Gaia Schiavon

PTEN Expression and KRAS Mutations on Primary Tumors and Metastases in the Prediction of Benefit From Cetuximab Plus Irinotecan for Patients With Metastatic Colorectal Cancer

PURPOSE PTEN, AKT, and KRAS are epidermal growth factor receptor (EGFR) downstream regulators. KRAS mutations confer resistance to cetuximab. This retrospective study investigated the role of PTEN loss, AKT phosphorylation, and KRAS mutations on the activity of cetuximab plus irinotecan in patients with metastatic colorectal cancer (mCRC). PATIENTS AND METHODS A cohort of patients with irinotecan-refractory mCRC who were treated with cetuximab plus irinotecan was tested for PTEN immunoreactivity (ie, immunohistochemistry; IHC), pAKT IHC, and KRAS mutations. Analyses were performed both on primary tumors and on related metastases, and the association among IHC, mutational results, and treatment outcomes was investigated. RESULTS One-hundred two patients were eligible. Ninety-six primary tumors, 59 metastases, and 53 paired samples were available. Forty-nine primary tumors (58% of assessable samples) had a preserved PTEN expression (PTEN-positive), whereas 35 (40% of assessable samples) were pAKT-positive. Levels of concordance between primary tumors and metastases were 60%, 68%, and 95% for PTEN, pAKT, and KRAS, respectively. PTEN status on primary tumors and pAKT status both on primary tumors and on metastases did not predict response or progression-free survival (PFS). On metastases, 12 (36%) of 33 patients with PTEN-positive tumors were responders compared with one (5%) of 22 who had PTEN-negative tumors (P = .007). The median PFS of patients with PTEN-positive metastases was 4.7 months compared with 3.3 months for those with PTEN-negative metastases (hazard ratio [HR], 0.49; P = .005). Patients with PTEN-positive metastases and KRAS wild type had longer PFS compared with other patients (5.5 months v 3.8 months; HR, 0.42; P = .001). CONCLUSION PTEN loss in metastases may be predictive of resistance to cetuximab plus irinotecan. The combination of PTEN IHC and KRAS mutational analyses could help to identify a subgroup of patients with mCRC who have higher chances of benefiting from EGFR inhibition.

Mutation tracking in circulating tumor DNA predicts relapse in early breast cancer.

Noninvasive mutation tracking in plasma can detect circulating tumor DNA arising from residual micrometastatic disease and thus identify patients at high risk of recurrence. Risk of recurrence Predicting whether a cancer patient will relapse remains a formidable challenge in modern medicine. Fortunately, circulating tumor DNA (ctDNA) present in the blood may give clues on residual disease—cancer cells left behind to seed new tumors even after treatment. Garcia-Murillas et al. developed a personalized ctDNA assay based on digital polymerase chain reaction to track mutations over time in patients with early-stage breast cancer who had received apparently curative treatments, surgery, and chemotherapy. Mutation tracking in serial samples accurately predicted metastatic relapse—in several instances, months before clinical relapse (median of ~8 months). Such unprecedented early prediction could allow for intervention before the reappearance of cancer in high-risk patients. In addition, the authors were able to shed light on the genetic events driving such metastases, by massively parallel sequencing of the ctDNA, which could inform new drug-based therapies on the basis of the patients’ individual mutations. The identification of early-stage breast cancer patients at high risk of relapse would allow tailoring of adjuvant therapy approaches. We assessed whether analysis of circulating tumor DNA (ctDNA) in plasma can be used to monitor for minimal residual disease (MRD) in breast cancer. In a prospective cohort of 55 early breast cancer patients receiving neoadjuvant chemotherapy, detection of ctDNA in plasma after completion of apparently curative treatment—either at a single postsurgical time point or with serial follow-up plasma samples—predicted metastatic relapse with high accuracy [hazard ratio, 25.1 (confidence interval, 4.08 to 130.5; log-rank P < 0.0001) or 12.0 (confidence interval, 3.36 to 43.07; log-rank P < 0.0001), respectively]. Mutation tracking in serial samples increased sensitivity for the prediction of relapse, with a median lead time of 7.9 months over clinical relapse. We further demonstrated that targeted capture sequencing analysis of ctDNA could define the genetic events of MRD, and that MRD sequencing predicted the genetic events of the subsequent metastatic relapse more accurately than sequencing of the primary cancer. Mutation tracking can therefore identify early breast cancer patients at high risk of relapse. Subsequent adjuvant therapeutic interventions could be tailored to the genetic events present in the MRD, a therapeutic approach that could in part combat the challenge posed by intratumor genetic heterogeneity.

Pharmacogenetic Profiling in Patients With Advanced Colorectal Cancer Treated With First-Line FOLFOX-4 Chemotherapy

PURPOSE The objective is to investigate whether polymorphisms with putative influence on fluorouracil/oxaliplatin activity are associated with clinical outcomes of patients with advanced colorectal cancer treated with first-line oxaliplatin, folinic acid, and fluorouracil palliative chemotherapy. MATERIALS AND METHODS Consecutive patients were prospectively enrolled onto medical oncology units in Central Italy. Patients were required to have cytologically/histologically confirmed metastatic disease with at least one measurable lesion. Peripheral blood samples were used for genotyping 12 polymorphisms in thymidylate synthase, methylenetetrahydrofolate reductase, xeroderma pigmentosum group D (XPD), excision repair cross complementing group 1 (ERCC1), x-ray cross complementing group 1, x-ray cross complementing protein 3, glutathione S-transferases (GSTs) genes. The primary end point of the study was to investigate the association between genotypes and progression-free survival (PFS). RESULTS In 166 patients, ERCC1-118 T/T, XPD-751 A/C, and XPD-751 C/C genotypes were independently associated with adverse PFS. The presence of two risk genotypes (ERCC1-118 T/T combined with either XPD-751 A/C or XPD-751 C/C) occurred in 50 patients (31%). This profiling showed an independent role for unfavorable PFS with a hazard ratio of 2.84% and 95% CI of 1.47 to 5.45 (P = .002). Neurotoxicity was significantly associated with GSTP1-105 A/G. Carriers of the GSTP1-105 G/G genotype were more prone to suffer from grade 3 neurotoxicity than carriers of GSTP1-105 A/G and GSTP1-105 A/A genotypes. CONCLUSION A pharmacogenetic approach may be an innovative strategy for optimizing palliative chemotherapy in patients with advanced colorectal cancer. These findings deserve confirmation in additional prospective studies.

Merlin/NF2 Suppresses Tumorigenesis by Inhibiting the E3 Ubiquitin Ligase CRL4DCAF1 in the Nucleus

Current models imply that the FERM domain protein Merlin, encoded by the tumor suppressor NF2, inhibits mitogenic signaling at or near the plasma membrane. Here, we show that the closed, growth-inhibitory form of Merlin accumulates in the nucleus, binds to the E3 ubiquitin ligase CRL4(DCAF1), and suppresses its activity. Depletion of DCAF1 blocks the promitogenic effect of inactivation of Merlin. Conversely, enforced expression of a Merlin-insensitive mutant of DCAF1 counteracts the antimitogenic effect of Merlin. Re-expression of Merlin and silencing of DCAF1 implement a similar, tumor-suppressive program of gene expression. Tumor-derived mutations invariably disrupt Merlins ability to interact with or inhibit CRL4(DCAF1). Finally, depletion of DCAF1 inhibits the hyperproliferation of Schwannoma cells from NF2 patients and suppresses the oncogenic potential of Merlin-deficient tumor cell lines. We propose that Merlin suppresses tumorigenesis by translocating to the nucleus to inhibit CRL4(DCAF1).

Repeated intermittent low-dose therapy with zoledronic acid induces an early, sustained, and long-lasting decrease of peripheral vascular endothelial growth factor levels in cancer patients.

Purpose: On the basis of stimulating data on animals reporting that weekly regimens of zoledronic acid (ZA) were effective in reducing skeletal tumor burden, we designed a study on humans to investigate the potential antiangiogenic role of a weekly low-dose therapy with ZA in patients with malignancies. Experimental Design: Twenty-six consecutive patients with advanced solid cancer and bone metastases received 1 mg of ZA every week for four times (days 1, 7, 14, and 21) followed by 4 mg of ZA with a standard 28-day schedule repeated thrice (days 28, 56, and 84). Patients were prospectively evaluated for circulating levels of vascular endothelial growth factor (VEGF) just before the beginning of drug infusion (0) and again at 7, 14, 21, 28, 56, and 84 days after the first ZA infusion. Results: The median VEGF basal value showed an early statistically significant (P = 0.038) decrease 7 days after the first 1-mg infusion of ZA. This effect on VEGF-circulating levels persisted also after the following 1-mg infusions at 14 (P = 0.002), 21 (P = 0.001), and 28 days (P = 0.008). Interestingly, the decrease of VEGF-circulating levels persisted also at each programmed time point during the second phase of the study (ZA 4 mg every 4 weeks). No significant differences were recorded in platelet levels, WBC count, or hemoglobin concentration before and after each ZA infusion. Conclusions: In the present study, we report that a repeated low-dose therapy with ZA is able to induce an early significant and long-lasting decrease of VEGF levels in cancer patients.

Analysis of ESR1 mutation in circulating tumor DNA demonstrates evolution during therapy for metastatic breast cancer.

ESR1 mutations evolve during the treatment of metastatic breast cancer. An evolving problem A large number of breast cancers express the estrogen receptor, making them susceptible to hormonal treatments. Unfortunately, these tumors can develop mutations in the estrogen receptor gene (ESR1) and become resistant to hormonal therapies that were previously effective. Schiavon et al. used three independent cohorts of breast cancer patients to demonstrate that these mutations only evolved in cases where hormonal therapy was started late in the course of the disease, after development of metastasis, and not during the initial course of treatment. If these findings are confirmed in prospective clinical trials, then they will explain why starting hormonal treatment early decreases the risk of subsequent resistance to hormonal therapy. Acquired ESR1 mutations are a major mechanism of resistance to aromatase inhibitors (AIs). We developed ultra high–sensitivity multiplex digital polymerase chain reaction assays for ESR1 mutations in circulating tumor DNA (ctDNA) and investigated the clinical relevance and origin of ESR1 mutations in 171 women with advanced breast cancer. ESR1 mutation status in ctDNA showed high concordance with contemporaneous tumor biopsies and was accurately assessed in samples shipped at room temperature in preservative tubes. ESR1 mutations were found exclusively in estrogen receptor–positive breast cancer patients previously exposed to AI. Patients with ESR1 mutations had a substantially shorter progression-free survival on subsequent AI-based therapy [hazard ratio, 3.1; 95% confidence interval (CI), 1.9 to 23.1; P = 0.0041]. ESR1 mutation prevalence differed markedly between patients who were first exposed to AI during the adjuvant and metastatic settings [5.8% (3 of 52) versus 36.4% (16 of 44), respectively; P = 0.0002]. In an independent cohort, ESR1 mutations were identified in 0% (0 of 32; 95% CI, 0 to 10.9) tumor biopsies taken after progression on adjuvant AI. In a patient with serial sampling, ESR1 mutation was selected during metastatic AI therapy to become the dominant clone in the cancer. ESR1 mutations can be robustly identified with ctDNA analysis and predict for resistance to subsequent AI therapy. ESR1 mutations are rarely acquired during adjuvant AI but are commonly selected by therapy for metastatic disease, providing evidence that mechanisms of resistance to targeted therapy may be substantially different between the treatment of micrometastatic and overt metastatic cancer.

Pharmacogenetic Profiling for Cetuximab Plus Irinotecan Therapy in Patients With Refractory Advanced Colorectal Cancer

PURPOSE Regulation of epidermal growth factor receptor (EGFR) signaling pathways may play a relevant role in determining the activity of cetuximab therapy in patients with metastatic colorectal cancer (MCRC). We investigated possible associations between genetic variants and clinical outcomes of MCRC patients treated with cetuximab-irinotecan salvage therapy. PATIENTS AND METHODS Patients who underwent cetuximab-irinotecan salvage therapy after disease progression during or after first-line bolus/infusional fluorouracil, leucovorin, and oxaliplatin chemotherapy and a second-line irinotecan-based regimen were considered eligible for analysis of polymorphisms with putative influence on cetuximab-related pathways. Epidermal growth factor (EGF) 61A>G, EGF receptor (EGFR) 216G>T, EGFR 497G>A, EGFR intron-1 (CA)(n) dinucleotide short (S)/long (L) variant, cyclin-D1 870A>G, immunoglobulin-G fragment-C receptors RIIIa 158G>T, and RIIa 131G>A were studied for a possible association with overall survival (OS) as the primary end point. Additional analyses were addressed at possible associations among polymorphisms and EGFR expression, toxicity, and response. RESULTS In 110 assessable patients, significant association with favorable OS was observed for EGFR intron-1 S/S and EGF 61 G/G genotypes. In the multivariate model, EGFR intron-1 S/S and EGF 61 G/G genotypes showed a hazard ratio of 0.41 (95% CI, 0.21 to 0.78; P = .006) and 0.44 (95% CI, 0.23 to 0.84; P = .01), respectively. EGFR intron-1 S/S carriers showed more frequent G2-G3 skin toxicity (chi(2) test = 12.7; P = .001) and treatment response (chi(2) test = 9.45; P = .008) than EGFR intron-1 L/L carriers. CONCLUSION Although additional studies are required for confirmation, our findings could optimize the use of cetuximab in MCRC patients.

Drug Transporters and Imatinib Treatment: Implications for Clinical Practice

Imatinib mesylate is approved for the treatment of chronic myeloid leukemia (CML) and advanced gastrointestinal stromal tumors (GIST). Unfortunately, in the course of treatment, disease progression occurs in the majority of patients with GIST. Lowered plasma trough levels of imatinib over time potentially cause disease progression, a phenomenon known as “acquired pharmacokinetic drug resistance.” This outcome may be the result of an altered expression pattern or activity of drug transporters. To date, the role of both efflux transporters (ATP-binding cassette transporters, such as ABCB1 and ABCG2) and uptake transporters [solute carriers such as organic cation transporter 1 (OCT1) and organic anion transporting polypeptide 1A2 (OATP1A2)] in imatinib pharmacokinetics and pharmacodynamics has been studied. In vitro experiments show a significant role of ABCB1 and ABCG2 in cellular uptake and retention of imatinib, although pharmacokinetic and pharmacogenetic data are still scarce and contradictory. ABCB1 and ABCC1 expression was shown in GIST, whereas ABCB1, ABCG2, and OCT1 were found in mononuclear cells in CML patients. Several studies have reported a clinical relevance of tumor expression or activity of OCT1 in CML patients. Further (clinical) studies are required to quantify drug transporter expression over time in organs involved in imatinib metabolism, as well as in tumor tissue. In addition, more pharmacogenetic studies will be needed to validate associations. Clin Cancer Res; 17(3); 406–15. ©2010 AACR.

Receptor Activator of NF-kB (RANK) Expression in Primary Tumors Associates with Bone Metastasis Occurrence in Breast Cancer Patients

Background Receptor activator of NFkB (RANK), its ligand (RANKL) and the decoy receptor of RANKL (osteoprotegerin, OPG) play a pivotal role in bone remodeling by regulating osteoclasts formation and activity. RANKL stimulates migration of RANK-expressing tumor cells in vitro, conversely inhibited by OPG. Materials and Methods We examined mRNA expression levels of RANKL/RANK/OPG in a publicly available microarray dataset of 295 primary breast cancer patients. We next analyzed RANK expression by immunohistochemistry in an independent series of 93 primary breast cancer specimens and investigated a possible association with clinicopathological parameters, bone recurrence and survival. Results Microarray analysis showed that lower RANK and high OPG mRNA levels correlate with longer overall survival (P = 0.0078 and 0.0335, respectively) and disease-free survival (P = 0.059 and 0.0402, respectively). Immunohistochemical analysis of RANK showed a positive correlation with the development of bone metastases (P = 0.023) and a shorter skeletal disease-free survival (SDFS, P = 0.037). Specifically, univariate analysis of survival showed that “RANK-negative” and “RANK-positive” patients had a SDFS of 105.7 months (95% CI: 73.9–124.4) and 58.9 months (95% CI: 34.7–68.5), respectively. RANK protein expression was also associated with accelerated bone metastasis formation in a multivariate analysis (P = 0.029). Conclusions This is the first demonstration of the role of RANK expression in primary tumors as a predictive marker of bone metastasis occurrence and SDFS in a large population of breast cancer patients.

Noninvasive Detection of HER2 Amplification with Plasma DNA Digital PCR

Purpose: Digital PCR is a highly accurate method of determining DNA concentration. We adapted digital PCR to determine the presence of oncogenic amplification through noninvasive analysis of circulating free plasma DNA and exemplify this approach by developing a plasma DNA digital PCR assay for HER2 copy number. Experimental Design: The reference gene for copy number assessment was assessed experimentally and bioinformatically. Chromosome 17 pericentromeric probes were shown to be suboptimal, and EFTUD2 at chromosome position 17q21.31 was selected for analysis. Digital PCR assay parameters were determined on plasma samples from a development cohort of 65 patients and assessed in an independent validation cohort of plasma samples from 58 patients with metastatic breast cancer. The sequential probability ratio test was used to assign the plasma DNA digital PCR test as being HER2-positive or -negative in the validation cohort. Results: In the development cohort, the HER2:EFTUD2 plasma DNA copy number ratio had a receiver operator area under the curve (AUC) = 0.92 [95% confidence interval (CI), 0.86–0.99, P = 0.0003]. In the independent validation cohort, 64% (7 of 11) of patients with HER2-amplified cancers were classified as plasma digital PCR HER2–positive and 94% (44 of 47) of patients with HER2-nonamplified cancers were classified as digital PCR HER2–negative, with a positive and negative predictive value of 70% and 92%, respectively. Conclusion: Analysis of plasma DNA with digital PCR has the potential to screen for the acquisition of HER2 amplification in metastatic breast cancer. This approach could potentially be adapted to the analysis of any locus amplified in cancer. Clin Cancer Res; 19(12); 3276–84. ©2013 AACR.