pCR In Nodes Research Articles

A cross-sectional study to unravel the cryptic epidemiology of bovine tropical theileriosis by comparing four traditional and molecular techniques in water buffaloes

A comparison of 4 techniques, viz. blood smear examination, lymph node examination, PCR on blood and PCR on lymph node aspirate for the accurate and sensitive diagnosis of cryptic cases of BTT in water buffaloes, was made. These tests were laboratory standardized and later validated on 85 field samples of suspected bubaline hosts. Keeping blood smear examination as a gold standard for detecting actual number of confirmed positive cases, PCR on blood was 100% sensitive and 88.6% specific. When blood PCR was kept as standard, the lymph node biopsy and lymph node PCR were 90 and 86.96% sensitive along with 77.33 and 90.32% specific, respectively, in detecting theileriosis from suspected samples. The described PCR-based assay provides a valuable tool to study the epidemiology of BTT in buffaloes and some vital data regarding epidemiology of theileriosis in water buffaloes from semi arid parts of India was generated.

Effect of clinical and biological factors on pathologic complete response (pCR) after neoadjuvant chemotherapy for patients (pts) with early-stage breast cancer (BC).

e11551 Background: To evaluate predictive factors that impact pathologic complete response (pCR) after neoadjuvant chemotherapy (NC) for BC pts. The identification of such factors may improve pt selection for neoadjuvant therapy to maximize the likelihood of breast conservation surgery (BCS) Methods: A total of 279 pts treated with NC (anthracyclines and/or taxanes based chemotherapy) for BC were analyzed. Pathologic response was evaluated using Sataloff classification. Student’s test and Fisher’s test were used for statistical analysis; OS and DFS rates were estimated by Kaplan-Meier analysis and log rank test for outcome. Results: According to the TNM classification, 87.4% of pts had T2 /T3 tumors, and 45.9% of pts had a lymph node involvement before NC. HER-2 over expression was found in 23.5 % and 18.6 % of pts had triple negative tumors. Forty three (43%) of pts achieved a clinical complete response. BCS was performed in 44.8%. pCR was 26.4% in the breast primary tumor and 32.8% in the axillary nodes. Univariate analysis showed that the following variables at diagnosis were correlated with a pCR: T2 vs T3/T4 (P<0.001), ductal invasive carcinoma (P=0.001), high grade (p<0.007), high mitotic index (P<0.001), estrogen receptor (ER) negative tumors (p=0.001), progesterone receptor negative tumors (p=0.001), chemotherapy with taxanes (p=0.0026), trastuzumab therapy (p<0.001) and the number of chemotherapy cycles (p=0.002). In multivariate analysis, ER negative, high mitotic index and HER-2 overexpression were significantly associated with a pCR (P<0.01). Lymph node pCR was a significant prognostic factor with 5 year RFS and DFS (respectively p<0.001 and p=0.016). At a median follow-up of 53.4 months, OS and DFS at 5 years were 86.1% and 24.2% respectively. Conclusions: Our data support the hypothesis that ER negative, high mitotic index and HER2 overexpression significantly predicted pCR in pts receiving neoadjuvant therapy for early BC. These results may suggest using such factors before deciding neoadjuvant chemotherapy in order to increase eligibility for BCS.

Phase I Trial Results Testing Adoptively Transferred T Cells Expressing CD20-Specific Chimeric Antigen Receptors Containing CD28 and CD137 Costimulatory Domains In Patients with Mantle Cell Lymphoma and Indolent Lymphoma

AbstractAbstract 561 Background:Mantle cell lymphoma and indolent B cell lymphomas are incurable with chemotherapy but are susceptible to the T cell-mediated graft-versus-lymphoma effect of allogeneic hematopoietic cell transplantation (HCT). However, HCT is associated with high treatment-related morbidity and mortality, and furthermore, many patients are not eligible due to age, comorbidities, and lack of a suitable donor. We have therefore pursued a novel immunotherapy for lymphoma using adoptive transfer of autologous patient-derived T lymphocytes genetically modified to express a chimeric antigen receptor (CAR) specific for the CD20 antigen, a well-established immunotherapy target expressed on B-cell lymphomas. We conducted a previous clinical trial that demonstrated this approach was safe and feasible, but revealed several areas needing improvement, including modest in vivo persistence of transferred cells and limited anti-lymphoma effect. We have attempted to address these shortcomings in the current follow-up trial. Methods:In this pilot phase I protocol, peripheral blood mononuclear cells were obtained from consenting subjects by apheresis, activated with OKT3 and IL-2, and electroporated on day 4–5 with a plasmid containing an SP163 translational enhancer and a NeoR gene and encoding a CAR consisting of a mouse anti-human CD20 scFv (Leu16), an IgG1 spacer, and CD4 transmembrane, intracellular CD28 and CD137 (4-1BB) costimulatory and CD3ζ signaling domains. Transfected cells were selected with G418 and expanded ex vivo by restimulation every 12–14 days using a rapid expansion protocol. Patients were lymphodepleted with 1000 mg/m2 cyclophosphamide (CY) two days prior to the first T cell infusion, and then received 3 infusions 2–5 days apart of 108, 109, and 3.3 × 109 cells/m2, followed by 14 days of low-dose IL-2 injections (250,000 U/m2 s.c. twice daily). Results:Four patients have been enrolled to date, and three patients received a total of 9 T cell infusions. The fourth patient, whose cells did not expand to the target level, opted to withdraw from the study rather than receive a reduced number of cells. Modified cells had an activated effector T cell phenotype (CD3+/CD45RAlow/CD45RO+/CD25+/CD27-/CD28-) and demonstrated in vitro cytotoxicity against CD20+ target cells. Toxicities related to T cell infusions occurred in 1 patient: grade 2 fever and orthostatic hypotension, and grade 3 hypoxia, which all resolved after overnight observation. Other toxicities were associated with CY and IL-2, and were mild and predictable. Modified T cells were detectable by PCR in lymph nodes and bone marrow in all treated patients, and persisted in peripheral blood for up to 5 months. Clinical responses to CY + T cell infusions + IL-2 included a complete remission in 1 patient lasting 10 months thus far, no evaluable disease in a second patient, who is progression-free after 7 months, and stable disease with a partial PET response lasting 3 months thus far in the third patient. Intermediate-dose CY resulted in significant depletion of circulating CD3+ T cells, including CD4+/FoxP3+ regulatory T cells, and CD20+ B cells in all patients, and led to increased IL-2, IL-7, and IL-15 levels in 1 patient. Conclusions:These results suggest that infusion of CD20-specific T cells expressing a CAR containing costimulatory domains is well-tolerated, and lymphodepletion with CY and inclusion of costimulatory domains in the CAR leads to improved T cell persistence and possibly enhanced anti-lymphoma activity compared with “first generation” CARs. (Supported by NIH Grants R21 CA117131 and M01-RR-00037, the Lymphoma Research Foundation, the Damon Runyon Cancer Research Foundation, the American Society of Clinical Oncology Foundation, David and Patricia Giuliani, Bezos Family Foundation, Hext Family Foundation, the Edson Foundation, and the Leukemia and Lymphoma Society). Disclosures:No relevant conflicts of interest to declare.

Adénopathies inguinales bilatérales et érythème noueux : une présentation originale de la maladie des griffes du chat

Cat scratch disease is usually revealed by a proximal lymphadenopathy related to the inoculation site. We report a 22-year-old female who presented with erythema nodosum and bilateral inguinal lymphadenopathy. Serologic test and lymph node PCR detection for Bartonella henselae were negative. Nevertheless, the patient received doxycycline and clinical manifestations rapidly resolved. A follow-up detection of IgM and IgG against Bartonella henselae performed 1month later was positive. This case report illustrates an original presentation of cat scratch disease and reminds us the lack of sensitivity of laboratory investigations.

Immunotherapy with the saponin enriched-Leishmune ® vaccine versus immunochemotherapy in dogs with natural canine visceral leishmaniasis

Leishmune ®, the first licensed vaccine for prophylaxis against canine visceral leishmaniasis (CVL) and is also immunotherapeutic when used with double saponin adjuvant concentration. The Leishmune ® therapeutic vaccine was assessed for immunotherapy (IT) in 31 infected dogs and for immunochemotherapy (ICT) in combination with allopurinol or amphotericinB/allopurinol, in 35 dogs. Compared to infected untreated control dogs, at month 3, both treatments increased the proportion of dogs showing intradermal response to Leishmania antigen to a similar extent (from 8 to 67%, in the IT and to 76%, in the ICT groups), and conversely reduced from 100 to 38% (IT) and to 18% (ICT) the proportion of symptomatic cases, from 54 to 12% (IT) and to 15% (ICT) the proportion of parasite evidence in lymph nodes and from 48 to 19% (IT) and 12% (ICT) the proportion of deaths, indicating that the immunotherapy with enriched-Leishmune ® vaccine promotes the control of the clinical and parasitological signs of CVL rendering most dogs asymptomatic although PCR positive. By month 8, negative lymph node PCR results were obtained in 80% of the ICT-treated dogs, but only in 33% of the IT group ( p = 0.0253), suggesting that the combination of additional chemotherapy with Leishmune ®-enriched saponin vaccination abolished, not only the symptoms but also the latent infection condition, curing the dogs. The animals were followed up until 4.5 years after the beginning of the experiment and, compared to the untreated control group at month 3 (12/25 dogs; 48%), a decrease in the rate of CVL deaths was only seen after ICT treatment (7/35 dogs; 20%; 0.0273) but not after IT treatment (10/31 dogs; 32%; p = 0.278), pointing out an additional advantage of the ICT treatment with the enriched-Leishmune ® in the control and cure of CVL.

Prognostic significance of occult axillary lymph node metastases after chemotherapy‐induced pathologic complete response of cytologically proven axillary lymph node metastases from breast cancer

Primary systemic chemotherapy has been a standard of care for the management of locally advanced breast cancer (LABC) patients and has increasingly been used for patients with large operable breast cancer. Pathologic complete response (pCR) of axillary lymph node metastases predicted an excellent probability of long-term disease-free and overall survival. Although the clinical significance of occult lymph node metastases in patients with breast cancer was extensively studied, their prognostic value in patients with LABC after primary chemotherapy was not known. This study evaluated the detection rate and clinical significance of occult lymph node metastases in lymph nodes that contained metastatic carcinoma at the time of initial diagnosis and converted to negative based on routine pathologic examination after primary systemic chemotherapy. Fifty-one patients with LABC and cytologically involved axillary lymph nodes that converted to negative after preoperative chemotherapy were identified from 2 prospective clinical trials. All lymph node sections were reviewed, 1 deeper level hematoxylin and eosin-stained section of each lymph node was obtained and immunohistochemical staining for cytokeratin (CK) was performed. A total of 762 lymph nodes were evaluated for occult metastases. Kaplan-Meier survival curves were used for calculating disease-free and overall survival times. Occult axillary lymph node metastases were identified in 8 of 51 (16%) patients. In 6 patients, occult metastases were found in only 1 lymph node. In 7 patients, only isolated CK-positive cells were identified. In all cases, occult carcinoma cells were embedded within areas of fibrosis, foreign body giant cell reaction, and extensive histiocytosis. Patients with occult lymph node metastases tended to have a higher frequency of residual primary breast tumors than those without occult metastases (4 of 8 vs 7 of 43, respectively). There was no statistically significant difference in disease-free or overall survival times between patients with and without occult metastases after a median follow-up 63 months. Persistent occult axillary lymph node metastases were not uncommon in patients with axillary lymph node-positive LABC who experienced a pCR in involved lymph nodes after preoperative chemotherapy. However, such occult metastases did not adversely affect the good prognosis associated with axillary lymph node pCR. Therefore, routine lymph node CK evaluation was not recommended after primary chemotherapy.

Remarkable complete pathologic response rate after preoperative paclitaxel, gemcitabine, and trastuzumab chemotherapy in HER2 positive stage II/III breast cancer: a phase II multicenter study.

Abstract Abstract #5105 Background: Preoperative paclitaxel (P) and gemcitabine (G) combination therapy given on D1/D8 every 3 weeks (wks) for 4 cycles was well tolerated and effective in stage II/III breast cancer (BC) in our previous phase II study, with an 18% pathological complete response (pCR) rate. Adding trastuzumab (H) to the preoperative chemotherapy increases both of the clinical and pathological response rates in HER2 positive BC patients (pts). Thus far, the highest pCR rate reported in the literature was obtained with anthracycline-based regimens in combination with trastuzumab. This study evaluated whether non-anthracycline combination chemotherapy with PGH could improve the pCR rate in HER2 positive BC. Methods: HER2 positive, stage II/III BC pts with cytologically confirmed axillary lymph node (LN), ≥ 18 years of age, with adequate organ function, and good performance status were eligible. No prior therapy was allowed. Pts received H intravenously (iv) at 4 mg/kg on D1 of the first cycle with subsequent weekly doses of 2 mg/kg in combination with P 80 mg/m2 and G 1,200 mg/m2, iv, on D1/D8 every 3 wks for 6 cycles. Within 2 wks postoperatively, patients received H 6 mg/kg every 3 wks for 11 cycles with tamoxifen or an aromatase inhibitor for 5 years if indicated. All pts received postoperative radiation therapy. Initial evaluation included sonogram and MRI of the breast, MUGA scan, or echocardiogram, and PET-CT. Results: All 53 planned pts were enrolled between April 2007 and February 2008. The median age was 43 years (range, 26–61 years), the median primary tumor size by sonogram was 5.3 cm (range, 2.0 to > 12 cm) with 89 % ≥ stage IIIA, 42% T3/T4, and 28% N3. Twenty four tumors (45%) were multiple and 20 tumors (38%) were ER positive. By May 2008, 47 patients completed surgery with a 74% breast conservation rate. Twenty-eight of 47 (60%; 95% CI, 45-72) patients achieved pCR in both the tumor and lymph node, with 68% (32/47; 95% CI, 54-80) pCR in the primary tumor, and 77% (36/47; 95% CI, 63-86) pCR in the axillary LN. Median metastatic focus in 21 positive LNs (n=11 pts) was 1 mm (range, <1–13mm). Grade III/IV adverse events (AE) were neutropenia (53%), febrile neutropenia (4%), and transient elevation of AST/ALT (9%). After 6 cycles of PGH chemotherapy, all patients maintained above normal LVEF. Conclusions: A remarkably high pCR was obtained by non-anthracyline based PGH combination therapy for HER2 positive stage II/III breast cancer. This combination is well tolerated with mild degree of AEs.
 Supported by NCC Grant No 0610240-3. Trastuzumab, paclitaxel, and gemcitabine were supplied by Roche, CJ Cheiljedang CO., and Eli Lilly and CO., respectively. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 5105.

A randomized phase-III trial of docetaxel/capecitabine versus doxorubicin/cyclophosphamide as primary chemotherapy for patients with stage II/III breast cancer

We aimed to determine the efficacies of a non-anthracycline-containing regimen, docetaxel/capecitabine (TX), in comparison with an anthracycline-containing regimen, doxorubicin/cyclophosphamide (AC), as primary chemotherapy for node-positive early stage breast cancer. In this phase-III single center randomized study, we randomized 209 women with axillary node positive, stage II/III breast cancer to receive four cycles of either TX or AC followed by surgery and cross-over to the other treatment as an adjuvant therapy. The primary endpoint was tumor pathologic complete response (pCR). Clinical response rates, toxicity profiles, disease free survival (DFS), and overall survival were secondary objectives. In total, 204 patients had clinical and radiological evaluation of response, and underwent surgery. Compared with AC, TX increased pCR in primary tumors (21% vs. 10%, respectively, P = 0.024) and clinical response (84% vs. 65%, P = 0.003). TX was associated with less nausea and vomiting, but more stomatitis, diarrhea, myalgia, and skin/nail changes than AC. With a median follow-up of 37 months, there was no significant difference in DFS by treatment groups (P = 0.932). Fewer patients developed recurrence who achieved pCR in lymph node (LN) (P = 0.025; hazard ratio, 0.189; 95% CI, 0.044-0.815) in the multivariate analysis. TX showed superior efficacies to AC with increased pathologic and clinical complete response rates. Although these findings did not translate into a gain in DFS, the patients who achieved pCR in LN developed significantly less recurrence.

HHV‐6 and EBV DNA quantitation in lymph nodes of 86 patients with Hodgkin's lymphoma

Human herpesvirus (HHV-6) and Epstein-Barr virus (EBV), are two ubiquitous human herpesviruses which share many common features although they belong to different sub-families. In particular, both viruses are found in lymph nodes of patients suffering from Hodgkin's lymphoma. The aim of this study was to detect and to quantify independently HHV-6 and EBV by a real-time PCR in lymph nodes from 86 patients with Hodgkin's lymphoma. EBV quantitative method was compared with LMP-1 protein detection among the same samples. EBV genome was detected for 61.6% of the patients (53/86) and the highest prevalence of this virus was observed in Hodgkin's lymphoma with mixed-cellularity histopathological type (80%). In contrast to that, HHV-6 genome was detected for 79.1% of the patients (68/86) and was most observed in the nodular-sclerosis group (83.6%). Among the 68 HHV-6 positive samples, 63 belonged to the B subtype. A large number of biopsies (47.7%) were positive for both viruses whereas a little number (7%) was negative for both. EBV quantitation and LMP-1 immunohistochemistry were correlated statistically but this latter technique was less sensitive. Among the nodular-sclerosis patients, HHV-6-/EBV+ patients were significatively older than HHV-6+/EBV- patients. Patients infected dually had higher values of quantitation for each virus than those positive for one virus. Data of the clinical follow-up obtained by diagnosis and during the treatment of 83 patients, were correlated with the virological findings.

Leishmune ® vaccine blocks the transmission of canine visceral leishmaniasis : Absence of Leishmania parasites in blood, skin and lymph nodes of vaccinated exposed dogs

Leishmune ® vaccine is the first licensed vaccine against canine visceral leishmaniasis. It contains the Fucose–Mannose-ligand (FML) antigen of Leishmania donovani. The potential Leishmune ® vaccine effect on the interruption of the transmission of the disease, was assayed by monitoring, in untreated ( n = 40) and vaccinated dogs ( n = 32) of a Brazilian epidemic area: the kala-azar clinical signs, the FML-seropositivity and the Leishmania parasite evidence by immunohistochemistry of skin and PCR for Leishmanial DNA of lymph node and blood samples. On month 11 after vaccination, untreated controls showed: 25% of symptomatic cases, 50% of FML-seropositivity, 56.7% of lymph node PCR, 15.7% of blood PCR and 25% of immunohistochemical positive reactions. The Leishmune ®-vaccinated dogs showed 100% of seropositivity to FML and a complete absence of clinical signs and of parasites (0%) in skin, lymph node and blood PCR samples ( p < 0.01). The positivity in FML-ELISA in untreated dogs significantly correlates with the PCR in lymph node samples ( p < 0.001) and with the increase in number of symptoms ( p = 0.006) being strong markers of infectiousness. The absence of symptoms and of evidence of Leishmania DNA and parasites in Leishmune ®-vaccinated animals indicates the non-infectious condition of the Leishmune ®-vaccinated dogs.

Experimental infection of rhesus and pig-tailed macaques with macaque rhadinoviruses.

The recognition of naturally occurring rhadinoviruses in macaque monkeys has spurred interest in their use as models for human infection with Kaposi sarcoma-associated herpesvirus (human herpesvirus 8). Rhesus macaques (Macaca mulatta) and pig-tailed macaques (Macaca nemestrina) were inoculated intravenously with rhadinovirus isolates derived from these species (rhesus rhadinovirus [RRV] and pig-tailed rhadinovirus [PRV]). Nine rhadinovirus antibody-negative and two rhadinovirus antibody-positive monkeys were used for these experimental inoculations. Antibody-negative animals clearly became infected following virus inoculation since they developed persisting antibody responses to virus and virus was isolated from peripheral blood on repeated occasions following inoculation. Viral sequences were also detected by PCR in lymph node, oral mucosa, skin, and peripheral blood mononuclear cells following inoculation. Experimentally infected animals developed peripheral lymphadenopathy which resolved by 12 weeks following inoculation, and these animals have subsequently remained free of disease. No increased pathogenicity was apparent from cross-species infection, i.e., inoculation of rhesus macaques with PRV or of pig-tailed macaques with RRV, whether the animals were antibody positive or negative at the time of virus inoculation. Coinoculation of additional rhesus monkeys with simian immunodeficiency virus (SIV) isolate SIVmac251 and macaque-derived rhadinovirus resulted in an attenuated antibody response to both agents and shorter mean survival compared to SIVmac251-inoculated controls (155.5 days versus 560.1 days; P < 0.019). Coinfected and immunodeficient macaques died of a variety of opportunistic infections characteristic of simian AIDS. PCR analysis of sorted peripheral blood mononuclear cells indicated a preferential tropism of RRV for CD20(+) B lymphocytes. Our results demonstrate persistent infection of macaque monkeys with RRV and PRV following experimental inoculation, but no specific disease was readily apparent from these infections even in the context of concurrent SIV infection.