The use of histotripsy as intratumoral immunotherapy beyond tissue ablation-the rationale for exploring the immune effects of histotripsy.

Mechanical high-intensity focused ultrasound (M-HIFU), which includes histotripsy, is a non-ionizing, non-thermal ablation technology that can be delivered by noninvasive methods. Because acoustic cavitation is the primary mechanism of tissue disruption, histotripsy is distinct from the conventional HIFU techniques resulting in hyperthermia and thermal injury. Phase I human trials have shown the initial safety and efficacy of histotripsy in treating patients with malignant liver tumors. In addition to tissue ablation, a promising benefit of M-HIFU has been stimulating a local and systemic antitumor immune response in preclinical models and potentially in the Phase I trial. Preclinical studies combining systemic immune therapies appear promising, but clinical studies of combinations have been complicated by systemic toxicities. Consequently, combining M-HIFU with systemic immunotherapy has been demonstrated in preclinical models and may be testing in future clinical studies. An additional alternative is to combine intratumoral M-HIFU and immunotherapy using microcatheter-placed devices to deliver both M-HIFU and immunotherapy intratumorally. The promise of M-HIFU as a component of anti-cancer therapy is promising, but as forms of HIFU are tested in preclinical and clinical studies, investigators should report not only the parameters of the energy delivered but also details of the preclinical models to enable analysis of the immune responses. Ultimately, as clinical trials continue, clinical responses and immune analysis of patients undergoing M-HIFU including forms of histotripsy will provide opportunities to optimize clinical responses and to optimize application and scheduling of M-HIFU in the context of the multi-modality care of the cancer patient.

A novel diagnostic examination for dropped head syndrome (DHS) (Prone position cervical extension test; DHS test).

BACKGROUND: Dropped head syndrome (DHS) is followed by severe cervical extension muscle weakness that results in chin-on chest deformity. However, maintaining a neutral cervical position can be temporarily possible, and the diagnosis of DHS might sometimes be difficult. The purpose of the present study is to examine a novel clinical test (DHS test) as the diagnostic utility for objective evaluation that focuses on cervical extension condition in the prone position. METHODS: One hundred subjects were diagnosed with isolated neck extensor myopathy (INEM)-DHS at our hospital (17 men and 83 women, mean age 75.0 ± 8.5 years), and 62 subjects were enrolled as age-matched controls. The DHS test consisted of three examinations; the first was "Ceiling gazing test" in standing position, the second was horizontal gazing in "Sphinx prone position test", and the third was horizontal gazing in "Hands and knees prone position test". We investigated the sensitivity and specificity of the DHS test for DHS. RESULTS: The patients showing positive in the INEM-DHS group were 63/100 in Ceiling gaze test, 73/100 in the Sphinx prone position test, and 91/100 in the Hands and knees prone position test. In the control group, 0/62 patients presented positive in the Ceiling gaze test, 4/62 in the Sphinx prone position test, and 0/62 in the Hands and knees prone position test. Sensitivity and specificity of the DHS test were 63.0%/100%, 73.0%/93.5%, and 91.0%/100% in the Ceiling gaze test, Sphinx position prone position test, and Hands and knees prone position test, respectively. CONCLUSION: The prone position cervical extension test (DHS test) would be useful as a novel objective diagnostic tool for INEM-DHS.

Niclosamide-induced Wnt signaling inhibition in colorectal cancer is mediated by autophagy.

The Wnt signaling pathway, known for regulating genes critical to normal embryonic development and tissue homeostasis, is dysregulated in many types of cancer. Previously, we identified that the anthelmintic drug niclosamide inhibited Wnt signaling by promoting internalization of Wnt receptor Frizzled 1 and degradation of Wnt signaling pathway proteins, Dishevelled 2 and ß-catenin, contributing to suppression of colorectal cancer growth in vitro and in vivo Here, we provide evidence that niclosamide-mediated inhibition of Wnt signaling is mediated through autophagosomes induced by niclosamide. Specifically, niclosamide promotes the co-localization of Frizzled 1 or ß-catenin with LC3, an autophagosome marker. Niclosamide inhibition of Wnt signaling is attenuated in autophagosome-deficient ATG5-/- MEF cells or cells expressing shRNA targeting Beclin1, a critical constituent of autophagosome. Treatment with the autophagosome inhibitor 3MA blocks niclosamide-mediated Frizzled 1 degradation. The sensitivity of colorectal cancer cells to growth inhibition by niclosamide is correlated with autophagosome formation induced by niclosamide. Niclosamide inhibits mTORC1 and ULK1 activities and induces LC3B expression in niclosamide-sensitive cell lines, but not in the niclosamide-resistant cell lines tested. Interestingly, niclosamide is a less effective inhibitor of Wnt-responsive genes (ß-catenin, c-Myc, and Survivin) in the niclosamide-resistant cells than in the niclosamide-sensitive cells, suggesting that deficient autophagy induction by niclosamide compromises the effect of niclosamide on Wnt signaling. Our findings provide a mechanistic understanding of the role of autophagosomes in the inhibition of Wnt signaling by niclosamide and may provide biomarkers to assist selection of patients whose tumors are likely to respond to niclosamide.

Evaluation of Yanagihara facial nerve grading system based on a muscle fiber analysis of human facial muscles.

PURPOSE: We morphometrically analyzed human facial muscles, and evaluated the Yanagihara facial nerve grading system using our data. METHODS: We used 15 types of human facial muscle, 2 types of masticatory muscle and 2 types of skeletal muscle. The materials were obtained from 11 Japanese male cadavers aged 43-86 years. We counted the muscle fibers and measured the transverse area of the muscle fibers (TAMF), and then calculated the number of muscle fibers (NMF) per mm2 and the average TAMF. RESULTS: We found a significant correlation between average TAMF and NMF (r = - 0.70; p < 0.01). We classified facial muscles into three types based on the correlational results. Type A had a low average TAMF and high NMF. Type C had a high average TAMF and low NMF. Masticatory and skeletal muscles were characterized as Type C. Type B was intermediate between Types A and C. CONCLUSIONS: Pathological changes in the facial muscles in facial nerve palsy seem to vary according to the type of facial muscle, because each facial muscle has a unique fiber-type composition. As the nine discrete facial expressive states evaluated in the Yanagihara system involve all three facial muscle types of our classification, the Yanagihara system is an outstanding system for grading facial nerve palsy in terms of the facial muscle morphology.

Polyfunctional anti-human epidermal growth factor receptor 3 (anti-HER3) antibodies induced by HER3 vaccines have multiple mechanisms of antitumor activity against therapy resistant and triple negative breast cancers.

BACKGROUND: Upregulation of human epidermal growth factor receptor 3 (HER3) is a major mechanism of acquired resistance to therapies targeting its heterodimerization partners epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2), but also exposes HER3 as a target for immune attack. We generated an adenovirus encoding full length human HER3 (Ad-HER3) to serve as a cancer vaccine. Previously we reported the anti-tumor efficacy and function of the T cell response to this vaccine. We now provide a detailed assessment of the antitumor efficacy and functional mechanisms of the HER3 vaccine-induced antibodies (HER3-VIAs) in serum from mice immunized with Ad-HER3. METHODS: Serum containing HER3-VIA was tested in complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) assays and for its effect on HER3 internalization and degradation, downstream signaling of HER3 heterodimers and growth of metastatic HER2+ (BT474M1), HER2 therapy-resistant (rBT474), and triple negative (MDA-MB-468) breast cancers. RESULTS: HER3-VIAs mediated CDC and ADCC, HER3 internalization, interruption of HER3 heterodimer-driven tumor signaling pathways, and anti-proliferative effects against HER2+ tumor cells in vitro and significant antitumor effects against metastatic HER2+ BT474M1, treatment refractory HER2+ rBT474 and triple negative MDA-MB-468 in vivo. CONCLUSIONS: In addition to the T cell anti-tumor response induced by Ad-HER3, the HER3-VIAs provide additional functions to eliminate tumors in which HER3 signaling mediates aggressive behavior or acquired resistance to HER2-targeted therapy. These data support clinical studies of vaccination against HER3 prior to or concomitantly with other therapies to prevent outgrowth of therapy-resistant HER2+ and triple negative clones.

Effects of Aerobic Cycling Training on O2 Dynamics in Several Leg Muscles in Early Post-myocardial Infarction.

The aim of this study was to test the effects of aerobic cycling training on O2 dynamics in several leg muscles in early post-myocardial infarction (post-MI). Fifteen post-MI patients were divided into a 12-week training group (TR, n = 9) or a control/non-training group (CON, n = 6). All participants performed ramp bicycle exercise until exhaustion at two times: within 12-35 days of their MI and then again 12 weeks later. Muscle O2 saturation (SmO2) and total hemoglobin concentration (∆total-Hb) were monitored continuously at thigh and lower leg muscles by near infrared spectroscopy. In CON, there were no significant alterations in muscle O2 dynamics between before and after 12 weeks at any measurement sites. In TR, after 12 weeks, lower SmO2 was observed at all measurement sites. In total-Hb, no significant changes were found after training at any measurement sites in TR. Moreover, the muscle deoxygenation after 12 weeks was related to an improvement of peak O2 uptake in all muscles. Our findings suggest that aerobic cycling training may be useful for early post-MI patients to improve peak aerobic capacity via enhancement of muscle deoxygenation and O2 extraction at several leg muscles.

Multi-site Measurements of Muscle O2 Dynamics During Cycling Exercise in Early Post-myocardial Infarction.

The aim of this study was to compare the muscle oxygen dynamics between early post-myocardial infarction (n = 12; MI) and age-matched elderly subjects without MI (n = 12; CON) in several leg muscles during ramp cycling exercise. Muscle oxygen saturation (SmO2), deoxygenated-hemoglobin concentration (∆deoxy-Hb), and total-hemoglobin concentration (∆total-Hb) were monitored continuously at the distal site of vastus lateralis (VLd), proximal site of the vastus lateralis (VLp), rectus femoris (RF), vastus medialis (VM), gastrocnemius medialis (GM), and tibialis anterior (TA) muscles by near infrared spatial resolved spectroscopy. At given absolute workloads, higher SmO2 was observed at VLd, VLp, RF, and VM in MI, compared to CON. Simultaneously, in MI, deoxy-Hb was lower at VLd, VLp, and VM than CON. In contrast to the thigh muscles, muscle oxygen dynamics were similar between groups in GM and TA. In total-Hb, no significant differences were found at any measurement sites. These results demonstrated that the absence of muscle deoxygenation was observed in MI muscles, especially in the thigh muscles, but not in the lower leg muscles.

Aerobic training enhances muscle deoxygenation in early post-myocardial infarction.

PURPOSE: Exercise-induced skeletal muscle deoxygenation is startling by its absence in early post-myocardial infarction (MI) patients. Exercise training early post-MI is associated with reduced cardiovascular risk and increased aerobic capacity. We therefore investigated whether aerobic training could enhance the muscle deoxygenation in early post-MI patients. METHODS: 21 ± 8 days after the first MI patients (n = 16) were divided into 12-week aerobic training (TR, n = 10) or non-training (CON, n = 6) groups. Before and after intervention, patients performed ramp bicycle exercise until exhaustion. Muscle deoxygenation was measured at vastus lateralis by near-infrared spectroscopy during exercise. RESULTS: Aerobic training significantly increased peak oxygen uptake (VO2) (18.1 ± 3.0 vs. 22.9 ± 2.8 mL/kg/min), decreased the change in muscle oxygen saturation from rest to submaximal and peak exercise (∆SmO2; 2.4 ± 5.7 vs. -7.0 ± 3.4 %), and increased the relative change in deoxygenated hemoglobin/myoglobin concentration from rest to submaximal (-1.5 ± 2.3 vs. 3.0 ± 3.6 µmol/L) and peak exercise (1.1 ± 4.5 vs. 8.2 ± 3.5 µmol/L). Change in total hemoglobin/myoglobin concentration in muscle was not significantly affected by training. In CON, no significant alterations were found after 12 weeks in either muscle deoxygenation or peak VO2 (18.6 ± 3.8 vs. 18.9 ± 4.6 mL/kg/min). An increase in peak VO2 was significantly negatively correlated with change in ∆SmO2 (r = -0.65) and positively associated with change in ∆deoxy-Hb/Mb at peak exercise (r = 0.64) in TR. CONCLUSIONS: In early post-MI patients, aerobic training enhanced skeletal muscle deoxygenation, and the enhancement was related to increased aerobic capacity.

The Effects of Passive Cycling Exercise for 20 min on Cardiorespiratory Dynamics in Healthy Men [corrected].

An increase in the incidence rate of cardiovascular disease is attributed to high daily sitting time, while a drop in risk of cardiovascular disease comes from a decrease in daily sitting time, rather than an increase in physical activity levels. Although short-duration passive exercise increases energy expenditure and blood flow, few studies have reported on the responses of cardiorespiratory dynamics to long-duration passive exercise. The purpose of this study was to consider the effect of long-duration passive exercise for 20 min on cardiorespiratory and muscle oxygen dynamics. Eight healthy men continuously performed passive exercise using a cycle ergometer for 20 min at 50 rpm. Changes in oxygen uptake, cardiac output and muscle oxygenation were measured during passive cycling exercise. The oxygen uptake at 1 min after the start of passive exercise was significantly increased, compared to resting level, but subsequently returned to the same as resting level. Cardiac output showed no change during passive cycling exercise. Tissue oxygen saturation increased after the start of passive exercise and subsequently maintained steady state. These results suggest that the effect of increases in energy expenditure was not maintained by passive exercise for 20 min. In addition, it is likely that passive cycling exercise for 20 min has an effect on peripheral circulation, although the exercise seems to have no effect on central circulation.

Effects of Low Volume Aerobic Training on Muscle Desaturation During Exercise in Elderly Subjects.

Aging enhances muscle desaturation responses due to reduced O2 supply. Even though aerobic training enhances muscle desaturation responses in young subjects, it is unclear whether the same is true in elderly subjects. Ten elderly women (age: 62±4 years) participated in 12-weeks of cycling exercise training. Training consisted of 30 min cycling exercise at the lactate threshold. The subjects exercised 15±6 sessions during training. Before and after endurance training, the subjects performed ramp cycling exercise. Muscle O2 saturation (SmO2) was measured at the vastus lateralis by near infrared spectroscopy during the exercise. There were no significant differences in SmO2 between before and after training. Nevertheless, changes in peak pulmonary O2 uptake were significantly negatively related to changes in SmO2 (r=-0.67, p<0.05) after training. Muscle desaturation was not enhanced by low volume aerobic training in this study, possibly because the training volume was too low. However, our findings suggest that aerobic training may potentially enhance muscle desaturation at peak exercise in elderly subjects.

Low Volume Aerobic Training Heightens Muscle Deoxygenation in Early Post-Angina Pectoris Patients.

The aim of this study was to investigate the effect of low volume aerobic exercise training on muscle O2 dynamics during exercise in early post-angina pectoris (AP) patients, as a pilot study. Seven AP patients (age: 72 ± 6 years) participated in aerobic exercise training for 12 weeks. Training consisted of continuous cycling exercise for 30 min at the individual's estimated lactate threshold, and the subjects trained for 15 ± 5 exercise sessions over 12 weeks. Before and after training, the subjects performed ramp cycling exercise until exhaustion. Muscle O2 saturation (SmO2) and relative changes from rest in deoxygenated hemoglobin concentration (∆Deoxy-Hb) and total hemoglobin concentration (∆Total-Hb) were monitored at the vastus lateralis by near infrared spatial resolved spectroscopy during exercise. The SmO2 was significantly lower and ∆Deoxy-Hb was significantly higher after training than before training, while there were no significant changes in ∆Total-Hb. These results indicated that muscle deoxygenation and muscle O2 extraction were potentially heightened by aerobic exercise training in AP patients, even though the exercise training volume was low.

Muscle Oxygen Dynamics During Cycling Exercise in Angina Pectoris Patients.

Muscle O2 dynamics during ramp cycling exercise were compared between angina pectoris patients (AP; n = 7, age: 73 ± 6 years) after coronary artery bypass grafting and age-, height-, and body weight-matched elderly control subjects (CON; n = 7, age: 74 ± 8 years). Muscle O2 saturation (SmO2) and relative change in deoxygenated (∆deoxy-Hb) and total hemoglobin concentration (∆total-Hb) were measured continuously during exercise in the vastus lateralis (VL) by near infrared spatial resolved spectroscopy. Pulmonary O2 uptake (VO2) was also monitored throughout exercise to determine peak VO2. In AP, SmO2 was significantly higher, and ∆deoxy-Hb was significantly lower during exercise, compared to CON. In all subjects, ∆SmO2 (values at peak exercise minus values at resting) was negatively correlated to peak VO2 (r = -0.52, p < 0.05), and ∆deoxy-Hb at peak exercise tended to be negatively associated with peak VO2 (r = 0.48, p = 0.07). Blunted skeletal muscle deoxygenation response was observed in AP patients, which may be related to lower aerobic capacity in AP patients.

Sex-Related Difference in Muscle Deoxygenation Responses Between Aerobic Capacity-Matched Elderly Men and Women.

Muscle O2 dynamics during ramp cycling exercise were compared between aerobic capacity-matched elderly men (n=8, age 65±2 years) and women (n=8, age 66±3 years). Muscle O2 saturation (SmO2) and relative change in deoxygenated (Δdeoxy-Hb) and total hemoglobin concentration (Δtotal-Hb) were monitored continuously during exercise in the vastus lateralis (VL) and gastrocnemius medialis (GM) by near infrared spatial resolved spectroscopy. SmO2 was significantly higher during exercise in women than in men in VL, but not in GM. In VL, Δdeoxy-Hb and Δtotal-Hb were significantly higher in men than in women, especially during high intensity exercise. However, no significant difference was observed in Δdeoxy-Hb or Δtotal-Hb in GM. Sex-related differences in muscle deoxygenation response may be heterogeneous among leg muscles in elderly subjects.

Muscle Deoxygenation and Its Heterogeneity Changes After Endurance Training.

The purpose of this study was to elucidate the time course of muscle deoxygenation and its heterogeneity changes through endurance training. Nine healthy untrained male participated in this study. The subjects performed a ramp incremental cycle exercise protocol to estimate VO2peak and muscle tissue oxygen saturation (SmO2) distribution in the VL muscle before and after 3 (3 wk-T) and 6 weeks of endurance training (6 wk-T). The probe of multi-channel near infrared spatially resolved spectroscopy was attached to the left vastus lateralis muscle along the direction of the long axis. The subjects performed cycle exercise at 60 % of VO2peak for 30 min/day, 3 days/week as the endurance training. After the training, VO2peak at 3 wk-T and 6 wk-T were significantly increased compared to pre-training (Pre-T) and VO2peak at 6 wk-T was significantly increased compared to 3 wk-T. Mean SmO2 within measurement sites at VO2peak was significantly decreased after 3 wk-T and 6 wk-T compared to Pre-T, but mean SmO2 was not significantly different between 3 wk-T and 6 wk-T. Conversely, the heterogeneity of the SmO2 during exercise was not significantly changed through endurance training. A significantly negative correlation was found between ΔVO2 and ΔSmO2 after the first 3 weeks of endurance training. In contrast, no correlation was found betweenΔVO2 and ΔSmO2 after the last 3 weeks of endurance training. These results suggest that the enhanced muscle O2 availability may be one of the primary factors in increasing VO2peak after the first 3 weeks of endurance training.

Perhexiline promotes HER3 ablation through receptor internalization and inhibits tumor growth.

INTRODUCTION: Human epidermal growth factor receptor HER3 has been implicated in promoting the aggressiveness and metastatic potential of breast cancer. Upregulation of HER3 has been found to be a major mechanism underlying drug resistance to EGFR and HER2 tyrosine kinase inhibitors and to endocrine therapy in the treatment of breast cancer. Thus, agents that reduce HER3 expression at the plasma membrane may synergize with current therapies and offer a novel therapeutic strategy to improve treatment. METHODS: We devised an image-based screening platform using membrane localized HER3-YFP to identify small molecules that promote HER3 internalization and degradation. In vitro and in vivo tumor models were used to characterize the signaling effects of perhexiline, an anti-anginal drug, identified by the screening platform. RESULTS: We found perhexiline, an anti-anginal drug, selectively internalized HER3, decreased HER3 expression, and subsequently inhibited signaling downstream of HER3. Consistent with these results, perhexiline inhibited breast cancer cell proliferation in vitro and tumor growth in vivo. CONCLUSIONS: This is the first demonstration that HER3 can be targeted with small molecules by eliminating it from the cell membrane. The novel approach used here led to the discovery that perhexiline ablates HER3 expression, and offers an opportunity to identify HER3 ablation modulators as innovative therapeutics to improve survival in breast cancer patients.

CEA/CD3-bispecific T cell-engaging (BiTE) antibody-mediated T lymphocyte cytotoxicity maximized by inhibition of both PD1 and PD-L1.

Bispecific T cell-engaging (BiTE) antibodies recruit polyclonal cytotoxic T cells (CTL) to tumors. One such antibody is carcinoembryonic antigen (CEA) BiTE that mediates T cell/tumor interaction by simultaneously binding CD3 expressed by T cells and CEA expressed by tumor cells. A widely operative mechanism for mitigating cytotoxic T cell-mediated killing is the interaction of tumor-expressed PD-L1 with T cell-expressed PD-1, which may be partly reversed by PD-1/PD-L1 blockade. We hypothesized that PD-1/PD-L1 blockade during BiTE-mediated T cell killing would enhance CTL function. Here, we determined the effects of PD-1 and PD-L1 blockade during initial T cell-mediated killing of CEA-expressing human tumor cell lines in vitro, as well as subsequent T cell-mediated killing by T lymphocytes that had participated in tumor cell killing. We observed a rapid upregulation of PD-1 expression and diminished cytolytic function of T cells after they had engaged in CEA BiTE-mediated killing of tumors. T cell cytolytic activity in vitro could be maximized by administration of anti-PD-1 or anti-PD-L1 antibodies alone or in combination if applied prior to a round of T cell killing, but T cell inhibition could not be fully reversed by this blockade once the T cells had killed tumor. In conclusion, our findings demonstrate that dual blockade of PD-1 and PD-L1 maximizes T cell killing of tumor directed by CEA BiTE in vitro, is more effective if applied early, and provides a rationale for clinical use.

Once-weekly muscle endurance and strength training prevents deterioration of muscle oxidative function and attenuates the degree of strength decline during 3-week forearm immobilization.

PURPOSE: Muscle unloading causes muscle function deterioration, but the extent to which training frequency or volume can be reduced while preserving muscle function during muscle unloading is unknown. We examined the effects of low-volume muscle endurance and strength training on forearm muscle oxidative capacity, endurance, and strength during a 3-week immobilization. METHODS: Twenty-seven, healthy, male volunteers were divided into four groups: immobilization only (IMM); immobilization with endurance and strength training, once-weekly (IMM + EST1) or twice-weekly (IMM + EST2); and control, without immobilization or training (CNT). Endurance training involved dynamic handgrip exercise, at 30% of maximal voluntary contraction (MVC), until exhaustion (~60 s). Strength training involved intermittent isometric handgrip exercise at 70% MVC (40 s). Muscle oxidative capacity was evaluated after exercise using the phosphocreatine recovery time constant using (31)phosphorus magnetic resonance spectroscopy. Endurance performance was evaluated according to the total work during dynamic handgrip exercise at 30% MVC at 1 Hz until exhaustion. RESULTS: Muscle oxidative capacity and total work deterioration was restricted to the IMM (P < 0.05) group. MVC decreased in the IMM and IMM + EST1 (P < 0.05) groups. However, the MVC amplitude decrease in the IMM + EST1 group was smaller than that in the IMM (P < 0.05) group. MVC remained unchanged in the other groups. CONCLUSION: During the 3-week immobilization, twice-weekly low-volume muscle endurance and strength training prevented deterioration in muscle strength, oxidative capacity, and endurance performance. Moreover, once-weekly muscle endurance and strength training prevented the deterioration of muscle oxidative capacity and endurance performance, and attenuated the degree of muscle strength decline.

An heregulin-EGFR-HER3 autocrine signaling axis can mediate acquired lapatinib resistance in HER2+ breast cancer models.

INTRODUCTION: The human epidermal growth factor receptor 2 (HER2) receptor tyrosine kinase (RTK) oncogene is an attractive therapeutic target for the treatment of HER2-addicted tumors. Although lapatinib, an FDA-approved small-molecule HER2 and epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), represents a significant therapeutic advancement in the treatment of HER2+ breast cancers, responses to lapatinib have not been durable. Consequently, elucidation of mechanisms of acquired therapeutic resistance to HER-directed therapies is of critical importance. METHODS: Using a functional protein-pathway activation mapping strategy, along with targeted genomic knockdowns applied to a series of isogenic-matched pairs of lapatinib-sensitive and resistant cell lines, we now report an unexpected mechanism of acquired resistance to lapatinib and similar TKIs. RESULTS: The signaling analysis revealed that whereas HER2 was appropriately inhibited in lapatinib-resistant cells, EGFR tyrosine phosphorylation was incompletely inhibited. Using a targeted molecular knockdown approach to interrogate the causal molecular underpinnings of EGFR-persistent activation, we found that lapatinib-resistant cells were no longer oncogene addicted to HER2-HER3-PI3K signaling, as seen in the parental lapatinib-sensitive cell lines, but instead were dependent on a heregulin (HRG)-driven HER3-EGFR-PI3K-PDK1 signaling axis. Two FDA-approved EGFR TKIs could not overcome HRG-HER3-mediated activation of EGFR, or reverse lapatinib resistance. The ability to overcome EGFR-mediated acquired therapeutic resistance to lapatinib was demonstrated through molecular knockdown of EGFR and treatment with the irreversible pan-HER TKI neratinib, which blocked HRG-dependent phosphorylation of HER3 and EGFR, resulting in apoptosis of resistant cells. In addition, whereas HRG reversed lapatinib-mediated antitumor effects in parental HER2+ breast cancer cells, neratinib was comparatively resistant to the effects of HRG in parental cells. Finally, we showed that HRG expression is an independent negative predictor of clinical outcome in HER2+ breast cancers, providing potential clinical relevance to our findings. CONCLUSIONS: Molecular analysis of acquired therapeutic resistance to lapatinib identified a new resistance mechanism based on incomplete and "leaky" inhibition of EGFR by lapatinib. The selective pressure applied by incomplete inhibition of the EGFR drug target resulted in selection of ligand-driven feedback that sustained EGFR activation in the face of constant exposure to the drug. Inadequate target inhibition driven by a ligand-mediated autocrine feedback loop may represent a broader mechanism of therapeutic resistance to HER TKIs and suggests adopting a different strategy for selecting more effective TKIs to advance into the clinic.

A randomized phase II study of immunization with dendritic cells modified with poxvectors encoding CEA and MUC1 compared with the same poxvectors plus GM-CSF for resected metastatic colorectal cancer.

OBJECTIVE: To determine whether 1 of 2 vaccines based on dendritic cells (DCs) and poxvectors encoding CEA (carcinoembryonic antigen) and MUC1 (PANVAC) would lengthen survival in patients with resected metastases of colorectal cancer (CRC). BACKGROUND: Recurrences after complete resections of metastatic CRC remain frequent. Immune responses to CRC are associated with fewer recurrences, suggesting a role for cancer vaccines as adjuvant therapy. Both DCs and poxvectors are potent stimulators of immune responses against cancer antigens. METHODS: Patients, disease-free after CRC metastasectomy and perioperative chemotherapy (n = 74), were randomized to injections of autologous DCs modified with PANVAC (DC/PANVAC) or PANVAC with per injection GM-CSF (granulocyte-macrophage colony-stimulating factor). Endpoints were recurrence-free survival overall survival, and rate of CEA-specific immune responses. Clinical outcome was compared with that of an unvaccinated, contemporary group of patients who had undergone CRC metastasectomy, received similar perioperative therapy, and would have otherwise been eligible for the study. RESULTS: Recurrence-free survival at 2 years was similar (47% and 55% for DC/PANVAC and PANVAC/GM-CSF, respectively) (χ P = 0.48). At a median follow-up of 35.7 months, there were 2 of 37 deaths in the DC/PANVAC arm and 5 of 37 deaths in the PANVAC/GM-CSF arm. The rate and magnitude of T-cell responses against CEA was statistically similar between study arms. As a group, vaccinated patients had superior survival compared with the contemporary unvaccinated group. CONCLUSIONS: Both DC and poxvector vaccines have similar activity. Survival was longer for vaccinated patients than for a contemporary unvaccinated group, suggesting that a randomized trial of poxvector vaccinations compared with standard follow-up after metastasectomy is warranted. (NCT00103142).

Novel adenoviral vector induces T-cell responses despite anti-adenoviral neutralizing antibodies in colorectal cancer patients.

First-generation, E1-deleted adenovirus subtype 5 (Ad5)-based vectors, although promising platforms for use as cancer vaccines, are impeded in activity by naturally occurring or induced Ad-specific neutralizing antibodies. Ad5-based vectors with deletions of the E1 and the E2b regions (Ad5 [E1-, E2b-]), the latter encoding the DNA polymerase and the pre-terminal protein, by virtue of diminished late phase viral protein expression, were hypothesized to avoid immunological clearance and induce more potent immune responses against the encoded tumor antigen transgene in Ad-immune hosts. Indeed, multiple homologous immunizations with Ad5 [E1-, E2b-]-CEA(6D), encoding the tumor antigen carcinoembryonic antigen (CEA), induced CEA-specific cell-mediated immune (CMI) responses with antitumor activity in mice despite the presence of preexisting or induced Ad5-neutralizing antibody. In the present phase I/II study, cohorts of patients with advanced colorectal cancer were immunized with escalating doses of Ad5 [E1-, E2b-]-CEA(6D). CEA-specific CMI responses were observed despite the presence of preexisting Ad5 immunity in a majority (61.3 %) of patients. Importantly, there was minimal toxicity, and overall patient survival (48 % at 12 months) was similar regardless of preexisting Ad5 neutralizing antibody titers. The results demonstrate that, in cancer patients, the novel Ad5 [E1-, E2b-] gene delivery platform generates significant CMI responses to the tumor antigen CEA in the setting of both naturally acquired and immunization-induced Ad5-specific immunity.