ChL6) has shown promise. Tumor cell response to low-dose rate (5-25 rads/h) irradiation from Y-90-ChL6 RIT, therefore, was correlated with levels of tumor cell mRNA for selected genes linked to programmed cell death (apoptosis). Three groups of 10-16 mice with 1-2 HBT 3477 xenograft tumors were treated with 100, 150, or 250 microCi Y-90-ChL6. Three tumors were taken before and two tumors each were taken 3, 6, and 24 h after injection of 150 microCi Y-90-ChL6. Tumor expression of mRNA was amplified by PCR for p53, PIC1, c-myc, and transforming growth factor-beta 1; quantitated; and standardized to N-ras. Tumors received radiation doses of 2000, 3000, and 5000 rads, respectively, for the groups of mice that received 100, 150, and 250 microCi Y-90-ChL6, and tumor regression occurred in each group, with mean tumor volumes decreased by 10, 50, and 95% at nadir after Y-90-ChL6 injection. At the highest dose level, 30% of mice had complete remissions, and no treatment deaths occurred, although tumors subsequently recurred. Continuous up-regulation of transforming growth factor-beta 1 and c-myc mRNA expression was observed from 3 to 24 h after treatment. Expression of p53 and PIC1 increased at 3 h and subsequently decreased to the untreated control levels. These observations are consistent with previous observations of early responses of p53 and PIC1 to cellular DNA damage and subsequent G1 cell cycle arrest or apoptosis. Apoptosis-associated gene expression patterns observed in this tumor model provide evidence that changes are initiated in the first 24 h of RIT associated with radiation doses of 100-700 rads. These preliminary data suggest that insight into the molecular basis of RIT-induced tumor regression may be gained by further studies using different radiation doses.

RB status as a determinant of response to UCN-01 in non-small cell lung carcinoma.

7-Hydroxystaurosporine (UCN-01), a protein kinase inhibitor in clinical development, demonstrates potent antineoplastic activity. To determine whether specific genetic abnormalities would modulate the response to UCN-01, a model of human non-small cell lung carcinoma (NSCLC) cell lines with differential abnormalities of p16CDKN2, RB, and p53 was used for these studies. Cell growth was measured by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay, and cell cycling was studied using flow cytometric analysis of DNA content. Changes in protein levels and phosphorylation were assessed by Western blotting. In cell lines expressing wild-type RB (A549 and Calul), UCN-01 treatment resulted in dose-dependent growth inhibition, arrest of cells in G1, and a reduction of cells in S phase. p16CDKN2-null cells showed similar growth inhibition to normal fetal lung fibroblasts. UCN-01-induced growth arrest was accompanied by induction of p21CDKN1 and a shift of Rb to the hypophosphorylated state in both p53 wild-type and mutant cell lines. In contrast, UCN-01 treatment of the RB-null cell line H596 resulted in less growth inhibition. To test the role of RB in response to UCN-01, effects of treatment were examined in two human isogenic models of RB expression: the bladder cancer cell line 5637 (RB-null) and the prostate cancer cell line DU-145 (RB-mutant). In the Rb-expressing 5637 subline (RB5), UCN-01 treatment resulted in Rb hypophosphorylation and an accumulation in G1 in contrast to the parent line. Similarly, the wild-type Rb-expressing DU-145 sublines (DU1.1 and B5) showed increased G1 arrest compared with the parent cells. We conclude that UCN-01-induced G1 arrest can occur in cells null for p53 and p16CDKN2, and that RB status influences the ability of UCN-01 to induce a G1 arrest. These data suggest that the molecular profile of cell cycle regulating genes in individual tumors may predict responsiveness and provide insight into optimal therapeutic application of this new antineoplastic agent.

Innovative Clinical Trials: The LUNG-MAP Study.

Although the proportion of patients with squamous cell carcinoma of the lung has declined over the last two decades, the disease is still fatal for tens of thousands of patients each year. The treatment of non-small cell lung cancer has advanced rapidly over the past decade, providing novel, targeted therapeutic options to patients, but has mostly been limited to the adenocarcinoma histology. Efforts are currently underway to bring squamous cell carcinoma of the lung into this new era of targeted therapy. This article reviews the rationale and trial design for the "LUNG-MAP: S1400 Phase II/III Biomarker-Driven Master Protocol for Second Line Therapy of Squamous Cell Lung Cancer" study. This multi-institutional, multi-cooperative group trial aims to individualize treatment for patients with metastatic squamous cell carcinoma to one of five arms based on the genomic profile of the tumor. The goal of this clinical trial is to rapidly identify new active drugs and bring them as soon as possible through a registration process for patients with squamous cell lung cancer by utilizing a novel trial design and involving all key stakeholders in drug development in a national effort. This could serve as a paradigm for drug development for malignancies with wide molecular heterogeneity.

Activity of high-dose toremifene plus cisplatin in platinum-treated non-small-cell lung cancer: a phase II California Cancer Consortium Trial.

PURPOSE: Although cisplatin is an important agent in non-small-cell lung cancer (NSCLC), de novo resistance is common and acquired resistance emerges rapidly during therapy. Proposed mediators of platinum resistance include the protein kinase C (PKC) signal transduction pathway and associated c-FOS overexpression. While estrogen administration has been reported to upregulate PKC and c-FOS expression, the triphenylethylenes tamoxifen and toremifene potentiate platinum cytotoxicity by inhibition of PKC. Downregulation of c-FOS expression has been reported to result from PKC inhibition. In view of these findings, we hypothesized that toremifene would reverse platinum resistance and that this interaction would be influenced by tumor estrogen receptor (ER) status. MATERIALS AND METHODS: A phase II trial of high-dose toremifene (600 mg orally daily on days 1-7) plus cisplatin (50 mg/m2 intravenously on days 4 and 11) every 28 days in NSCLC patients was conducted. A group of 30 patients with metastatic NSCLC who had been previously treated with platinum-based therapy were enrolled. RESULTS: All of the 30 patients were assessable for toxicity and 28 for tumor response. Therapy was well tolerated with minimal hematologic and non-hematologic toxicity. Common toxicity criteria grade 3 hematologic toxicity was seen in only three patients. Five patients achieved a partial response for an overall response rate of 18% (95% CI 6-37). Median overall survival was 8.1 months (95% CI 5.4-17). To assess PKC, ER, and c-Fos expression by immunohistochemistry, 12 informative pretreatment patient tumor specimens were obtained. Four patient tumor specimens were positive for one or both PKC isoforms (alpha and epsilon) while c-Fos was overexpressed in three. None of the responding patient tumors exhibited c-FOS or PKC-epsilon overexpression. ER expression was found to be infrequent (8%), contrasting with previous reports in this tumor type. CONCLUSION: While this phase II study indicates that high-dose toremifene plus cisplatin is feasible, active, and well tolerated in NSCLC patients previously treated with platinum compounds, the mechanism of action remains unclear. Further study of this regimen is warranted.

Abnormalities of apoptotic and cell cycle regulatory proteins in distinct histopathologic components of benign prostatic hyperplasia.

INTRODUCTION: Benign prostatic hyperplasia (BPH) is a slowly progressive abnormal glandular enlargement with heterogeneous morphology. Disruption of apoptotic pathways has been suggested as an important regulatory mechanism in this common and significantly morbid disease. METHODS: Prostatic tissue from 20 patients with BPH and no prior or subsequent prostatic carcinoma was obtained by transurethral prostatectomy (TURP) at the University of California Davis. Apoptotic regulatory proteins: BCL2, BAX and p27 were analyzed by immunohistochemistry and evaluated for expression in four distinct histologic patterns: hyperplastic epithelium, nodules, dilated glands and atrophic/inflammatory glands. RESULTS: Particularly striking was the decreased expression of BAX and an abnormal BCL2 : BAX ratio within all nodules relative to expression in other epithelial patterns. p27 expression was decreased in 35% of the hyperplastic epithelial areas and 10% of the nodules. DISCUSSION: Overall, abnormal expression of BCL2, BAX and/or p27 was identified in the hyperplastic epithelium of 19 (90%) of specimens and all 12 (100%) of the hyperplastic nodules. The high frequency of abnormalities in apoptosis regulatory genes, suggests that alteration of apoptotic pathways is important for the development of this condition.

Apoptosis-related gene and protein expression in human lymphoma xenografts (Raji) after low dose rate radiation using 67Cu-2IT-BAT-Lym-1 radioimmunotherapy.

Despite low radiation dose rates, radioimmunotherapy (RIT) has proven particularly effective in the treatment of malignancies, such as lymphoma. Apoptosis has been suggested to be a major mechanism for cell death from continuous low-dose rate radiation from radioimmunotherapy. The goal of this study was to examine Raji lymphoma xenografts for induction of apoptosis and modulation of apoptosis-related gene and protein expression in response to 67Cu-2IT-BAT-Lym-1 RIT. In preclinical and clinical trials, 67Cu-2IT-BAT-Lym-1 has shown an exceptionally long tumor residence time associated with substantial cumulated radiation doses. The Raji model mirrors human lymphomas that have mutant p53 and increased BCL2 expression. Untreated athymic BALB/c nu/nu mice and mice treated with 400 micrograms Lym-1, or 335-500 microCi 67Cu on less than 400 micrograms Lym-1 antibody, were observed for toxicity and response over 84 days. Subgroups of 4-5 mice were sacrificed at 3, 6 and 24 h after therapy so that tumors could be examined for poly(ADP-ribose) polymerase (PARP) and DNA ladder evidence for apoptosis and for BCL2, p53, p21, GADD45, TGF-beta 1 and c-MYC gene and protein expression. Untreated tumors had little evidence of apoptosis and Lym-1 had no effect on apoptosis or gene expression. 67Cu-2IT-BAT-Lym-1 RIT induced an overall response rate of 50% with tolerable toxicity, and 29% of the tumors were cured at cumulated tumor radiation doses of about 1800 cGy. Apoptosis was greatly increased in the RIT treated Raji xenografts as evidenced by cleavage of PARP to the characteristic 85 kD fragment at 3 and 6 h and by the DNA cleavage pattern. BCL2 gene and protein expression were substantially decreased at 3 and 24 h, respectively, after 67Cu-2IT-BAT-Lym-1 RIT despite only modest cumulated radiation doses (56 cGy at 3 h). Evidence for apoptosis preceded tumor regression by 4-6 days. In these therapy-resistant, human lymphoma tumors treated with 67Cu-2IT-BAT-Lym-1, apoptosis was convincingly demonstrated to be a major mechanism for the effectiveness of RIT and occurred by p53-independent mechanisms.

KRAS mutations in non-small-cell lung cancer and colorectal cancer: implications for EGFR-targeted therapies.

BACKGROUND: KRAS mutations are associated with diverse biologic functions as well as prognostic and predictive impact in non-small cell-lung cancer (NSCLC) and colorectal cancer (CRC). In CRC, benefit from monoclonal antibody therapies targeting EGFR is generally limited to patients whose tumors have wild-type (WT) KRAS, whereas data suggest that this association is not present for NSCLC. We hypothesized that the unique tobacco-related carcinogenesis of NSCLC results in a divergence of KRAS MT genotype compared with CRC, contributing to differences in outcomes from EGFR-targeted therapies. MATERIAL AND METHODS: Tumor from 2603 patients (838 CRC and 1765 NSCLC) was analyzed for KRAS mutations. DNA was extracted from microdissected formalin-fixed-paraffin-embedded specimens (FFPE) and 7 different base substitutions in codons 12 and 13 of KRAS were determined. RESULTS: KRAS mutation genotype differed significantly between NSCLC and CRC in frequency (25% vs. 39%; p<0.001), smoking-associated G>T transversions (73% versus 27%; p<0.001), and ratio of transversions to transitions (3.5 vs. 0.79; p<0.001). In NSCLC GLY12Cys mutations, resulting from a codon 12 GGT>TGT substitution, were observed in 44% compared to 10% for CRC. In contrast, codon 12 or 13 GLY>ASP substitutions (resulting in a G>A transition) were more frequent in CRC (42%) compared with NSCLC (21%). CONCLUSION: In this large dataset, KRAS mutation patterns are quantitatively and qualitatively distinct between NSCLC and CRC, reflecting in part differences in tobacco-related carcinogenesis. In light of differences in predictive value for EGFR-directed monoclonal antibody therapy and prognosis for specific KRAS mutations between NSCLC and CRC, these data provide an underlying biologic rationale.

Inhibition of Akt pathways in the treatment of prostate cancer.

Akt is a serine/threonine kinase mediating multiple intracellular pathways involved in prostate cancer (CaP) biology. Increased understanding of the molecular mechanisms of Akt activation and signaling have led to the development of an increasing number of Akt inhibitors. These biologic agents demonstrate activity against a wide range of cancers in preclinical studies. Clinical studies of Akt inhibition in CaP are in progress, including agents such as celecoxib, perifosine and genistein. How best to integrate Akt inhibitors with standard CaP therapy or select patients most likely to benefit is the subject of ongoing research.

Increased RB1 abnormalities in human primary prostate cancer following combined androgen blockade.

BACKGROUND: The RB1 proliferation control pathway is a critical determinant of cell cycle progression. Abnormalities of RB1 are found in a variety of cancers, and the association with human prostate cancer (CaP) was examined here. METHODS: RNA expression levels of RB1 in CaPs were examined by RT-PCR. RNA integrity was assessed by evaluating expression of an endogenous gene standard. RESULTS: Abnormally low RB1 mRNA expression was found in 12/33 (36%) of CaPs from patients who had received combined androgen blockade (CAB) treatment. In contrast, 6/48 (13%) untreated CaPs showed abnormally low expression. This difference was statistically significant (P = 0.015). In the samples from untreated patients, a higher frequency of abnormal RB1 was found in specimens with a higher Gleason grade (P = 0.038). In addition, one untreated stage C, grade 9 specimen was found to express RB1 transcripts lacking exon 22, as determined by sequencing of DNA from the truncated transcripts. CONCLUSIONS: These findings suggest that abnormalities of RB1 may contribute to hormone-withdrawal-related survival of CaP cells.

Decreased C-MYC and BCL2 expression correlates with methylprednisolone-mediated inhibition of Raji lymphoma growth.

Methylprednisolone (MP) and related corticosteroids are a fundamental part of regimens used to treat lymphoma and leukemia. In many of these malignancies, oncogenic activation of C-MYC and BCL2 is seen. Abnormalities of the tumor suppressor p53, which exerts growth-suppressing and apoptosis-enhancing functions through the transcriptional regulation of downstream genes including CDKN1, GADD45, and BCL2, are also often found. The goal was to determine the modulation of expression of the oncogenes (C-MYC and BCL2), the p53 pathway described above, and the apoptosis marker TGF-beta 1 in the human Raji lymphoma following MP treatment. Raji xenografts were grown in nude mice and growth curves characterized by sequential measurement. Mice were treated daily for 8 days with MP. Tumors were harvested untreated, or at 1 or 8 days after cessation of MP treatment, and the RNA was extracted. RT-PCR was used to determine the level of mRNA expression of the genes. Tumor growth was greatly reduced in the MP-treated mice. Gene expression levels for C-MYC and BCL2 were reduced at 1 day following MP and approached control levels 8 days after MP treatment. Expression levels of p53, CDKN1, and GADD45 were moderately and coordinately decreased at 1 day after cessation of MP treatment and remained repressed a week later. TGF-beta 1 exhibited no change in expression levels. These results suggest that decreased expression of C-MYC and BCL2 may play a role in the molecular events that initiate and are responsible for the growth inhibition of Raji lymphoma xenografts by MP.

Overexpression of cyclin D1 is rare in human prostate carcinoma.

BACKGROUND: Overexpression of cyclin D1 has been documented in a number of human cancers. Increased expression of cyclin D1 can contribute to cellular transformation and abnormal proliferation. METHODS: Quantitative RT-PCR and/or Western blot analysis were used to determine the level of cyclin D1 expression in 96 human prostate tumors, 15 benign prostate hyperplasias, 4 prostate cancer cell lines, and 3 xenografts. RESULTS: Our results demonstrate that 4.2% of the prostate tumors examined overexpressed cyclin D1 transcripts. In the cell lines, expression was normal, with the exception that reduced levels of cyclin D1 transcript and protein were observed in the DU145 cell line, as expected from cells with mutant RB. Normal levels of cyclin D1 were found in all xenograft tumors and BPH specimens examined. CONCLUSIONS: These data show that overexpression of cyclin D1 occurs rarely in human prostate tumors. However, when overexpression of cyclin D1 does occur, it may identify a subset of tumors with a different molecular biology.