Checkpoint molecule expression by B and T cell lymphomas in dogs.

Immunotherapies targeting checkpoint molecule programmed cell death 1 (PD-1) protein were shown to be effective for treatment of non-Hodgkin lymphoma in people, but little is known about the expression of PD-1 or its ligand PD-L1 by canine lymphoma. Therefore, flow cytometry was used to analyse expression of PD-1 and PD-L1 in canine lymphoma, using fine-needle aspirates of lymph nodes from 34 dogs with B cell lymphoma (BCL), 6 dogs with T cell lymphoma (TCL) and 11 dogs that had relapsed. Furthermore, fine-needle aspirates were obtained from 17 healthy dogs for comparison. Lastly, the impact of chemotherapy resistance on expression of PD-1 and PD-L1 was assessed in vitro. These studies revealed increased expression of PD-L1 by malignant B cells compared to normal B cells. In the case of TCL, tumour cells and normal T cells both showed low to negative expression of PD-1 and PD-L1. In addition, tumour infiltrating lymphocytes from both BCL and TCL had increased expression of both PD-1 and PD-L1 expression compared to B and T cells from lymph nodes of healthy animals. In vitro, chemotherapy-resistant BCL and TCL cell lines exhibited increases in both PD-1 and PD-L1 expression, compared to non-chemotherapy selected tumour cells. These findings indicate that canine lymphomas exhibit upregulated checkpoint molecule expression, though the impact of checkpoint molecule expression on tumour biological behaviour remains unclear.

User survey of Nanny Angel Network, a free childcare service for mothers with cancer.

PURPOSE: The purpose of the present study was to determine user satisfaction with Nanny Angel Network (nan), a free childcare service for mothers undergoing cancer treatment. METHODS: All 243 living mothers who had used the nan service were invited by telephone to participate in an online research survey; 197 mothers (81%) consented to participate. The survey, sent by e-mail, consisted of 39 items divided into these categories: demographics, supports, use, satisfaction, and general comments. RESULTS: Of the 197 mothers who consented to receive the e-mailed survey, 104 (53%) completed it. More than 90% of the mothers were very satisfied with the help and support from their Nanny Angel. Many mothers mentioned that the Nanny Angel was most helpful during treatment and medical appointments, with 75% also mentioning that their Nanny Angel helped them to adhere to their scheduled medical appointments. However, 64% felt that they had not received enough visits from their Nanny Angel. CONCLUSIONS: Satisfaction with the nan childcare provider was high, but mothers wished the service had been available to them more often. Our study highlights the importance of providing childcare to mothers with inadequate support systems, so as to allow for greater adherence to treatment and medical appointments, and for more time to recover.

PD-1 expression by canine T cells and functional effects of PD-1 blockade.

The co-inhibitory checkpoint molecule programmed death receptor 1 (PD-1) can trigger T cell functional exhaustion upon binding to its ligand PD-L1 expressed on tumour cells or macrophages. PD-1 blocking antibodies have generated remarkable results in human cancer patients, including inducing durable responses in a number of advanced cancers. Therefore, monoclonal antibodies specific for canine PD-1 were assessed for T cell binding and induction of functional activation. A total of 5-10% of CD4 T cells and 20-25% of CD8 T cells from healthy dogs expressed PD-1, and PD-1 expression was upregulated on T cells from dogs with cancer. Functionally, PD-1 antibodies significantly enhanced T-cell activation, as assessed by proliferation and interferon-gamma (IFN-γ) production. PD-1 antibodies also reversed T-cell suppression induced by canine soluble PD-L1 and by tumour cells and tumour explant fragments. These findings indicate that PD-1 antibodies have potential for use in cancer immunotherapy in dogs.

Phase I lead-in and subsequent randomized trial assessing safety and modulation of regulatory T cell numbers following a maximally tolerated dose doxorubicin and metronomic dose cyclophosphamide combination chemotherapy protocol in tumour-bearing dogs.

Maximally tolerated dose (MTD) and metronomic dose chemotherapeutic approaches alter the immune system and the angiogenic process in different yet potentially complementary ways. A combination of MTD doxorubicin (MTD-DOX) and metronomic cyclophosphamide (mCTX) protocol was evaluated for safety and effect on circulating regulatory T (Treg) cells. We found that mCTX can be safely administered with MTD-DOX in tumour-bearing dogs. Both combination DOX/mCTX and single-agent DOX resulted in significant depletions of circulating lymphocytes throughout the chemotherapy cycle without apparent selectivity for Tregs. The indiscriminant lymphocyte depletions were similar between dogs randomized to receive DOX and dogs randomized to receive DOX/mCTX, suggesting this effect is because of DOX alone. These findings may have implications as to the therapeutic benefit (or lack thereof) of concurrent combination MTD and metronomic protocols. Future investigations are required to determine the effects and indeed the efficacy of concurrent versus sequential applications of MTD and metronomic chemotherapy protocols.

Immune regulation of canine tumour and macrophage PD-L1 expression.

Expression of programmed cell death receptor ligand 1 (PD-L1) on tumor cells has been associated with immune escape in human and murine cancers, but little is known regarding the immune regulation of PD-L1 expression by tumor cells and tumor-infiltrating macrophages in dogs. Therefore, 14 canine tumor cell lines, as well as primary cultures of canine monocytes and macrophages, were evaluated for constitutive PD-L1 expression and for responsiveness to immune stimuli. We found that PD-L1 was expressed constitutively on all canine tumor cell lines evaluated, although the levels of basal expression were very variable. Significant upregulation of PD-L1 expression by all tumor cell lines was observed following IFN-γ exposure and by exposure to a TLR3 ligand. Canine monocytes and monocyte-derived macrophages did not express PD-L1 constitutively, but did significantly upregulate expression following treatment with IFN-γ. These findings suggest that most canine tumors express PD-L1 constitutively and that both innate and adaptive immune stimuli can further upregulate PD-L1 expression. Therefore the upregulation of PD-L1 expression by tumor cells and by tumor-infiltrating macrophages in response to cytokines such as IFN-γ may represent an important mechanism of tumor-mediated T-cell suppression in dogs as well as in humans.

Supraclavicular artery island flap in head and neck reconstruction.

Due to the complex anatomy of the head and neck, a wide range of pedicled or free flaps must be available to ensure optimal reconstruction of the various defects resulting from cancer surgery. The supraclavicular artery island flap is a fasciocutaneous flap harvested from the supraclavicular and deltoid regions. The blood supply of this flap is derived from the supraclavicular artery, a direct cutaneous branch of the transverse cervical artery in 93% of cases or the supraclavicular artery in 7% of cases. The supraclavicular artery is located in a triangle delineated by the posterior border of the sternocleidomastoid muscle medially, the external jugular vein posteriorly, and the median portion of the clavicle anteriorly. This pedicled flap is thin, malleable, and is easily and rapidly harvested with a reliable pedicle and minimal donor site morbidity. It can be used for one-step innervated reconstruction of many types of head and neck defects. It constitutes an alternative to local flaps, while providing equivalent functional results and must be an integral part of the cancer surgeon's therapeutic armamentarium.

Comparison of radial forearm flap and thoracodorsal artery perforator flap donor site morbidity for reconstruction of oral and oropharyngeal defects in head and neck cancer.

INTRODUCTION: Reconstruction of the oral cavity and oropharynx after tumour resection often involves the use of free flaps, but donor site morbidity must be taken into account. The radial forearm flap, the flap most commonly used in this setting, leaves a readily visible scar on an exposed region of the body. The thoracodorsal artery perforator flap (TDAP), which possesses the same plastic qualities as the radial forearm flap, leaves a scar that is hidden in the axilla. The purpose of this study was to evaluate the cosmetic results of radial forearm and thoracodorsal artery perforator free flap donor sites. MATERIAL AND METHODS: The medical charts of all patients undergoing reconstruction by a radial forearm or thoracodorsal artery perforator free flap between January 2011 and December 2011 were retrospectively reviewed. The Patient and Observer Scar Assessment Scales and the Vancouver Scar Scale were used to evaluate the quality of the scars. RESULTS: Reconstruction was performed by radial forearm flap in 4 cases and TDAP flap in 7 cases. The PSAS score was significantly lower in the TDAP group than in the radial forearm group (P=0.03), and the OSAS score was higher in the radial forearm group (21.5 versus 14). The Vancouver Scar Scale was significantly higher for radial forearm flap scars than for TDAP scars (8 versus 2.7, P=0.005). CONCLUSION: This is the first study to compare radial forearm and thoracodorsal artery perforator free flap donor site scars. It demonstrates the minimal TDAP donor site morbidity and the high level of patient satisfaction.

Velopharyngeal function after free thoracodorsal artery perforator flap in lateral and superior oropharyngeal cancer.

The purpose of this study was to report our experience with the free thoracodorsal artery perforator flap for reconstruction of the oropharynx and soft palate in head and neck cancer using a retrospective review of the medical charts of all patients undergoing oropharyngeal reconstruction by free thoracodorsal artery perforator flap during the same procedure as cancer resection between January 2011 and April 2013. Evaluation of speech, feeding and the presence of nasal emissions was performed 6 months after treatment in accordance with the Declaration of Helsinki. Nine patients were evaluated. Clear understanding of the patient was reported by the family and the examiner for seven patients, while understanding difficulties were reported for two patients (1 case of flap dehiscence and 1 technical error of flap fixation). The results indicated that, due to its complex anatomy and function, reconstruction of the soft palate remains a delicate procedure. The free thoracodorsal artery perforator flap allows functional soft palate reconstruction, while limiting donor site morbidity.

L-dex ratio in detecting breast cancer-related lymphedema: reliability, sensitivity, and specificity.

Advances in bioelectrical impedance analysis (BIA) permit the assessment of lymphedema by directly measuring lymph fluid changes. The objective of the study was to examine the reliability, sensitivity, and specificity of cross-sectional assessment of BIA in detecting lymphedema in a large metropolitan clinical setting. BIA was used to measure lymph fluid changes. Limb volume by sequential circumferential tape measurement was used to validate the presence of lymphedema. Data were collected from 250 women, including healthy female adults, breast cancer survivors with lymphedema, and those at risk for lymphedema. Reliability, sensitivity, specificity and area under the ROC curve were estimated. BIA ratio, as indicated by L-Dex ratio, was highly reliable among healthy women (ICC=0.99; 95% CI = 0.99 - 0.99), survivors at-risk for lymphedema (ICC=0.99; 95% CI = 0.99 - 0.99), and all women (ICC=0.85; 95% CI = 0.81 - 0.87); reliability was acceptable for survivors with lymphedema (ICC=0.69; 95% CI = 0.54 to 0.80). The L-Dex ratio with a diagnostic cutoff of >+7.1 discriminated between at-risk breast cancer survivors and those with lymphedema with 80% sensitivity and 90% specificity (AUC=0.86). BIA ratio was significantly correlated with limb volume by sequential circumferential tape measurement. Cross-sectional assessment of BIA may have a role in clinical practice by adding confidence in detecting lymphedema. It is important to note that using a cutoff of L-Dex ratio >+7.1 still misses 20% of true lymphedema cases, it is important for clinicians to integrate other assessment methods (such as self-report, clinical observation, or perometry) to ensure the accurate detection of lymphedema.

Liposomal clodronate treatment for tumour macrophage depletion in dogs with soft-tissue sarcoma.

Increased numbers of tumour-associated macrophages correlate with rapid tumour growth and metastasis in tumours. Thus, macrophage depletion has potential as a novel cancer therapy and positive responses have been reported in rodent tumour models. To investigate the effectiveness of this approach in dogs with cancer, we evaluated the effects of the macrophage-depleting agent liposomal clodronate (LC) in dogs with soft-tissue sarcoma (STS). To this end, we conducted a clinical trial of LC therapy in 13 dogs with STS. Repeated LC administration was well tolerated clinically. Preliminary examination of tumour biopsy sets from 5 of the 13 dogs demonstrated that the density of CD11b(+) macrophages was significantly decreased after LC treatment. Circulating concentrations of interleukin-8 were also significantly reduced. These preliminary studies are the first to suggest that LC can be used as a systemic macrophage-depleting agent in dogs to reduce numbers of tumour-associated macrophages.

The effects of symptomatic seroma on lymphedema symptoms following breast cancer treatment.

It has been speculated that symptomatic seroma, or seroma requiring needle aspiration, is one of the risk factors for lymphedema symptoms following breast cancer treatment. These symptoms exert tremendous impact on patients' quality of life and include arm swelling, chest/breast swelling, heaviness, tightness, firmness, pain, numbness, stiffness, or impaired limb mobility. Our aim was to explore if symptomatic seroma affects lymphedema symptoms following breast cancer treatment. Data were collected from 130 patients using a Demographic and Medical Information interview tool, Lymphedema and Breast Cancer Questionnaire, and review of medical record. Arm swelling was verified by Sequential Circumferential Arm Measurements and Bioelectrical Impedance Spectroscopy. Data analysis included descriptive statistics, Chi-squared tests, regression, exploratory factor analysis and exploratory structural equation modeling. Thirty-five patients (27%) developed symptomatic seroma. Locations of seroma included axilla, breast, and upper chest. Significantly, more women with seroma experienced more lymphedema symptoms. A well-fit exploratory structural equation model [X2(79) = 92.15, p = 0.148; CFI = 0.97; TLI = 0.96] revealed a significant unique effect of seroma on lymphedema symptoms of arm swelling, chest/breast swelling, tenderness, and blistering (beta = 0.48, p < 0.01). Patients who developed symptomatic seroma had 7.78 and 10.64 times the odds of developing arm swelling and chest/breast swelling versus those who did not, respectively (p < 0.001). Symptomatic seroma is associated with increased risk of developing lymphedema symptoms following breast cancer treatment. Patients who develop symptomatic seroma should be considered at higher risk for lymphedema symptoms and receive lymphedema risk reduction interventions.

[Radiofrequency treatment of lung tumours]. / Le traitement par radiofréquence des tumeurs pulmonaires.

Formal surgical resection is the standard treatment for patients with an operable non-small cell lung tumour and for selected patients with limited lung metastases, even if only a small number of patients are suitable for formal surgical resection due to comorbidities. CT-guided radiofrequency treatment is a minimally invasive therapeutic option that has been successfully applied to different organs, and for the lung is considered to be an alternative to surgery for patients who are not candidates for surgery. The procedure is well-tolerated and the complication rate is acceptable.

Higher Volume at Time of Breast Conserving Surgery Reduces Re-Excision in DCIS.

Purpose. The purpose of this study was to compare the surgical and pathological variables which impact rate of re-excision following breast conserving therapy (BCS) with or without concurrent additional margin excision (AM). Methods. The pathology database was queried for all patients with DCIS from January 2004 to September 2008. Pathologic assessment included volume of excision, subtype, size, distance from margin, grade, necrosis, multifocality, calcifications, and ER/PR status. Results. 405 cases were identified and 201 underwent BCS, 151-BCS-AM, and 53-mastectomy. Among the 201 BCS patients, 190 underwent re-excision for close or involved margins. 129 of these were treated with BCS and 61 with BCS-AM (P < .0001). The incidence of residual DCIS in the re-excision specimens was 32% (n = 65) for BCS and 22% (n = 33) for BCS-AM (P < .05). For both the BCS and the BCS-AM cohorts, volume of tissue excised is inversely correlated to the rate of re-excision (P = .0284). Multifocality (P = .0002) and ER status (P = .0382) were also significant predictors for rate of re-excision and variation in surgical technique was insignificant. Conclusions. The rate of positive margins, re-excision, and residual disease was significantly higher in patients with lower volume of excision. The performance of concurrent additional margin excision increases the efficacy of BCS for DCIS.

Decreased ratio of CD8+ T cells to regulatory T cells associated with decreased survival in dogs with osteosarcoma.

BACKGROUND: Increased numbers of regulatory T cells (Treg) and decreased ratios of CD8+ T cells to Treg have been shown to correlate with decreased survival times (ST) in humans with certain malignancies. A possible connection between Treg and ST in dogs with cancer has not been investigated previously. HYPOTHESIS: The purpose of this study was to compare numbers of Treg and T lymphocyte subsets in dogs with osteosarcoma (OSA) to those of healthy dogs and to determine whether pretreatment values were associated with disease-free interval or with ST. We hypothesized that Treg numbers would be increased in dogs with cancer and that dogs with a high percentage of Treg would have a poorer prognosis. ANIMALS: Twelve client-owned dogs with appendicular OSA were entered into a prospective clinical trial. Twenty-two healthy dogs were used as controls. METHODS: The percentages and numbers of Treg and CD4+ and CD8+ T cells in blood, lymph nodes, and tumors were determined with flow cytometry and compared between dogs with OSA and control dogs. RESULTS: Dogs with OSA had significantly fewer circulating CD8+ T cells and significantly more Treg compared with healthy dogs. The CD8/Treg ratio also was significantly lower in dogs with OSA compared with control dogs. In dogs with OSA, a decreased CD8/Treg ratio was associated with significantly shorter STs. CONCLUSIONS: These data support a role for Treg in the immune control of canine OSA and suggest that determination of the CD8/Treg ratio may be useful for assessing outcomes.

Changes in regulatory T cells in dogs with cancer and associations with tumor type.

BACKGROUND: Regulatory T cells (Treg) have been shown to suppress antitumor immunity and often are increased in humans and rodents with cancer. However, Tregs have not been well studied in dogs with cancer and it is not known if certain tumor types are associated with increased Tregs. HYPOTHESIS: We hypothesized that Treg percentages would be increased in dogs with cancer and that Treg percentages would be higher in dogs with certain types of cancer. ANIMALS: The percentages and numbers of Tregs and nonregulatory T cells and B cells were assessed in 34 dogs with cancer and 9 age-matched control dogs. Dogs evaluated included 14 dogs with sarcoma, 7 dogs with carcinoma, 7 dogs with lymphoma, and 6 dogs with mast cell tumor. METHODS: Numbers and percentages of Tregs, CD4+, and CD8+ T cells and B cells were determined using flow cytometry and compared between control dogs and dogs with cancer. RESULTS: The percentage of Tregs was significantly increased overall in dogs with cancer compared with control dogs. When tumor types were compared, Treg percentages were significantly increased in dogs with carcinoma. The Treg/CD8 T cell ratio was significantly higher in dogs with cancer compared with control dogs and was also significantly increased in 2 dogs with T-cell lymphoma. CONCLUSIONS: Treg percentages in blood were increased in dogs with cancer, particularly in dogs with carcinoma. The Treg/CD8 ratio also identified tumor-specific abnormalities in dogs with cancer. These findings indicate that tumor-specific factors may affect Tregs in dogs.

Anti-erythrocyte antibodies and disease associations in anemic and nonanemic dogs.

BACKGROUND: Flow cytometry has been used to detect anti-red blood cell (RBC) antibodies in dogs with immune-mediated hemolytic anemia (IMHA), but the prevalence of anti-RBC antibodies in anemic and nonanemic dogs with a variety of different diseases has not been assessed previously. HYPOTHESIS: We hypothesized that anti-RBC antibodies would be more common in anemic dogs and in dogs with immune-mediated disorders and cancer. ANIMALS: Blood samples from 292 dogs were analyzed prospectively by flow cytometry for anti-RBC antibodies. METHODS: Blood samples from 147 anemic and 145 nonanemic dogs were evaluated by flow cytometry to detect surface-bound immunoglobulin (Ig) G and IgM antibodies on RBC. Disease associations with RBC antibodies were determined, as was the correlation between disease status and the percentage of Ig(+) RBC. The specificity and sensitivity of flow cytometry and clinical variables for the diagnosis of IMHA were compared by Bayesian analysis. RESULTS: Anemic dogs were significantly more likely to be positive for anti-RBC antibodies (IgG, IgM, or both) than nonanemic dogs. Anemic dogs also had significantly higher percentages of Ig(+) RBC than nonanemic dogs, whereas dogs with IMHA had significantly higher percentages of Ig(+) RBC than dogs with all other diseases. Dogs with IMHA, infectious diseases, and immune-mediated thrombocytopenia were significantly more likely to have anti-RBC antibodies than dogs with other medical or surgical diseases. CONCLUSIONS: Anemic dogs with immune-mediated diseases and infectious diseases were at the highest risk for the development of anti-RBC antibodies, and flow cytometry for the detection of IgG on RBC was highly sensitive and specific for the diagnosis of IMHA.

Liposome-DNA complexes infused intravenously inhibit tumor angiogenesis and elicit antitumor activity in dogs with soft tissue sarcoma.

Intravenous gene delivery using liposome-DNA complexes (LDC) has previously been shown to elicit antitumor activity, but only in rodent tumor models. Therefore, we conducted a study to determine in a large animal spontaneous tumor model whether intravenous infusions of LDC could target gene expression to cutaneous tumor tissues and whether repeated treatments had an effect on tumor growth or angiogenesis. A total of 13 dogs with cutaneous soft tissue sarcomas were enrolled in the study and were randomized to receive a series of 6 weekly infusions of LDC containing either canine endostatin DNA or DNA encoding an irrelevant gene (luciferase). Serial tumor biopsies were obtained to assess transgene expression, tumor microvessel density (MVD), and intratumoral leukocyte inflammatory responses. We found that intravenous infusion of LDC did not result in detectable gene expression in cutaneous tumor tissues. However, two of 13 treated dogs had objective tumor responses and eight dogs had stable disease during the treatment period. In addition, a significant decrease in tumor MVD was noted in six of 12 treated dogs at the completion of six treatments. These results suggest that intravenous infusions of LDC may elicit nonspecific antitumor activity and inhibit tumor angiogenesis.

A receptor presentation hypothesis for T cell help that recruits autoreactive B cells.

To uncover mechanisms that drive spontaneous expansions of autoreactive B cells in systemic lupus erythematosus, we analyzed somatic mutations in variable region genes expressed by a panel of (NZB x SWR)F(1) hybridomas representing a large, spontaneously arising clone with specificity for chromatin. A single mutation within the Jkappa intron that was shared by all members of the lineage indicated that the clone emanated from a single mutated precursor cell and led to the prediction that a somatic mutation producing a functionally decisive amino acid change in the coding region would also be universally shared. Upon cloning and sequencing the corresponding germline V(H) gene, we found that two replacement somatic mutations in FR1 and CDR2 were indeed shared by all seven clone members. Surprisingly, neither mutation influenced Ab binding to chromatin; however, one of them produced a nonconservative amino acid replacement in a mutationally "cold" region of FR1 and created an immunodominant epitope for class II MHC-restricted T cells. The epitope was restricted by IA(q) (SWR), and the SWR MHC locus is associated with systemic lupus erythematosus in (NZB x SWR)F(1) mice. These, and related findings, provoke the hypothesis that autoreactive B cells may be recruited by a "receptor presentation" mechanism involving cognate interactions between T cells and somatically generated V region peptides that are self-presented by B cells.