Recent studies demonstrate an increased risk of second primary malignancies (SPM) in multiple myeloma (MM) patients on maintenance lenalidomide following autologous stem cell transplantation (ASCT). There may be other risk factors driving SPM post-ASCT in MM, so we explored this possibility through analysis of our large transplant database in conjunction with our long-term followup program. We conducted a retrospective cohort study of 841 consecutive MM patients who underwent ASCT from 1989–2009 at City of Hope, as well as a nested case-control analysis evaluating the role of all therapeutic exposures before, during and after ASCT. Median length of follow-up for the entire cohort was 3.4 years (range 0.3–19.9). Sixty cases with seventy SPM were identified. The overall cumulative incidence of SPM was 7.4% at five years and 15.9% at ten years if non-melanoma skin cancers (NMSCs) were included, and 5.3% at 5 years and 11.2% at 10 years if NMSCs were excluded. Multivariate analysis of the entire cohort revealed association of both older age (≥55yrs) (RR 2.3 p<0.004) and race (non-Hispanic white) (RR 2.4 p=0.01) with an increased risk of SPM. Furthermore, thalidomide exposure demonstrated a trend towards increased risk (OR=3.5, p=0.15); however, not enough patients were treated with lenalidomide to accurately assess the risk of this agent. Exclusion of NMSCs retained the association with these variables, but was accompanied with loss of statistical significance. This large single-institution analysis identified race and older age to be associated with increased risk of developing SPM. The trend toward increased risk with thalidomide exposure suggests a class effect from immunomodulatory drugs that may not be restricted to lenalidomide.
second primary malignancy; transplantation; myeloma
TBI plays an important role in patients undergoing HCT, but is associated with significant toxicities. Targeted TBI using helical tomotherapy (HT) results in reduced doses to normal organs, which predict for reduced toxicities compared to standard TBI.
Methods and Materials
Thirteen patients with multiple myeloma (MM) were treated on an autologous tandem transplant Phase I trial with high dose melphalan, followed 6 weeks later by TMI to skeletal bone. Doses levels were 10, 12, 14, and 16 Gy at 2 Gy QD/BID. On a separate allogeneic HCT trial, 8 patients (5 AML, 1 ALL, 1 NHL, 1 MM) were treated with TMI+TLI + splenic RT to 12 Gy (1.5 Gy BID) combined with fludarabine/melphalan.
For the 13 patients on the tandem autoHCT trial, median age was 54 (42–66). Median organ doses were 15–65% that of the GTV dose. Grade 1–2 acute toxicities were primarily observed. Six reported no vomiting, 9 no mucositis, 6 no fatigue, and 8 no diarrhea. For the 8 patients on the alloHCT trial, the median age was 52 (24–61). Grade 2–3 nausea, vomiting, mucositis and diarrhea were observed. On both trials no grade 4 non-hematologic toxicity was observed and all patients engrafted successfully.
This study demonstrates that TMI using HT is clinically feasible. Reduced acute toxicities observed compare favorably to those seen with standard TBI. Initial results are encouraging and warrant further evaluation as a method to dose escalate with acceptable toxicity or to offer TBI containing regimens to patients unable to tolerate standard approaches.
helical tomotherapy; IMRT; bone marrow transplantation; multiple myeloma; leukemia
We analysed the long-term outcomes of 60 multiple myeloma patients who underwent reduced intensity allogeneic stem cell transplantation between August 2000 and March 2008. Regimens included fludarabine and melphalan conditioning (flu-mel regimen) for allogeneic haematopoietic cell transplant (HCT) or a planned tandem regimen consisting of high-dose melphalan conditioning for autograft followed by low-dose total body irradiation conditioning for allogeneic HCT (auto-allo regimen). Donors included human leucocyte antigen-matched siblings (n=55) or matched unrelated donors (n=5). With a median follow-up of 9.8 years, 7-year overall survival (OS) and progression-free survival (PFS) were 60% and 31%, respectively. By multivariate Cox regression analysis, disease status of complete response (CR) or partial response (PR) at transplant and the presence of chronic graft-versus-host disease were significantly associated with improved OS. Only disease status was significantly associated with improved PFS. We noted a surprising number of very late relapses, with six patients (10%) relapsing between 6 and 12 years post-transplant. Among the six late relapse patients, all were transplanted within 14 months of diagnosis, 5 had normal karyotypes, and 5 were in CR/PR. Our data provide additional evidence that, while survival may be extended by reduced intensity allogeneic transplant, ultimately, it may not offer a cure.
multiple myeloma; reduced intensity; allogeneic; tandem auto-allo; transplantation
Docetaxel and vinorelbine have demonstrated Single-agent activity in breast cancer. Preclinical studies suggest potential synergy between these antitubulin chemotherapy agents. This study evaluates these drugs in combination in metastatic breast cancer. Taxane-naive patients with HER-2 negative, stage IV breast cancer without prior chemotherapy for metastatic disease, were eligible. Docetaxel (60 mg/m2) was given intravenously on Day 1, vinorelbine (27.5 mg/m2) intravenously on Days 8 and 15, and filgrastim on Days 2–21 of a 21-day cycle. The primary study outcome was one-year overall survival (OS), with secondary outcomes of progression-free survival (PFS), response rate (RR), and toxicity. Of 95 patients registered, 92 were eligible and received treatment. One-year OS was 74 % (95 % CI 64–82 %) with a median OS of 22.3 months (95 % CI 18.8–31.4 months). One-year PFS was 34 % (95 % CI 24–43 %) with median of 7.2 months (95 % CI 6.4–10.3). OS at 2 and 3 years were 49 % (95 % CI 38–59 %) and 30 % (95 % CI 21–40 %), respectively. OS was poorer for women with estrogen-receptor negative disease (n = 32) compared to estrogen-receptor positive (n = 60) (log-rank p = 0.031), but PFS was not significantly different (p = 0.11). RR was 59 % among the 74 patients with measurable disease. Grade 3 and 4 adverse events were 48 and 16 %, respectively. Grade 4 neutropenia was 12 % and grade 3/4 febrile neutropenia was 3 %. Common grade 3/4 nonhematologic toxicities were fatigue (14 %), pneumonitis (10 %), and dyspnea (9 %). The combination of docetaxel and vinorelbine is an active first-line chemotherapy in HER-2 nonoverexpressing, metastatic breast cancer. This combination is associated with significant hematologic and nonhematologic toxicity. The safety profile and expense of the filgrastim limit recommendations for routine use.
Breast cancer; Vinorelbine; Docetaxel; Metastatic
The probability of survival is conventionally calculated from autologous hematopoietic cell transplantation (aHCT). Conditional survival takes into account the changing probability of survival with time survived, but is not known for aHCT populations. We determined disease-, and cause-specific conditional survival for 2388 patients treated with aHCT over a period of 20 years at a single institution. A total of 1054 deaths (44% of the cohort) were observed: 78% attributed to recurrent disease; 9% to subsequent malignancies; and 6% to cardiopulmonary disease. Estimated probability of relative survival was 62% at 5 and 50% at 10 years from aHCT. On the other hand, 5-year relative survival was 70%, 75%, 81%, and 88% after having survived 1, 2, 5, and 10 years after aHCT, respectively. The cohort was at a 13.9-fold increased risk of death compared with the general population (95%CI=13.1–14.8). The risk of death approached that of the general population for 10-year survivors (SMR=1.4, 95%CI=0.9–1.9), with the exception of female Hodgkin lymphoma patients transplanted before 1995 at age 40 years or younger (SMR=6.0, 95%CI=1.9–14.0). Among those who had survived 10 years, non-relapse-related mortality rates exceeded relapse-related mortality rates. This study provides clinically relevant survival estimates after aHCT, and helps inform interventional strategies.
conditional survival; autologous HCT; cause-specific mortality
These NCCN Guidelines Insights highlight the important updates/changes specific to the management of metastatic breast cancer in the 2012 version of the NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines) for Breast Cancer. These changes/updates include the issue of retesting of biomarkers (estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2) on recurrent disease, new information regarding first-line combination endocrine therapy for metastatic disease, a new section on monitoring of patients with metastatic disease, and new information on endocrine therapy combined with an mTOR inhibitor as a subsequent therapeutic option.
To establish feasibility, maximum tolerated dose, and potential efficacy of ablative dose total marrow irradiation (TMI) delivered by helical tomotherapy, in patients with multiple myeloma (MM).
Patients with responding or stable MM received tandem autologous transplants (TASCT), first with melphalan 200 mg/m2, and ≥ 60 days later, with TMI. TMI doses were to be escalated from 1000 cGy, by increments of 200 cGy. All patients received thalidomide and dexamethasone maintenance.
Twenty two of 25 enrolled patients (79%) received TASCT: TMI was administered a median of 63.5 days (44–119) after melphalan. Dose limiting toxicities at level 5 (1800 cGy) included reversible grade 3 pneumonitis, congestive heart failure, and enteritis (1), and grade 3 hypotension (1). The estimated median radiation dose to normal organs was 11–81% of the prescribed marrow dose. Late toxicities included reversible enteritis (1), and lower extremity deep venous thrombosis during maintenance therapy (2). The complete and very good partial response rates were 55% and 27%, following TASCT and maintenance therapy. At a median of 35 months of follow-up (21-50+ months) progression-free and overall survival for all patients are 49 % (95% CI 0.27-0.71) and 82% (0.67-1.00).
Ablative dose TMI as part of TASCT is feasible, and the complete response rate is encouraging. Careful monitoring of late toxicities is needed. Further assessment of this modality is justified at the 1600 cGy maximum tolerated dose level in MM patients who are candidates for ASCT.
multiple myeloma; total marrow irradiation; tandem autologous transplantation
Cancer stem cells (CSCs) play critical roles in cancer initiation, progression, and therapeutic refractoriness. Although many studies have focused on the genes and pathways involved in stemness, characterization of the factors in the tumor microenvironment that regulate CSCs is lacking. In this study, we investigated the effects of stromal fibroblasts on breast cancer (BC) stem cells. We found that compared to normal fibroblasts, primary cancer-associated fibroblasts (CAFs) and fibroblasts activated by co-cultured BC cells produce higher levels of chemokine (C-C motif) ligand 2 (CCL2), which stimulates the stem cell-specific, sphere-forming phenotype in BC cells and CSC self-renewal. Increased CCL2 expression in activated fibroblasts required STAT3 activation by diverse BC-secreted cytokines, and in turn, induced NOTCH1 expression and the CSC features in BC cells, constituting a “cancer-stroma-cancer” signaling circuit. In a xenograft model of paired fibroblasts and BC tumor cells, loss of CCL2 significantly inhibited tumorigenesis and NOTCH1 expression. In addition, upregulation of both NOTCH1 and CCL2 was associated with poor differentiation in primary BCs, further supporting the observation that NOTCH1 is regulated by CCL2. Our findings therefore suggest that CCL2 represents a potential therapeutic target that can block the cancer-host communication that prompts CSC-mediated disease progression.
breast cancer; cancer stem cells; chemokine (C-C motif) ligand 2; tumor microenvironment; stromal fibroblasts
Triple-negative breast cancer (TNBC) is one of the hardest subtypes of breast cancer to treat due to the heterogeneity of the disease and absence of well-defined molecular targets. Emerging evidence has shown the role of cohesin in the formation and progression of various cancers including colon and lung cancer but the role of cohesin in breast cancer remains elusive. Our data showed that structural maintenance of chromosome 1 (SMC1), a subunit of the cohesin protein complex, is differentially overexpressed both at RNA and protein level in a panel of TNBC cell lines as compared to normal epithelial or luminal breast cancer cells, suggesting that the amplified product of this normal gene may play role in tumorigenesis in TNBC. In addition, our results show that induced overexpression of SMC1 through transient transfection enhanced cell migration and anchorage independent growth while its suppression with targeted small interfering RNA (siRNA) reduced the migration ability of TNBC cells. Increased expression of SMC1 also lead to increase in the mesenchymal marker vimentin and decrease in the normal epithelial marker, E-cadherin. Immunocytochemical studies along with flow cytometry and cell fractionation showed the localization of SMC1 in the nucleus, cytoplasm and also in the plasma membrane. The knockdown of SMC1 by siRNA sensitized the TNBC cells towards a PARP inhibitor (ABT-888) and IC50 was approximately three fold less than ABT-888 alone. The cytotoxic effect of combination of SMC1 suppression and ABT-888 was also confirmed by the colony propagation assay. Taken together, these studies report for the first time that SMC1 is overexpressed in TNBC cells where it plays a role in cell migration and drug sensitivity, and thus provides a potential therapeutic target for this highly invasive breast cancer subtype.
Autologous hematopoietic cell transplantation (HCT) improves survival in patients with multiple myeloma, but disease progression remains a challenge. Allogeneic HCT (alloHCT) has the potential to reduce disease progression through graft-versus-myeloma effects. The aim of the BMT CTN 0102 trial was to compare outcomes of autologous HCT (autoHCT) followed by alloHCT with non-myeloablative conditioning (auto-allo) to tandem autoHCT (auto-auto) in patients with standard risk myeloma. Patients in the auto-auto arm were randomized to one year of thalidomide and dexamethasone (Thal-Dex) maintenance therapy or observation (Obs).
Patients with multiple myeloma within 10 months from initiation of induction therapy were classified as standard (SRD) or high risk (HRD) disease based on cytogenetics and beta-2-microglobulin levels. Assignment to auto-allo HCT was based on availability of an HLA-matched sibling donor. Primary endpoint was three-year progression-free survival (PFS) according to intent-to-treat analysis.
710 patients were enrolled completed a minimum of 3-year follow up. Among 625 SRD patients, 189 and 436 were assigned to auto-allo and auto-auto, respectively. Seventeen percent (33/189) of SR patients in the auto-allo arm and 16% (70/436) in the auto-auto arm did not receive a second transplant. Thal-Dex was not completed in 77% (168/217) of assigned patients. PFS and overall survival (OS) did not differ between the Thal-Dex (49%, 80%) and Obs (41%, 81%) cohorts and these two arms were pooled for analysis. Three year PFS was 43% and 46% (p=0·671) and three-year OS was 77% and 80 % (p=0·191) with auto-allo and auto-auto, respectively. Corresponding progression/relapse rates were 46% and 50% (p=0·402); treatment-related mortality rates were 11% and 4% (p<0·001), respectively. Auto/allo patients with chronic graft-vs-host disease had a decreased risk of relapse. Most common grade 3 to 5 adverse events in auto-allo was hypebilirubenemia (21/189) and in the auto-auto was peripheral neuropathy (52/436). Among 85 HRD patients (37 auto-allo), three PFS was 40% and 33% (p=0·743) and three-year OS was 59% and 67% (p=0·460) with auto-allo and auto-auto, respectively.
Thal-Dex maintenance was associated with poor compliance and did not improve PFS or OS. At three years there was no improvement in PFS or OS with auto-allo compared to auto-auto transplantation in patients with standard risk myeloma. Decisions to proceed with alloHCT after an autoHCT in patients with standard risk myeloma should take into consideration results of the current trial. Future investigation of alloHCT in myeloma should focus to minimize TRM and maximize graft-versus myeloma effects. This trial was registered in Clinicaltrials.gov (NCT00075829) and was funded by the National Heart, Lung and Blood Institute and National Cancer Institute.
breast cancer; circulating tumor cells; HER2; ER; ERCC1; discordance
The primary goal of this trial was to determine the response rate of single-agent vorinostat in patients with metastatic breast cancer. The secondary goals included assessment of time to progression, evaluation of toxicities, and overall survival.
From June 2005 to March 2006, fourteen patients received vorinostat, 200 mg orally, twice daily for 14 days of each 21 day cycle. Response and progression were evaluated using RECIST criteria.
The median age for all patients was 60.5 years (range: 37–88). Eight patients were ER and/or PR positive, four were Her-2 positive. Sites of metastatic disease included brain, liver, lungs, bones, pelvis, pleura, chest wall, and distant lymph nodes. Patients received a median of 1.5 prior (range: 0–2) chemotherapeutic regimens for metastatic disease. Fatigue, nausea, diarrhea, and lymphopenia were the most frequent clinically significant adverse effects. The median number of cycles delivered was two (range: 1–20). There were no complete or partial responses and the study was terminated after the first stage, however 4 patients were observed with stable disease with time to progression of 4,8,9 and 14 months. The median number of months that patients received treatment on this study was 1.7 (range: 0.5–14).
While not meeting the RECIST response criteria for adequate single-agent activity, the observed tolerable toxicities and the potential for clinical benefit in terms of stable disease suggest that further assessment of vorinostat as a part of combination therapy with either chemotherapeutic or targeted agents in metastatic breast might be undertaken.
Data and ongoing research on new cytotoxic and targeted therapies for the treatment of patients with metastatic breast cancer are outlined, and new developments regarding approved but relatively new classes of cytotoxic and targeted agents and also new classes of targeted therapy that are undergoing clinical evaluation are highlighted.
Newer treatments have improved survival for patients with metastatic breast cancer over the last two decades, and a battery of new cytotoxic and targeted therapies is continuing to enhance this trend. This review outlines recent data and ongoing research in this area, by highlighting new developments (regarding approved but relatively new classes of cytotoxic and targeted agents) and also new classes of targeted therapy that are undergoing clinical evaluation. Mechanisms for synergy between agents are discussed where data are available, as is information on the rationale behind the development of agents that inhibit angiogenesis, DNA repair, histone deacetylases, heat shock proteins, or various signaling pathways in tumor proliferation. The abundance of clinical research surrounding anticancer agents, together with ongoing cancer biology research, is expected to further increase the available pool of therapeutic options for metastatic breast cancer. Concomitantly, in the absence of an effective targeted monotherapy, a better understanding of the interplay between biologic and cytotoxic anticancer agents will improve our ability to rationally design combination regimens with better efficacy and tolerability.
Metastatic breast cancer; Combination therapy; Targeted therapy; Monoclonal antibodies; Small-molecule inhibitors
MicroRNAs (miRNAs) have been recently detected in the circulation of cancer patients, where they are associated with clinical parameters. Discovery profiling of circulating small RNAs has not been reported in breast cancer (BC), and was carried out in this study to identify blood-based small RNA markers of BC clinical outcome.
The pre-treatment sera of 42 stage II-III locally advanced and inflammatory BC patients who received neoadjuvant chemotherapy (NCT) followed by surgical tumor resection were analyzed for marker identification by deep sequencing all circulating small RNAs. An independent validation cohort of 26 stage II-III BC patients was used to assess the power of identified miRNA markers.
More than 800 miRNA species were detected in the circulation, and observed patterns showed association with histopathological profiles of BC. Groups of circulating miRNAs differentially associated with ER/PR/HER2 status and inflammatory BC were identified. The relative levels of selected miRNAs measured by PCR showed consistency with their abundance determined by deep sequencing. Two circulating miRNAs, miR-375 and miR-122, exhibited strong correlations with clinical outcomes, including NCT response and relapse with metastatic disease. In the validation cohort, higher levels of circulating miR-122 specifically predicted metastatic recurrence in stage II-III BC patients.
Our study indicates that certain miRNAs can serve as potential blood-based biomarkers for NCT response, and that miR-122 prevalence in the circulation predicts BC metastasis in early-stage patients. These results may allow optimized chemotherapy treatments and preventive anti-metastasis interventions in future clinical applications.
Breast cancer; miRNA; Biomarker; Neoadjuvant chemotherapy; Metastasis
This study was designed to ascertain the dose-limiting toxicities (DLT) and maximally tolerated doses of the combination of fixed-dose tamoxifen and carboplatin, with escalating doses of topotecan, and to determine the pharmacokinetics of topotecan in the plasma and cerebrospinal fluid.
Tamoxifen 100 mg po bid, topotecan 0.25, 0.5, 0.75, or 1.0 mg/m2/d IV, administered as a 72 h continuous infusion on days 1–3, followed by carboplatin AUC = 3, IV on day 3. Cycles were repeated every 4 weeks.
Seventeen patients received 39 cycles of treatment: median 2, (range 1–5). The tumors included glioblastoma (6), anaplastic astrocytoma (2), metastatic non-small cell (3), small cell lung (2), and one each with medulloblastoma, ependymoma, and metastatic breast or colon carcinoma. The median Karnofsky performance status was 70% (range 60–90%) and age: 52 (range 24–75). Eleven patients were female and six male. Toxicities included thrombocytopenia (2), neutropenia without fever lasting 6 days (1), DVT (2), and emesis (1). Topotecan levels, total and lactone, were measured prior to the end of infusion in plasma and cerebrospinal fluid (CSF). At 1.0 mg/m2/d, the median CSF/plasma ratio was 19.4% (range 15.1–59.1%). The total plasma topotecan in two pts with DLTs was 4.63 and 5.87 ng/ml, in three without DLTs at the same dose level the mean total plasma topotecan was 3.4 ng/ml (range 3.02–3.83). Plasma lactone levels were 33% of the total; CSF penetration was 20% of the total plasma levels. 4/8 pts with high-grade gliomas had stable disease (median: 3 cycles (range 2–5)). Two had minor responses. One patient with metastatic non-small cell and one with small cell lung cancer had objective PRs.
The recommended phase II doses are: tamoxifen 100 mg po bid, topotecan 0.75 mg/m2/d IV continuous infusion for 72 h, followed by carboplatin AUC = 3 IV on day 3. Measurable topotecan levels, both total and lactone, are observed in the CSF.
Topotecan; Carboplatin; Tamoxifen; Brain tumors; Chemotherapy
Papillary carcinoma of the breast represents approximately 0.5% of all newly diagnosed cases of breast cancer. The prevalence of both invasive and in situ papillary carcinoma seems to be greater older postmenopausal women, and -in relative terms-in males. Histologic features of the tumor include cellular proliferations surrounding fibrovascular cores, with or without invasion. In this review, characteristics of both in situ and invasive disease are outlined. Immunohistochemical analyses of papillary carcinoma suggest the utility of markers such as smooth muscle myosin heavy chain, calponin, p63 and high molecular weight keratins, which can characterize the myoepithelial cell layer. With respect to radiographic evaluation of papillary carcinoma, ultrasonography is the most extensively studied imaging modality, though magnetic resonance mammography has potential utility. Available data suggest improved outcome for papillary carcinoma as compared to invasive ductal carcinoma. Treatment-related information for patients with papillary carcinoma is limited, and patterns noted in available series suggest a variable approach to this disease. The scarcity of information underscores the need for further treatment- and outcome-related studies in papillary carcinoma of the breast.
papillary; breast carcinoma; male breast cancer; breast ultrasonography; breast magnetic resonance mammography
The purposes of this study were to evaluate the organ biodistribution, pharmacokinetics, immunogenicity, and tumor uptake of 111Indium (111In)-MxDTPA-trastuzumab in patients with human epidermal growth factor receptor 2 (HER2)-overexpressing breast cancers and to determine whether 90Y-MxDTPA-trastuzumab should be evaluated in subsequent clinical therapy trials.
Patients with HER2-overexpressing breast cancers who were to undergo planned trastuzumab therapy first received unlabeled trastuzumab (4–8 mg/kg IV), followed 4 hours later by 5 mCi 111In-MxDTPA-trastuzumab (10 mg antibody). Serial blood samples, 24-hour urine collections, and nuclear scans were performed at defined time points for 7 days.
Eight (8) patients received 111In-MxDTPA-trastuzumab, which was well tolerated with no adverse side-effects. Three (3) of 7 patients with known lesions demonstrated positive imaging on nuclear scans. No antiantibody responses were observed for 2 months postinfusion. Organ doses (cGy/mCi) assuming radiolabeling with 90Y were 19.9 for heart wall, 17.6 for liver, 4.6 for red marrow, and 2.8 for the whole body. Tumor doses ranged from 24 to 172 cGy/mCi.
In summary, results from this study indicate that 90Y-MxDTPA-trastuzumab is an appropriate agent to evaluate in therapy trials. No evidence of an immune response to 111In-MxDTPA-trastuzumab was detected, predicting for the ability to administer multiple cycles. With the exception of cardiac uptake, pharmacokinetics and organ biodistribution were comparable to other 90Y-labeled monoclonal antibodies previously evaluated in the clinic. Cardiac uptake was comparable to hepatic uptake and therefore predicted to not be prohibitively high as to result in dose-limiting cardiotoxicity.
breast cancer; indium-111; radioimmunotherapy; radiolabeled antibody; trastuzumab
Although diffuse large B-cell lymphoma (DLBCL) usually occurs in the lymph nodes, approximately 30–40% of the time it can have an extranodal site of involvement and it can arise in nearly every body site such as intestine, bone, breast, liver, skin, lung, and central nervous system. Muscle involvement of DLBCL is especially uncommon, comprising 0.5% of extranodal NHL. We report a case of a 72-year-old man with extranodal DLBCL of a unique manifestation in the calf muscle, involving predominantly the gastrocnemius muscle. The patient achieved complete response and remained free of local recurrence or metastasis following diagnosis.
IL-6/Jak2 signaling is viewed critical for persistent Stat3 activation in cancer. However, IL-6-induced Stat3 activity is transient in normal physiology. Here we identify a mechanism important for persistent Stat3 activation in tumor cells and the tumor microenvironment. We show that sphingosine-1-phosphate receptor 1 (S1PR1), a G-protein-coupled receptor for lysophospholipid sphingosine-1-phosphate (S1P), is elevated in Stat3-positive tumors. Stat3 is a transcription factor for the S1pr1 gene. Enhanced S1pr1 expression activates Stat3 and upregulates Il6 gene expression, thereby accelerating tumor growth and metastasis. Conversely, silencing S1pr1 in tumor cells or immune cells inhibits tumor Stat3 activity, tumor growth and metastasis. S1P/S1PR1-induced Stat3 activation is persistent, in contrast to transient Stat3 activation by IL-6. S1PR1 activates Stat3 in part by upregulating Jak2 tyrosine kinase activity. We demonstrate that Stat3-induced S1pr1 expression, as well as S1P/S1PR1 pathway, is important for persistent Stat3 activation in cancer cells and the tumor microenvironment and for malignant progression.
GTI-2040 is a 20-mer antisense oligonucleotide targeting the mRNA of ribonucleotide reductase M2. It was combined with oxaliplatin and capecitabine in a phase I trial in patients with advance solid tumors based on previous studies demonstrating potentiation of chemotherapy with ribonucleotide reductase inhibitors.
Patients at least 18 years of age with advanced incurable solid tumors and normal organ function as well as a Karnofsky performance status of ≥60% were eligible. One prior chemotherapy regimen for advanced disease or relapse within 12 months of adjuvant chemotherapy was required. Patients could have received prior fluoropyrimidines, including capecitabine, but not oxaliplatin. Treatment cycles were 21 days. In each cycle, GTI-2040 was given as a continuous intravenous infusion over 14 days, oxaliplatin as a 2-h intravenous infusion on day 1, and capecitabine orally twice a day for 14 days. In cycle 1 only, oxaliplatin and capecitabine were started on day 2 to allow ribonucleotide reductase mRNA levels to be measured with and without oxaliplatin and capecitabine. Doses were escalated in cohorts of three patients using a standard 3 + 3 design until the maximum tolerated dose was established, defned as no more than one first-cycle dose-limiting toxicity among six patients treated at a given dose level.
The maximum tolerated dose was estimated to be the combination of GTI-2040 3 mg/kg per day for 14 days, capecitabine 600 mg/m2 twice daily for 14 days, and oxaliplatin 100 mg/m2 every 21 days. Dose-limiting toxicities were hematologic. GTI-2040 pharmacokinetics, obtained at steady-state on days 7 and 14, showed the high inter-patient variability previously reported. Two of six patients had stable disease at the maximum tolerated dose and one patient, with heavily pre-treated non-small cell lung cancer, had a partial response at a higher dose level. In samples from a limited number of patients, there was no clear decrease in ribonucleotide reductase expression in peripheral blood mononuclear cells during treatment.
A combination of GTI-2040, capecitabine and oxaliplatin is feasible in patients with advanced solid tumors.
GTI-2040; Oxaliplatin; Capecitabine; Ribonucleotide reductase; Pharmacokinetics
Trastuzumab is a monoclonal antibody targeted to the Her2 receptor and approved for treatment of Her2-positive breast cancer. Among patients who initially respond to trastuzumab therapy, resistance typically arises within one year. BT/HerR cells are trastuzumab-resistant variants of Her2-positive BT474 breast cancer cells. The salient feature of BT/HerR cells is failure to down-regulate PI3K/Akt signaling upon trastuzumab binding. The current work addresses the mechanism of sustained signaling in BT/HerR cells, focusing on the protein kinase A (PKA) pathway.
We performed microarray analysis on BT/HerR and BT474 cell lines to identify genes that were up- or down-regulated in trastuzumab resistant cells. Specific genes in the PKA pathway were quantified using RT-PCR and Western hybridization. SiRNA transfection was used to determine the effects of gene knockdown on cellular response to trastuzumab. Electrophoretic mobility shift assays were used to measure cAMP-responsive element binding activity under defined conditions. Immunohistochemistry was used to analyze protein expression in clinical samples.
BT/HerR cells had elevated PKA signaling activity and several genes in the PKA regulatory network had altered expression in these cells. Down-regulation of one such gene, the PKA-RIIα regulatory subunit, conferred partial trastuzumab resistance in Her2-positive BT474 and SK-Br-3 cell lines. Forskolin activation of PKA also produced significant protection against trastuzumab-mediated Akt dephosphorylation. In patient samples, PKA signaling appeared to be enhanced in residual disease remaining after trastuzumab-containing neoadjuvant therapy.
Activation of PKA signaling may be one mechanism contributing to trastuzumab resistance in Her2-positive breast cancer. We propose a molecular model by which PKA confers its effects.
PKA; PKA-RIIα; Herceptin; breast cancer; drug resistance
Human breast tumors contain a breast cancer stem cell (BCSC) population with properties reminiscent of normal stem cells. We found 37 microRNAs that were differentially expressed between human BCSCs and non-tumorigenic cancer cells. Three clusters, miR-200c-141, miR-200b-200a-429 and miR-183-96-182 were down-regulated in human BCSCs, normal human and murine mammary stem/progenitor cells and embryonal carcinoma cells. Expression of BMI1, a known regulator of stem cell self-renewal, was modulated by miR-200c. MiR-200c inhibited the clonogenicity of breast cancer cells and suppressed the growth of embryonal carcinoma cells in vitro. Most importantly, miR-200c strongly suppressed the ability of normal mammary stem cells to form mammary ducts and tumor formation driven by human BCSCs in vivo. The coordinated down-regulation of three microRNA clusters and the similar functional regulation of clonogenicity by miR-200c provide a molecular link that connects breast cancer stem cells with normal stem cells.
Cisplatin and gemcitabine have single-agent activity in metastatic breast cancer, and preclinical data support synergy of the combination. Two parallel, phase II trials were conducted to evaluate the response rate, response duration, and toxicities of the combination. Genetic polymorphisms were analyzed for correlation with outcomes.
Patients and Methods
Eligible women had measurable disease and heavily or minimally pretreated metastatic breast cancer. The heavily pretreated protocol required prior anthracycline and taxane therapy; cisplatin as part of high-dose therapy was allowed. All patients received cisplatin 25 mg/m2 on days 1 through 4 and gemcitabine 1,000 mg/m2 on days 2 and 8 of a 21-day cycle with prophylactic granulocyte colony-stimulating factor in the heavily pretreated group. Sera from a subset of patients were evaluated by polymerase chain reaction restriction fragment length polymorphism for polymorphisms in 10 genes of interest.
Of 136 women enrolled, 74 were heavily pretreated. Both protocols accrued to their two-stage design. The response rate for both the heavily and minimally pretreated cohorts was 26%, and the median durations of response were 5.3 and 5.9 months, respectively. In a multivariate analysis, hormone receptor–negative disease was associated with a higher response rate. The most common grades 3 or 4 toxicities were thrombocytopenia (71%), neutropenia (66%), and anemia (38%). In a subset of 55 patients, the xeroderma pigmentosum group D (XPD)–751, x-ray cross-complementing group 3 (XRCC3) and cytidine deaminase polymorphisms were significantly associated with clinical outcomes.
Combination cisplatin and gemcitabine is active in metastatic breast cancer regardless of prior therapy. Genetic polymorphisms may tailor which patients benefit from this regimen.
Metabolism of oxygen, while central to life, also produces reactive oxygen species (ROS) that have been implicated in processes as diverse as cancer, cardiovascular disease, and aging. It has recently been shown that central nervous system stem cells1, 2 and hematopoietic stem cells and early progenitors3-6 contain lower levels of ROS than their more mature progeny and that these differences appear to be critical for maintaining stem cell function. We hypothesized that epithelial tissue stem cells and their cancer stem cell (CSC) counterparts may also share this property. Here we show that normal mammary epithelial stem cells contain lower concentrations of ROS than their more mature progeny cells. Congruently, subsets of CSCs in some human and murine breast tumors contain lower ROS levels than corresponding non-tumorigenic cells (NTCs). Consistent with ROS being critical mediators of ionizing radiation-induced cell killing7, 8, CSCs in these tumors develop less DNA damage and are preferentially spared after irradiation compared to NTCs. Lower ROS levels in CSCs are associated with increased expression of free radical scavenging systems. Pharmacologic depletion of ROS scavengers in CSCs significantly decreases their clonogenicity and results in radiosensitization. These results indicate that, similar to normal tissue stem cells, subsets of CSCs in some tumors contain lower ROS levels and enhanced ROS defenses compared to their non-tumorigenic progeny, which may contribute to tumor radioresistance.
Tumor DNA has been shown to be present both in circulating tumor cells in blood and as fragments in the plasma of metastatic cancer patients. The identification of ultra-rare tumor-specific mutations in blood would be the ultimate marker to measure efficacy of cancer therapy and/ or early recurrence. Herein we present a method for detecting microinsertions/deletions/indels (MIDIs) at ultra-high analytical selectivity. MIDIs comprise about 15% of mutations.
Methods and Findings
We describe MIDI-Activated Pyrophosphorolysis (MAP), a method of ultra-high analytical selectivity for detecting MIDIs. The high analytical selectivity of MAP is putatively due to serial coupling of two rare events: heteroduplex slippage and mis-pyrophosphorolysis. MAP generally has an analytical selectivity of one mutant molecule per >1 billion wild type molecules and an analytical sensitivity of one mutant molecule per reaction. The analytical selectivity of MAP is about 100,000-fold better than that of our previously described method of Pyrophosphorolysis Activated Polymerization-Allele specific amplification (PAP-A) for detecting MIDIs. The utility of this method is illustrated in two ways. 1) We demonstrate that two EGFR deletions commonly found in lung cancers are not present in tissue from four normal human lungs (107 copies of gDNA each) or in blood samples from 10 healthy individuals (107 copies of gDNA each). This is inconsistent, at least at an analytical sensitivity of 10−7, with the hypotheses of (a) hypermutation or (b) strong selection of these growth factor-mutated cells during normal lung development leads to accumulation of pre-neoplastic cells with these EGFR mutations, which sometimes can lead to lung cancer in late adulthood. Moreover, MAP was used for large scale, high throughput “gene pool” analysis. No germline or early embryonic somatic mosaic mutation was detected (at a frequency of >0.3%) for the 15/18 bp EGFR deletion mutations in 6,400 individuals, suggesting that early embryonic EGFR somatic mutation is very rare, inconsistent with hypermutation or strong selection of these deletions in the embryo. 2) The second illustration of MAP utility is in personalized monitoring of therapy and early recurrence in cancer. Tumor-specific p53 mutations identified at diagnosis in the plasma of six patients with stage II and III breast cancer were undetectable after therapy in four women, consistent with clinical remission, and continued to be detected after treatment in two others, reflecting tumor progression.
MAP has an analytical selectivity of one part per billion for detection of MIDIs and an analytical sensitivity of one molecule. MAP provides a general tool for monitoring ultra-rare mutations in tissues and blood. As an example, we show that the personalized cancer signature in six out of six patients with non-metastatic breast cancer can be detected and that levels over time are correlated with the clinical course of disease.