Targeting CXCR4-dependent immunosuppressive Ly6Clow monocytes improves antiangiogenic therapy in colorectal cancer.

Antiangiogenic therapy with antibodies against VEGF (bevacizumab) or VEGFR2 (ramucirumab) has been proven efficacious in colorectal cancer (CRC) patients. However, the improvement in overall survival is modest and only in combination with chemotherapy. Thus, there is an urgent need to identify potential underlying mechanisms of resistance specific to antiangiogenic therapy and develop strategies to overcome them. Here we found that anti-VEGFR2 therapy up-regulates both C-X-C chemokine ligand 12 (CXCL12) and C-X-C chemokine receptor 4 (CXCR4) in orthotopic murine CRC models, including SL4 and CT26. Blockade of CXCR4 signaling significantly enhanced treatment efficacy of anti-VEGFR2 treatment in both CRC models. CXCR4 was predominantly expressed in immunosuppressive innate immune cells, which are recruited to CRCs upon anti-VEGFR2 treatment. Blockade of CXCR4 abrogated the recruitment of these innate immune cells. Importantly, these myeloid cells were mostly Ly6Clow monocytes and not Ly6Chigh monocytes. To selectively deplete individual innate immune cell populations, we targeted key pathways in Ly6Clow monocytes (Cx3cr1-/- mice), Ly6Chigh monocytes (CCR2-/- mice), and neutrophils (anti-Ly6G antibody) in combination with CXCR4 blockade in SL4 CRCs. Depletion of Ly6Clow monocytes or neutrophils improved anti-VEGFR2-induced SL4 tumor growth delay similar to the CXCR4 blockade. In CT26 CRCs, highly resistant to anti-VEGFR2 therapy, CXCR4 blockade enhanced anti-VEGFR2-induced tumor growth delay but specific depletion of Ly6G+ neutrophils did not. The discovery of CXCR4-dependent recruitment of Ly6Clow monocytes in tumors unveiled a heretofore unknown mechanism of resistance to anti-VEGF therapies. Our findings also provide a rapidly translatable strategy to enhance the outcome of anti-VEGF cancer therapies.

Esophageal Carcinosarcoma with Basaloid Squamous Cell Carcinoma: A Case Report and Review of the Literature.

Esophageal carcinosarcoma is a rare tumor composed of neoplastic squamous epithelium and sarcomatous spindle cells. The origin of spindle cells remains unknown; however, the majority of sarcomatous components are currently considered to be derived from existing carcinomatous cells via epithelial-mesenchymal transition (EMT). We report a case of esophageal carcinosarcoma harboring basaloid squamous cell carcinoma successfully treated with preoperative chemotherapy. A 78-year-old man complaining dysphagia was diagnosed as esophageal carcinosarcoma. After two courses of preoperative chemotherapy with cisplatin and 5-fluorouracil, curative esophagectomy with lymph node dissection was performed thoracoscopically. Histopathological findings of the resected specimen revealed the mixture of basaloid squamous cell carcinoma and sarcomatous spindle cells. A transitional zone between both components was also detected. As fibrosis was identified around both two components, the findings indicated that both carcinomatous and sarcomatous neoplasms disappeared by preoperative chemotherapy. Final pathological diagnosis was esophageal carcinosarcoma with basaloid squamous cell carcinoma. No recurrent lesions have been detected for 25 months after the surgery. Sarcomatous spindle cells could be derived from the components of basaloid squamous cell carcinoma in our present case due to the presence of histological transition between two components. In addition, the marked immunoreactivity of vimentin (an EMT marker) detected in the tumor cells of basaloid squamous cell carcinoma could be consistent with the concept of monoclonal origin via EMT. The regimen targeting squamous cell carcinoma could also be effective in the treatment of sarcomatous components. Preoperative therapy might achieve the improvement of clinical outcome of patients with esophageal carcinosarcoma.

Definitive chemoradiotherapy with docetaxel, cisplatin, and 5-fluorouracil (DCF-R) for advanced cervical esophageal cancer.

BACKGROUND: Recently, definitive chemoradiotherapy (dCRT) has become one of the essential treatment strategies for esophageal squamous cell carcinoma (ESCC) and has been especially gaining prevalence for cervical ESCC to preserve the larynx. Our department recently introduced dCRT concomitant with docetaxel, cisplatin, and 5-fluorouracil (DCF-R) for treating advanced cervical ESCC. This study aims to assess the safety and outcomes of DCF-R in patients with advanced cervical ESCC. METHODS: We retrospectively assessed 11 patients with advanced cervical ESCC (clinical stage: II-IV, including T4b and/or M1 lymph node) who received DCF-R as the first-line treatment between December 2010 and February 2015. RESULTS: Our patient cohort comprised 8 males and 3 females (median age 68 years; range 54-76 years). The pretreatment clinical stage included stage II (1), stage III (7), and stage IV (3) cases [including 3 patients with T4b (2 trachea and 1 thyroid) and 3 patients with M1 lymph node]. We attained complete response (CR) in 10 patients and stable disease in 1 patient. Of 10 patients with CR, 5 experienced recurrence and 5 continued exhibiting CR. Furthermore, grade 3 or more adverse events included leucopenia (91%), neutropenia (91%), febrile neutropenia (45%), and pharyngeal pain (55%). While the 2-year overall survival rate was 72%, the 2-year recurrent-free survival rate was 64%, respectively. CONCLUSIONS: DCF-R treatment for advanced cervical esophageal cancer could be completed by the careful administration; although a strong blood toxicity might occur, this treatment may provide the chance to obtain favorable prognosis with larynx preservation.

Endogenous angiogenesis inhibitor vasohibin1 exhibits broad-spectrum antilymphangiogenic activity and suppresses lymph node metastasis.

During cancer progression, the angiogenesis that occurs is involved in tumor growth and hematogenous-distant metastasis, whereas lymphangiogenesis is involved in regional lymph node metastasis. Angiogenesis is counterregulated by various endogenous inhibitors; however, little is known about endogenous inhibitors of lymphangiogenesis. We recently isolated vasohibin1 as an angiogenesis inhibitor intrinsic to the endothelium and further demonstrated its anticancer activity through angiogenesis inhibition. Here, we examined the effect of vasohibin1 on lymphangiogenesis. Vasohibin1 exhibited broad-spectrum antilymphangiogenic activity in the mouse cornea induced by factors including VEGF-A, VEGF-C, FGF2, and PDGF-BB. We then inoculated highly lymph node-metastatic cancer cells into mice and examined the effect of vasohibin1 on lymph node metastasis. Tail-vein injection of adenovirus containing the human vasohibin1 gene inhibited tumor lymphangiogenesis and regional lymph node metastasis. Moreover, local injection of recombinant vasohibin1 inhibited lymph node metastasis. These results suggest vasohibin1 to be the first known intrinsic factor having broad-spectrum antilymphangiogenic activity and indicate that it suppresses lymph node metastasis.

Impairment of VEGF-A-stimulated lamellipodial extensions and motility of vascular endothelial cells by chondromodulin-I, a cartilage-derived angiogenesis inhibitor.

Chondromodulin-I (ChM-I) is a cartilage-derived angiogenesis inhibitor that has been identified as inhibitory to the growth activity of vascular endothelial cells. In our present study, we demonstrate the anti-angiogenic activity of recombinant human ChM-I (rhChM-I) in mouse corneal angiogenesis and examine its action. We focus on the VEGF-A-induced migration of vascular endothelial cells, a critical regulatory step in angiogenesis. In a modified Boyden chamber assay, nanomolar concentrations of rhChM-I inhibited the chemotactic migration of human umbilical vein endothelial cells (HUVECs) induced by VEGF-A as well as by FGF-2 and IGF-I. The ChM-I action was found to be endothelial cell-specific and independent of cell adhesions. Time-lapse analysis further revealed that rhChM-I markedly reduces VEGF-A-stimulated motility of HUVECs and causes frequent alterations of the moving front due to the appearance of multiple transient protrusions. This action involved the inhibition of cell spreading and the disrupted reorganization of the actin cytoskeleton upon VEGF-A stimulation. Consistent with these observations, rhChM-I was found to significantly reduce the activity of Rac1/Cdc42 during cell spreading, and the VEGF-A-induced Rac1 activity but not its basal activity in quiescent cells. Taken together, our present data suggest that ChM-I impairs the VEGF-A-stimulated motility of endothelial cells by destabilizing lamellipodial extensions.

Vasohibin-1 expression in endothelium of tumor blood vessels regulates angiogenesis.

In this study, we characterized the significance of the vascular endothelial growth factor-inducible angiogenesis inhibitor vasohibin-1 to tumors. In pathological sections of non-small cell lung carcinoma, vasohibin-1 was present in the endothelial cells of blood vessels of the tumor stroma, but not in the lymphatics. In cancer cells, the presence of vasohibin-1 was associated with hypoxia-inducible factor 1alpha/vascular endothelial growth factor and fibroblast growth factor-2 expression. We then examined the function of vasohibin-1 in the mouse by subcutaneously inoculating with Lewis lung carcinoma cells. Resultant tumors in vasohibin-1(-/-) mice contained more immature blood vessels and fewer apoptotic tumor cells than tumors in wild-type mice. In wild-type mice that had been inoculated with Lewis lung carcinoma cells, tail vein injection of adenovirus containing the human vasohibin-1 gene inhibited tumor growth and tumor angiogenesis. Moreover, the remaining tumor vessels in adenoviral human vasohibin-1 gene-treated mice were small, round, and mature, surrounded by mural cells. The addition of adenoviral human vasohibin-1 gene to cisplatin treatment improved cisplatin's antitumor activity in mice. These results suggest that endogenous vasohibin-1 is not only involved in tumor angiogenesis, but when sufficient exogenous vasohibin-1 is supplied, it blocks sprouting angiogenesis by tumors, matures the remaining vessels, and enhances the antitumor effect of conventional chemotherapy.

A comparative analysis of clinicopathological factors between esophageal small cell and basaloid squamous cell carcinoma.

Esophageal small cell carcinoma (E-SmCC) and basaloid squamous cell carcinomas (BSCCs) are both highly aggressive malignancies, but their detailed differences in clinical behaviors have remained virtually unknown. In addition, treatment strategies of the patients with E-SmCC have not been established. 29 cases of E-SmCC and 39 with BSCC were examined in this study to clarify the clinical features and outcome of the patients with E-SmCC and to compare the findings with those of BSCC. E-SmCCs presented a more advanced status than BSCC (TNM Stage: P = .002). Esophagectomy was performed in 15 small cell carcinoma patients and 14 were treated with non-surgical/systemic therapy. The clinical outcome of the small cell carcinoma cases was significantly worse than those with BSCC (P = .001), but results of a stage-stratified analysis revealed that the Stage I small cell carcinoma patients presented favorable prognosis (3-year survival rate 100%, n = 4). In contrast, among those with Stage II-IV, clinical outcome tended to be better in the systemic therapy group (3-year survival rate 49%, n = 13) than the surgically treated group (3-year survival rate 0%, n = 12). E-SmCC was a more aggressive neoplasm than BSCC. However, early detection could possibly improve the clinical outcome of patients with E-SmCC. Systemic therapy could also benefit the patients with advanced disease (Stage II-IV).

Hiatal hernia involving prolapse of the entire stomach into the mediastinum after distal gastrectomy: a case report.

BACKGROUND: Prolapse of a small part of the proximal stomach through the hiatus into the mediastinum is relatively common. Hiatal hernia involving the postoperative stomach has been reported previously, but the degree of hernia was not so severe, and hiatal hernia involving the prolapse of the entire stomach following gastrectomy into the mediastinum has never been reported. We describe a very rare case of large hiatal hernia involving the entire postoperative stomach. CASE PRESENTATION: A 79-year-old man with a history of distal gastrectomy for submucosal benign tumor 40 years ago was referred to our hospital because of dysphagia and weight loss. Computed tomography revealed prolapse of the entire postoperative stomach into the mediastinum, and a radical operation was performed. There was a strong adhesion in the hernial sac of the mediastinum, but only little adhesion due to a previous open surgery in the abdominal cavity was present. After the stomach was pulled into the abdominal cavity, suture cruroplasty and Toupet fundoplication without dissection of the short gastric artery were performed. The patient experienced postoperative paralytic ileus, but the rest of the postoperative course was uneventful and the symptom of dysphagia improved. CONCLUSIONS: We presented a very rare large hiatal hernia involving the entire postoperative stomach. Toupet fundoplication preserving the short gastric artery could be one of the optimal surgeries to prevent postoperative regurgitation of the remnant stomach.

Curative resection of advanced esophageal cancer with metachronous stage IV breast cancer: A case report.

INTRODUCTION: Recent studies have shown the safety and efficacy of curative resection of esophageal cancer with multiple primary cancers. However, our literature search revealed no curative surgery cases for esophageal cancer in patients with multiple primary cancers with distant metastasis. CASE PRESENTATION: A 75-year-old woman visited our hospital with dysphagia. She had a history of breast cancer with multiple bone metastasis. Esophagogastroduodenoscopy revealed a circumferential mass in the upper intrathoracic esophagus. Histopathological examination of the biopsy showed squamous cell carcinoma. Other imaging findings revealed multiple nodules in the liver. The nodules were thought to have originated from the breast, but metastasis of esophageal cancer was considered a possibility. Intraoperative frozen sections of the liver and peritoneal nodules showed adenocarcinoma. Thoracoscopic esophagectomy was then performed. Following surgery, the patient received fulvestrant therapy, followed by capecitabine therapy, and the liver tumors decreased in size. She is currently alive after 1.5 years of the surgery without local recurrence of esophageal cancer. DISCUSSION: Although the patient had metastatic breast cancer, her relapse-free interval of 20 years and good response to hormone therapy for 15 years were favorable prognostic factors. Her life expectancy was estimated to be a few years and surgery was performed. CONCLUSION: Curative resection could be considered for patients with esophageal cancer who have an additional cancer with distant metastasis when the prognosis of the additional cancer is not poor.

Different strategy of salvage esophagectomy between residual and recurrent esophageal cancer after definitive chemoradiotherapy.

BACKGROUND: Clinical outcomes appear to differ between patients with residual or recurrent esophageal cancer after definitive chemoradiotherapy. We aimed to identify the patients most likely to benefit from this high-risk surgery, divided by the patients whose cancer was residual and recurrent groups, respectively. METHODS: We retrospectively examined 100 cases of patients who failed to respond to definitive chemoradiotherapy for thoracic esophageal squamous cell carcinoma and subsequently underwent salvage transthoracic esophagectomy. RESULTS: In-hospital morbidity was similar in both groups. T status prior to administration of chemoradiotherapy correlated with survival in the group with residual cancer (P=0.010), but this relationship was not significant in the group with recurrent cancer (P=0.635). On the other hand, pathological T status showed a significant correlation with survival in both the residual (P<0.001) and recurrent groups (P=0.001). Patients with T3 disease in the recurrent group showed better survival, similar to T0-2 patients, while worse survival was demonstrated in the residual group. In the recurrent group, N status before chemoradiotherapy did not correlate with survival (P=0.895). CONCLUSIONS: Patients with residual cancer would have good prognosis by salvage esophagectomy in cases in which the cancer had not invaded to the adventitia at the time of chemoradiotherapy and surgery. Conversely, patients whose cancer was recurrent might benefit from salvage surgery if the cancer appears to be resectable. T and N status before chemoradiotherapy are not important factors in consideration of salvage esophagectomy in cases of recurrent cancer.

Treatment of spontaneous esophageal rupture (Boerhaave syndrome) using thoracoscopic surgery and sivelestat sodium hydrate.

BACKGROUND: The mortality rate of spontaneous esophageal rupture remains 20% to 40% due to severe respiratory failure. We have performed thoracoscopic surgery for esophageal disease at our department since 1994. Sivelestat sodium hydrate reportedly improves the pulmonary outcome in the patients with acute lung injury (ALI). METHODS: We retrospectively evaluated the usefulness of thoracoscopic surgery and perioperative administration of sivelestat sodium hydrate for spontaneous esophageal rupture in 12 patients who underwent thoracoscopy at our department between 2002 and 2014. RESULTS: The patient cohort included 11 males and one female (median age, 61 years). The lower left esophageal wall was perforated in all patients. Surgical procedures consisted of thoracoscopic suture and thoracic drainage in six patients, transhiatal suture and thoracoscopic thoracic drainage in five, and thoracoscopic esophagectomy and thoracic drainage in one. The median time from onset to surgery was 8 hours with a surgical duration of 210 minutes, blood loss 260 mL, postoperative ventilator management 1 day, intensive care unit (ICU) stay 5 days, and interval to restoration of oral ingestion 13 days. Postoperative complications included respiratory failure in four patients, pyothorax in three, and leakage in one. There was no instance of perioperative mortality. Regarding perioperative administration of sivelestat sodium hydrate, the postoperative arterial oxygen partial pressure-to-fractional inspired oxygen ratio (P/F) and C-reactive protein (CRP) levels in the administration group were significantly better than those in the non-administration group on postoperative days 4 (P=0.035) and 5 (P=0.037), respectively. In contrast, there was no significant difference between the groups in median time of ventilator management, ICU stay, oral ingestion following surgery, or hospital stay. CONCLUSIONS: Thoracoscopic surgery obtained acceptable results in all patients, including two with a significant time elapse from onset to treatment. Furthermore, sivelestat sodium hydrate was suggested to help improve postoperative respiration and inflammatory response.

Bilateral approach for thoracoscopic esophagectomy with lymph node dissection in the dorsal area of the thoracic aorta in patients with esophageal cancer: A report of two cases.

INTRODUCTION: The incidence of lymph node metastasis in the dorsal area of the thoracic aorta (DTA) is relatively low in patients with esophageal cancer. It is difficult to approach the DTA using surgical procedures, such as an open thoracotomy and thoracoscopy in the left decubitus position. CASE PRESENTATION: Case 1: A 70-year-old man with esophageal cancer underwent thoracoscopic esophagectomy with mediastinal lymph node dissection via a right thoracoscopic approach, followed by lymphadenectomy in the DTA via left thoracoscopy in the prone position. Microscopic findings revealed two metastatic lymph nodes in the DTA. The definitive diagnosis was squamous cell carcinoma of the esophagus, and the pathological stage was T2N3M0 (Union for International Cancer Control [UICC], 7th edition). The patient showed lung metastasis 8 months after the surgery. Case 2: A 72-year-old man with esophageal cancer underwent esophagectomy via a bilateral approach in the prone position, using a similar procedure as in case 1. The definitive diagnosis was squamous cell carcinoma of the esophagus, and the pathological stage was T3N2M0. The patient showed a metastatic mediastinal lymph node 4 months after the surgery. CONCLUSION: Bilateral thoracoscopic esophagectomy in the prone position can be safely performed, and it might be an alternative curative surgery for esophageal cancer. However, both our cases showed metastasis in the early postoperative period. The long-term outcome and significance of dissection of lymph nodes in the DTA in patients with esophageal cancer remains controversial. Further studies are required to establish the indications and efficacy of this therapeutic approach.

Combined thoracoscopic and endoscopic surgery for a large esophageal schwannoma.

A 47-year-old woman presented to our hospital with complaints of dysphagia. Esophagogastroduodenoscopy identified a submucosal tumor in the left wall of the esophagus that was diagnosed as a benign schwannoma on biopsy. Computed tomography revealed a tumor of length 60 mm in the thoracic esophagus, with its cranial edge at the level of the aortic arch. On endoscopy, a submucosal tunnel was created 40 mm proximal to the cranial edge of the tumor, and its oral end was dissected from the mucosal and muscular layers. This was followed by the resection of the entire tumor by left-sided thoracoscopy. The esophageal defect was closed in layer by continuous suture from the thoracic side. Endoscopic closure was achieved by using clips. No postoperative complications were observed. Oral diet was resumed from postoperative day 7 and the patient was discharged on postoperative day 9. This combined approach has not been described for similar tumors. Our experience demonstrated that large esophageal tumors can be safely excised with minimally invasive surgery by using a combination of thoracoscopy and endoscopy.

Ly6Clo monocytes drive immunosuppression and confer resistance to anti-VEGFR2 cancer therapy.

Current anti-VEGF therapies for colorectal cancer (CRC) provide limited survival benefit, as tumors rapidly develop resistance to these agents. Here, we have uncovered an immunosuppressive role for nonclassical Ly6Clo monocytes that mediates resistance to anti-VEGFR2 treatment. We found that the chemokine CX3CL1 was upregulated in both human and murine tumors following VEGF signaling blockade, resulting in recruitment of CX3CR1+Ly6Clo monocytes into the tumor. We also found that treatment with VEGFA reduced expression of CX3CL1 in endothelial cells in vitro. Intravital microscopy revealed that CX3CR1 is critical for Ly6Clo monocyte transmigration across the endothelium in murine CRC tumors. Moreover, Ly6Clo monocytes recruit Ly6G+ neutrophils via CXCL5 and produce IL-10, which inhibits adaptive immunity. Preventing Ly6Clo monocyte or Ly6G+ neutrophil infiltration into tumors enhanced inhibition of tumor growth with anti-VEGFR2 therapy. Furthermore, a gene therapy using a nanoparticle formulated with an siRNA against CX3CL1 reduced Ly6Clo monocyte recruitment and improved outcome of anti-VEGFR2 therapy in mouse CRCs. Our study unveils an immunosuppressive function of Ly6Clo monocytes that, to our knowledge, has yet to be reported in any context. We also reveal molecular mechanisms underlying antiangiogenic treatment resistance, suggesting potential immunomodulatory strategies to enhance the long-term clinical outcome of anti-VEGF therapies.

Anti-VEGF therapy induces ECM remodeling and mechanical barriers to therapy in colorectal cancer liver metastases.

The survival benefit of anti-vascular endothelial growth factor (VEGF) therapy in metastatic colorectal cancer (mCRC) patients is limited to a few months because of acquired resistance. We show that anti-VEGF therapy induced remodeling of the extracellular matrix with subsequent alteration of the physical properties of colorectal liver metastases. Preoperative treatment with bevacizumab in patients with colorectal liver metastases increased hyaluronic acid (HA) deposition within the tumors. Moreover, in two syngeneic mouse models of CRC metastasis in the liver, we show that anti-VEGF therapy markedly increased the expression of HA and sulfated glycosaminoglycans (sGAGs), without significantly changing collagen deposition. The density of these matrix components correlated with increased tumor stiffness after anti-VEGF therapy. Treatment-induced tumor hypoxia appeared to be the driving force for the remodeling of the extracellular matrix. In preclinical models, we show that enzymatic depletion of HA partially rescued the compromised perfusion in liver mCRCs after anti-VEGF therapy and prolonged survival in combination with anti-VEGF therapy and chemotherapy. These findings suggest that extracellular matrix components such as HA could be a potential therapeutic target for reducing physical barriers to systemic treatments in patients with mCRC who receive anti-VEGF therapy.

Effects of vascular-endothelial protein tyrosine phosphatase inhibition on breast cancer vasculature and metastatic progression.

BACKGROUND: The solid tumor microvasculature is characterized by structural and functional abnormality and mediates several deleterious aspects of tumor behavior. Here we determine the role of vascular endothelial protein tyrosine phosphatase (VE-PTP), which deactivates endothelial cell (EC) Tie-2 receptor tyrosine kinase, thereby impairing maturation of tumor vessels. METHODS: AKB-9778 is a first-in-class VE-PTP inhibitor. We examined its effects on ECs in vitro and on embryonic angiogenesis in vivo using zebrafish assays. We studied the impact of AKB-9778 therapy on the tumor vasculature, tumor growth, and metastatic progression using orthotopic models of murine mammary carcinoma as well as spontaneous and experimental metastasis models. Finally, we used endothelial nitric oxide synthase (eNOS)-deficient mice to establish the role of eNOS in mediating the effects of VE-PTP inhibition. All statistical tests were two-sided. RESULTS: AKB-9778 induced ligand-independent Tie-2 activation in ECs and impaired embryonic zebrafish angiogenesis. AKB-9778 delayed the early phase of mammary tumor growth by maintaining vascular maturity (P < .01, t test); slowed growth of micrometastases (P < .01, χ(2) test) by preventing extravasation of tumor cells (P < 0.01, Fisher exact test), resulting in a trend toward prolonged survival (27.0 vs 36.5 days; hazard ratio of death = 0.33, 95% confidence interval = 0.11 to 1.03; P = .05, Mantel-Cox test); and stabilized established primary tumor blood vessels, enhancing tumor perfusion (P = .03 for 4T1 tumor model and 0.05 for E0771 tumor model, by two-sided t tests) and, hence, radiation response (P < .01, analysis of variance; n = 7 mice per group). The effects of AKB-9778 on tumor vessels were mediated in part by endothelial nitric oxide synthase activation. CONCLUSIONS: Our results demonstrate that pharmacological VE-PTP inhibition can normalize the structure and function of tumor vessels through Tie-2 activation, which delays tumor growth, slows metastatic progression, and enhances response to concomitant cytotoxic treatments.