PURPOSE: Our objective was to evaluate operating characteristics, particularly specificity and positive predictive value (PPV), by mapping plasma miR371 expression to actual clinical events in patients with a history of germ cell tumor. PATIENTS AND METHODS: One hundred eleven male patients with a history of or newly diagnosed germ cell tumors were evaluable. Biospecimens obtained before confirmed clinical events were analyzed for miR371 expression with blinding of providers and laboratory personnel to analytic results or clinical status, respectively. Cases (patients with clinically confirmed active germ cell malignancy [aGCM]) and controls (patients with no clinically confirmed aGCM) were assigned over the course of the management. Patients were assigned risk status (high, low, or moderate) based on the composite clinical picture at time points in management. RESULTS: Considering all cases and controls and results of prospectively obtained biosamples analyzed for miR371 expression, 46 (35%) of 132 samples had clinically confirmed aGCM over the course of management; 44 (96%) of these 46 patients had plasma miR371 expression (true positives) with no false positives. Two (4%) of 46 patients had no miRNA expression despite pathologic confirmation of aGCM (false negatives). Plasma miR371 expression in confirmed aGCM had a specificity, sensitivity, positive predictive value, and negative predictive value of 100%, 96%, 100%, and 98%, respectively. Interpretation of sensitivity and negative predictive value is limited by modest follow-up. Specificity and sensitivity were 100% and 98%, 100% and 92%, and 100% and 97% in the low-, moderate-, and high-risk groups, respectively, with a median follow-up time of 15 months. CONCLUSION: Plasma miR371 expression predicts aGCM with high specificity and positive predictive value. Although other operating characteristics of miR371 await longer follow-up for more complete definition, the findings of a highly specific liquid biopsy strongly support moving forward with large-scale, real-world clinical trials to further define full operating characteristics and to identify clinical utility and areas of patient benefit.

PURPOSE: The long-term toxicities of chemotherapy and radiotherapy can represent a significant burden to testicular cancer survivors. Retroperitoneal lymph node dissection (RPLND) is an established treatment for testicular germ cell tumors with minimal late morbidity although little data exist on its efficacy in early metastatic seminoma. Surgery in early metastatic seminoma is a prospective phase II single-arm, multi-institutional trial of RPLND as first-line treatment for testicular seminoma with clinically low-volume retroperitoneal lymphadenopathy. PATIENTS AND METHODS: Twelve sites in the United States and Canada prospectively enrolled adult patients with testicular seminoma and isolated retroperitoneal lymphadenopathy (1-3 cm). Open RPLND was performed by certified surgeons with a primary end point of 2-year recurrence-free survival (RFS). Complication rates, pathologic up/downstaging, recurrence patterns, adjuvant therapies, and treatment-free survival were assessed. RESULTS: A total of 55 patients were enrolled, with a median (IQR) largest clinical lymph node size of 1.6 cm (1.3-1.9). RPLND pathology demonstrated a median (IQR) largest lymph node size of 2.3 cm (0.9-3.5); nine patients (16%) were pN0, 12 (22%) pN1, 31 (56%) pN2, and 3 (5%) pN3. One patient received adjuvant chemotherapy. With a median (IQR) follow-up of 33 months (12.0-61.6), 12 patients experienced recurrence, with a 2-year RFS of 81% and a recurrence rate of 22%. Of the patients who experienced recurrence, 10 were treated with chemotherapy and two underwent additional surgery. At last follow-up, all patients who experienced a recurrence were disease-free and the 2-year overall survival was 100%. Four patients (7%) experienced short-term complications, and four patients experienced long-term complications including incisional hernia (1) and anejaculation (3). CONCLUSION: RPLND is a treatment option for testicular seminoma with clinically low-volume retroperitoneal lymphadenopathy and is associated with low long-term morbidity.

Purpose: Although the anti–EGF receptor (EGFR) monoclonal antibody cetuximab is an effective strategy in colorectal cancer therapy, its clinical use is limited by intrinsic or acquired resistance. Alterations in the “sphingolipid rheostat”—the balance between the proapoptotic molecule ceramide and the mitogenic factor sphingosine-1-phosphate (S1P)—due to sphingosine kinase 1 (SphK1) overactivation have been involved in resistance to anticancer-targeted agents. Moreover, cross-talks between SphK1 and EGFR-dependent signaling pathways have been described. Experimental design: We investigated SphK1 contribution to cetuximab resistance in colorectal cancer, in preclinical in vitro/in vivo models, and in tumor specimens from patients. Results: SphK1 was found overexpressed and overactivated in colorectal cancer cells with intrinsic or acquired resistance to cetuximab. SphK1 contribution to resistance was supported by the demonstration that SphK1 inhibition by N,N-dimethyl-sphingosine or silencing via siRNA in resistant cells restores sensitivity to cetuximab, whereas exogenous SphK1 overexpression in sensitive cells confers resistance to these agents. Moreover, treatment of resistant cells with fingolimod (FTY720), a S1P receptor (S1PR) antagonist, resulted in resensitization to cetuximab both in vitro and in vivo, with inhibition of tumor growth, interference with signal transduction, induction of cancer cells apoptosis, and prolongation of mice survival. Finally, a correlation between SphK1 expression and cetuximab response was found in colorectal cancer patients. Clin Cancer Res; 19(1); 138–47. ©2012 AACR.

Therapy-induced neuroendocrine prostate cancer (t-NEPC) is a highly aggressive subtype of prostate cancer with poor patient survival. Emerging evidence indicates that t-NEPC can develop when prostate adenocarcinoma cells acquire cancer stem-like cell signaling in the presence of androgen receptor inhibition, followed by redifferentiation toward neuroendocrine lineage and subsequent t-NEPC progression. Whether the stem-like signaling is controlled by the core pluripotency stem cell genes (e.g., LIN28 and SOX2) remains unknown. Here, we report that the transcription of the LIN28B isoform and SOX2 were co-upregulated in t-NEPC patient tumors, patient-derived xenografts, transgenic mice, and cell models. Immunohistochemistry validated that LIN28B and SOX2 protein expression were elevated in t-NEPC patient biopsies. Using prostate adenocarcinoma and t-NEPC cell models, we demonstrated that LIN28B induced a stem-like gene network, neuroendocrine biomarkers, and neuroendocrine cell morphology. LIN28B depletion by CRISPR inhibited t-NEPC tumorigenesis and xenograft growth. These LIN28B functions were mediated mainly through the suppression of let-7 miRNA expression, resulting in de-repression of the transcription factor HMGA2 and HMGA2-mediated SOX2 expression. This study revealed a mechanism by which t-NEPC can develop through the LIN28B/let-7/SOX2 axis that regulates a cancer cell stem-like gene network, highlighting LIN28B as a potential therapeutic target in t-NEPC.

Cetuximab is the only targeted agent approved for the treatment of head and neck squamous cell carcinomas (HNSCC), but low response rates and disease progression are frequently reported. As the phosphoinositide 3-kinase (PI3K) and the mammalian target of rapamycin (mTOR) pathways have an important role in the pathogenesis of HNSCC, we investigated their involvement in cetuximab resistance. Different human squamous cancer cell lines sensitive or resistant to cetuximab were tested for the dual PI3K/mTOR inhibitor PF-05212384 (PKI-587), alone and in combination, both in vitro and in vivo. Treatment with PKI-587 enhances sensitivity to cetuximab in vitro, even in the condition of epidermal growth factor receptor (EGFR) resistance. The combination of the two drugs inhibits cells survival, impairs the activation of signalling pathways and induces apoptosis. Interestingly, although significant inhibition of proliferation is observed in all cell lines treated with PKI-587 in combination with cetuximab, activation of apoptosis is evident in sensitive but not in resistant cell lines, in which autophagy is pre-eminent. In nude mice xenografted with resistant Kyse30 cells, the combined treatment significantly reduces tumour growth and prolongs mice survival. Phosphoinositide 3-kinase/mammalian target of rapamycin inhibition has an important role in the rescue of cetuximab resistance. Different mechanisms of cell death are induced by combined treatment depending on basal anti-EGFR responsiveness.

Introduction: Src tyrosine kinase overactivation has been correlated with a poor response to human epidermal growth factor receptor 2 (HER2) inhibitors in breast cancer. To identify the mechanism by which Src overexpression sustains this resistance, we tested a panel of breast cancer cell lines either sensitive or resistant to lapatinib. Methods: To determine the role of Src in lapatinib resistance, we evaluated the effects of Src inhibition/silencing in vitro on survival, migration, and invasion of lapatinib-resistant cells. In vivo experiments were performed in JIMT-1 lapatinib-resistant cells orthotopically implanted in nude mice. We used artificial metastasis assays to evaluate the effect of Src inhibition on the invasiveness of lapatinib-resistant cells. Src-dependent signal transduction was investigated with Western blot and ELISA analyses. Results: Src activation was higher in lapatinib-resistant than in lapatinib-sensitive cells. The selective small-molecule Src inhibitor saracatinib combined with lapatinib synergistically inhibited the proliferation, migration, and invasion of lapatinib-resistant cells. Saracatinib combined with lapatinib significantly prolonged survival of JIMT-1-xenografted mice compared with saracatinib alone, and impaired the formation of lung metastases. Unexpectedly, in lapatinib-resistant cells, Src preferentially interacted with epidermal growth factor receptor (EGFR) rather than with HER2. Moreover, EGFR targeting and lapatinib synergistically inhibited survival, migration, and invasion of resistant cells, thereby counteracting Src-mediated resistance. These findings demonstrate that Src activation in lapatinib-resistant cells depends on EGFR-dependent rather than on HER2-dependent signaling. Conclusions: Complete pharmacologic EGFR/HER2 inhibition is required to reverse Src-dependent resistance to lapatinib in breast cancer.