High PCR Research Articles

Development and Validation of KASP Markers for Stv-bi, a Rice Stripe Virus Resistance Gene in Rice (Oryza sativa L.)

The application of molecular markers in rice breeding facilitates the rapid screening of genotypes in early growth stages without phenotypic assessment. In the present study, we developed and validated high throughput Kompetitive Allele Specific PCR (KASP) assays for rice stripe virus (RSV) resistance genes. The newly developed RSV-KASP markers were compared with the gel-based InDel marker, Indel7. The results of the RSV-KASP assay and the Indel7 analysis were consistent. Due to their high accuracy, time saving attribute, high throughput features, and cost-effectiveness, KASP could be more suitable for RSV genotyping than other methods.

Tolerance of Honey Bees to Varroa Mite in the Absence of Deformed Wing Virus.

The global spread of the parasitic mite Varroa destructor has emphasized the significance of viruses as pathogens of honey bee (Apis mellifera) populations. In particular, the association of deformed wing virus (DWV) with V. destructor and its devastating effect on honey bee colonies has led to that virus now becoming one of the most well-studied insect viruses. However, there has been no opportunity to examine the effects of Varroa mites without the influence of DWV. In Papua New Guinea (PNG), the sister species, V. jacobsoni, has emerged through a host-shift to reproduce on the local A. mellifera population. After initial colony losses, beekeepers have maintained colonies without chemicals for more than a decade, suggesting that this bee population has an unknown mite tolerance mechanism. Using high throughput sequencing (HTS) and target PCR detection, we investigated whether the viral landscape of the PNG honey bee population is the underlying factor responsible for mite tolerance. We found A. mellifera and A. cerana from PNG and nearby Solomon Islands were predominantly infected by sacbrood virus (SBV), black queen cell virus (BQCV) and Lake Sinai viruses (LSV), with no evidence for any DWV strains. V. jacobsoni was infected by several viral homologs to recently discovered V. destructor viruses, but Varroa jacobsoni rhabdovirus-1 (ARV-1 homolog) was the only virus detected in both mites and honey bees. We conclude from these findings that A. mellifera in PNG may tolerate V. jacobsoni because the damage from parasitism is significantly reduced without DWV. This study also provides further evidence that DWV does not exist as a covert infection in all honey bee populations, and remaining free of this serious viral pathogen can have important implications for bee health outcomes in the face of Varroa.

Study of efficacy and tolerability of paclitaxel and carboplatin as a concurrent chemotherapeutic radiation sensitizer regimen in patients of esophageal cancer: Prospective observational study from tertiary center in northern India.

e16526 Background: Neo-adjuvant chemotherapy followed by surgery is the standard of care in non metastatic oesophageal cancers. After the overall survival benefit observed in Cross trial, Paclitaxel and Carboplatin based neo-adjuvant chemoradiation has become the preferred treatment modality. There is paucity of data of its efficacy and tolerability in Indian patients. So this prospective study was done to assess the same. Methods: We enrolled 64 patients aged 18-75 years with histologically confirmed, potentially resectable squamous-cell carcinoma of the esophagus or esophagogastric junction from Aug 2017 to Jan 2019. On days 1, 8, 15, 22, and 29, carboplatin AUC 2 and paclitaxel 50 mg/m2 intravenously and a total radiation dose of 41.4 Gy was given in 23 fractions of 1.8 Gy each by external-beam radiation. Pathological complete response rate, Clinical response based on PET-CT scan and tolerability of paclitaxel and carboplatin as a concurrent chemotherapeutic radiation sensitizer regimen was studied. Results: 23 out 64 patients underwent surgery while others were either lost to follow up or they refused for surgery. Of these 23, 87.5% could complete all 5 planned cycles of chemotherapy. Complete pathological response was found in 73.9% patients. Statistically significant correlation was found between pathological and PET-CT based response (p-0.044). 4 patients showed distant progression on PET-CT based evaluation after neoadjuvant chemo radiotherapy before surgery. The most common major hematologic toxic effects were leukopenia (14%) and neutropenia (12.5%). Febrile neutropenia and grade 3 anemia was observed 2 and 4 patients respectively. The most common major non-hematologic toxic effects were vomiting (4.6%), nausea (3.1%), bronchoesophageal fistula(3.1%). Post operative mortality was seen in 1(4.3%) patient. Grade 3 pulmonary complications, atrial fibrillation and anastomotic leak was observed in 1 patient each. Conclusions: Concurrent chemoradiotherpy with weekly paclitaxel and caboplatin is well tolerated in Indian patients. High complete resection rate and PCR rate can be achieved with this regime with manageable adverse effect profile. Response evaluation with PET-CT after completion of preoperative therapy has correlation with pathological complete response.

Incidence of brain metastases in patients receiving neoadjuvant chemotherapy (NAC) with trastuzumab and pertuzumab (HP) in HER2-positive early breast cancer (BC).

e12653 Background: The addition of pertuzumab in the neoadjuvant setting has become a standard of care in stage II-III HER2-positive BC. In the Katherine study the incidence of brain metastases (BM) at the follow-up of 3-years was 5.9 % in patients (pts) with residual disease who received TDM1. The aim of this study was to assess the incidence of BM in pts who received NAC with HP at a single center who were found to have pathological complete response (pCR) (ypT0/is ypN0) versus non-pCR at the time of surgery. Methods: Chart review on HER2-positive pts treated with NAC and HP between September 1, 2013 to May 1, 2018 was conducted. Only surgical specimens of pts whose pre-NAC specimens were internally reviewed for HER2 status by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) were included in this analysis. Data on BM as component of distant recurrence along with invasive disease-free (IDFS) and overall survival (OS) were collected. Results: 540 pts were identified. Cases with no internally verified-HER2 status (387), equivocal status (10) and discordant internal assessment (13) were excluded. 130 pts with preoperative HER2 status confirmed by dedicated breast pathologists were included. Clinicopathological features are described in Table. pCR was achieved in 77/130 (60%) of cases, residual disease in 53/130 (40%). The median follow-up was 2.83 (0.35-5.3) years. The rate of BM as first presentation of distant disease was 3.8 % (3/77) and 3.7 % (2/53) in pts with pCR and non-pCR, respectively. Median time to development BM was 35 (13-58) months and 11.7 (9-14) months in the pCR group and non-pCR, respectively. Conclusions: In our cohort, the combination of HP was associated with a high pCR rate. At a median follow-up of 2.8 years, the incidence of BM appeared to be similar in pts who achieved pCR versus non-pCR. The data of IDFS and OS will be reported. Further data are needed to validate our findings before designing clinical trials to test prophylactic strategies to prevent BM. [Table: see text]

A phase I safety and feasibility study of neoadjuvant chemoradiation plus pembrolizumab followed by consolidation pembrolizumab in resectable stage IIIA non-small cell lung cancer.

9009 Background: Patients (pts) with resectable stage IIIA non-small cell lung cancer (NSCLC) have high rates of recurrence despite concurrent chemoradiation (CRT) followed by surgery. Immune checkpoint inhibitor consolidation has improved outcomes in unresectable stage III pts. Here we report the addition of concurrent neoadjuvant pembrolizumab (P) to CRT in stage IIIA patients to determine the safety and feasibility of this approach. Methods: Pts with stage IIIA NSCLC deemed resectable by a thoracic surgeon received neoadjuvant CRT consisting of cisplatin, etoposide, and concurrent P (200mg every 3 weeks x 3) with 45 Gy in 25 fractions. Pts without progression underwent resection followed by 6 months of consolidation P. The primary objective was feasibility and safety (defined as ≤30% grade 3 or higher pulmonary toxicity or any grade 4/5 nonhematologic toxicity). Ten pts were to be enrolled in Part 1, and if 2 or fewer pts had events then an additional 10 pts were to be enrolled. Secondary objectives were progression free survival (PFS), overall response rate (ORR), and pathologic complete response rate (pCR). Results: The median age of 9 enrolled pts was 66 years (range 49-76). 67% were female. 8 pts were assessable for radiographic response with an ORR of 75%. One pt came off study for progression prior to surgery and one had pleural metastases found during surgery so resection was aborted. Six pts underwent complete resection with a pCR rate of 67% (4/6). Consolidation P was started on 4 pts, with 3 completing treatment and 1 declined further treatment after 3 cycles. Median follow-up is 19.6 months and median PFS has not been reached. None of the patients who underwent resection have recurred. Serious adverse events were reported in all 9 pts with most significant being 2 grade 5 events: 1 due to pneumocystis pneumonia after resection but prior to consolidation, and 1 due to cardiac arrest during the neoadjuvant phase. Grade 3 events included 1 episode each of pneumonitis, bronchopleural fistula, acute kidney injury, colon perforation, and febrile neutropenia. Conclusions: The addition of P to neoadjuvant CRT in resectable stage IIIA pts resulted in a high pCR rate at resection. Although the relationship between grade 5 events and the addition of P was not clear, the stopping rule for infeasibility was met. As other larger studies are underway, the trial was halted rather than amended. This investigator initiated trial was funded by Merck. Clinical trial information: NCT02987998.

A multicenter phase II trial of the combination cisplatin/ docetaxel/durvalumab/tremelimumab as single-cycle induction treatment in locally advanced HNSCC (CheckRad-CD8 trial).

6519 Background: PD-1/PD-L1 inhibitors are efficient in head and neck squamous cell cancer (HNSCC). Combination with anti-CTLA4 agents may enhance anti-tumor activity compared to anti-PD-1/PD-L1 monotherapy in different tumor types. In the CheckRad-CD8 trial the typical induction treatment consisting of Cisplatin/Docetaxel was combined with Durvalumab/Tremelimumab. Patients with pathological complete response (pCR) in the re-biopsy after induction treatment or at least 20% increase of intratumoral CD8 density in the re-biopsy compared to baseline entered radioimmunotherapy with concomitant Durvalumab/Tremelimumab. Methods: In this prospective multicenter phase II trial, patients with HNSCC stage III-IVB received a single cycle of Cisplatin 30mg/m² d1-3, Docetaxel 75mg/m² d1, Durvalumab 1500mg fix dose d5 and Tremelimumab 75mg fix dose d5. Objectives of this interim analysis were to quantify the effect of the induction treatment on intratumoral CD8 density and the pCR rate and to generate safety data. Results: Between Sep 2018 and Dec 2019, 57 patients were enrolled. Median age was 59 years, 22 patients (37%) were current smokers, 27 patients (47%) had oropharyngeal tumors (52% p16 positive). The median pre-treatment intratumoral CD8 density was 335 CD8+ cells/mm². After induction treatment 27 patients (47%) had a pCR in the re-biopsy and further 25 patients (44%) had a relevant increase of intratumoral CD8+ cells (median increase by factor 3.0). Response according to RECIST criteria was CR in 1 (2%), PR in 19 (33%) and SD in 20 patients (35%) (17 patients not evaluable). Adverse events (AE) grade 3-4 appeared in 39 patients (68%) and mainly consisted of leucopenia (43%) and infections (28%). 6 patients (11%) developed grade 3-4 immune-related AEs. In multivariable analysis the intratumoral CD8 density was the only independently significant predictor of pCR (odds ratio 1.0013 per cell/mm², 95%-CI 1.00023-1.0023, p=0.017). 42 patients (74%) continued with Durvalumab/ Tremelimumab concomitant to radiotherapy. Conclusions: Single cycle induction treatment with Cisplatin/Docetaxel/Durvalumab/Tremelimumab is feasible and achieves a high pCR rate. CD8 density may have a predictive role for further treatment planning in locally advanced HNSCC. Clinical trial information: NCT03426657 .

Short-course radiotherapy followed by chemotherapy before TME in locally advanced rectal cancer: The randomized RAPIDO trial.

4006 Background: Local control in locally advanced rectal cancer (LARC) has improved. However, systemic relapses remain high even with postoperative chemotherapy, possibly due to low compliance. Short-course radiotherapy (SCRT) followed by delayed surgery with, in the waiting period, chemotherapy, may lead to better compliance, downstaging and fewer distant metastases. The main objective of the international multicenter phase III RAPIDO trial is to decrease Disease-related Treatment Failure (DrTF), defined as locoregional failure, distant metastasis, a new primary colon tumor or treatment-related death, by reducing the risk of systemic relapse without compromising local control. Methods: MRI-diagnosed LARC patients with either cT4a/b, extramural vascular invasion, cN2, involved mesorectal fascia or enlarged lateral lymph nodes considered to be metastatic were randomly assigned to SCRT (5x5 Gy) with subsequent six cycles of CAPOX or nine cycles of FOLFOX4 followed by total mesorectal excision (TME) (experimental arm) or, capecitabine-based chemoradiotherapy (25-28 x 2.0-1.8 Gy) followed by TME and optional, predefined by hospital policy, postoperative eight cycles of CAPOX or twelve cycles of FOLFOX4 (standard arm). Results: Between June 2011 and June 2016, 920 patients were randomized. Pathological complete response rates were 27.7% vs 13.8% (OR 2.40 [1.70 – 3.39]; p < 0.001) in the experimental and standard arms, respectively. At three years, cumulative probability of DrTF was 23.7% in the experimental arm and 30.4% in the standard arm (HR 0.76 [0.60 – 0.96]; p = 0.02). Probability at three years of distant metastasis and locoregional failure were, in the experimental and standard arms, 19.8% vs 26.6% (HR 0.69 [0.53 – 0.89]; p = 0.004) and 8.7% vs 6.0% (HR 1.45 [0.93 – 2.25]; p = 0.10), respectively. No differences in DrTF between hospitals with or without policy for postoperative chemotherapy were found (p = 0.37). Overall health ( p = 0.192), quality of life ( p = 0.125) and low anterior resection syndrome score ( p = 0.136) were comparable between the two treatment arms. Conclusions: A lower rate of DrTF, as a result of a lower rate of distant metastases, in high-risk LARC patients can be achieved with preoperative short-course radiotherapy, followed by chemotherapy and TME than by conventional chemoradiotherapy. In addition, the high pCR rate, achieved with the experimental treatment regimen can contribute to organ preservation. This treatment can be considered as a new standard of care. Clinical trial information: NCT01558921 .

The impact of neoadjuvant and/or adjuvant treatment modalities in resectable gastric cancer (rGC).

e16564 Background: In rGC ( > cT2), we investigated the impact of various neoadjuvant and/or adjuvant treatment modalities on pathologic complete response (pCR), surgical margins, and overall survival (OS). Methods: The National Cancer Database (NCDB) was interrogated to identify rGC patients (pts) between 2004-2015. Gastric adenocarcinoma cases that were cT2-T4b, any N, M0 and underwent definitive surgery were included. We analyzed the association of 9 treatment groups: neoadjuvant chemoradiation only (nCRT), neoadjuvant chemo only (nCT), adjuvant chemo only (aCT), adjuvant chemoradiation only (aCRT), neoadjuvant chemo and adjuvant radiation (nCTaRT), received any chemo at all (any CT), received any chemoradiation at all (any CRT), received any radiation at all (any RT), and no perioperative therapy (NT) across 3 endpoints: pCR, margin status, and OS using logistic regression and Cox proportional hazards models with adjustment for baseline characteristics. Results: From 183,204 GC cases screened, a total 3061 pts were available with a median follow-up of 41.6 mos and median OS of 29.0 mos. On multivariable analyses, nCRT was associated with the greatest odds of having a pCR (odds ratio or OR 59.6, 95% confidence interval (CI) 10.6-334.1, p < 0.001) with NT as the reference. Having received any RT (OR 0.42, 0.10-1.86), nCRT (OR 0.68, 0.33-1.37), or nCT (OR 0.83, 0.60-1.15) was associated with the lowest odds for having positive surgical margins although none reached p < 0.05. For OS, having received any CT (hazard ratio or HR 0.41, 0.35-0.48) was associated with the lowest risk of death followed by nCRT (HR 0.48, 0.35-0.66), aCT (HR 0.52, 0.43-0.62), aCRT (HR 0.55, 0.48-0.63), any CRT (HR 0.61, 0.41-0.91), nCT (HR 0.62, 0.54-0.71), and nCTaRT (HR 0.67, 0.52-0.87, all p < 0.05). Median OS was greatest in pts treated with any CT (53.9 mos) followed by nCRT (39.1 mos) and aCT (36.1 mos) with 2-year OS rates being 65.6% (95% CI 61.3-69.5%), 63.6% (52.3-73.0%), and 59.7% (54.2-64.7%), respectively. Conclusions: Although nCRT had a high pCR rate, receipt of any CT (neoadjuvant and/or adjuvant) afforded the greatest OS in this modality-by-modality comparison in a large cohort of rGC pts.

Best Foot Forward: Neoadjuvant Systemic Therapy as Standard of Care in Triple-Negative and HER2-Positive Breast Cancer.

Neoadjuvant systemic treatment of early-stage breast cancer has been used to improve resectability and reduce the extent of breast and axillary surgery. More recently, several other merits of neoadjuvant systemic treatment have emerged, including the ability to tailor clinically available adjuvant systemic therapy options based on pathologic response and to serve as a platform for early assessment of novel agents and response biomarkers and as an avenue for treatment optimization investigations (local and systemic therapy escalation and de-escalation trials guided by pathologic response). Attainment of a pathologic complete response (pCR) is associated with excellent long-term outcomes; conversely, the presence of residual disease is associated with a high risk of recurrence for patients with HER2-positive breast cancer and triple-negative breast cancer (TNBC). Treatment strategies in early-stage HER2-positive breast cancer include regimens incorporating trastuzumab, pertuzumab, ado-trastuzumab emtansine, and neratinib, resulting in high pCR rates and overall excellent long-term outcomes. Currently available cytotoxic regimens yield pCR for 35% to 55% of patients with TNBC, and immune checkpoint inhibition is showing early promise for this subtype. New drug and predictive biomarker evaluations in the neoadjuvant setting aim to develop optimal treatment strategies for the individual patient, with the ultimate goal of maximizing efficacy and minimizing toxicity. Research efforts involving novel agents are being undertaken to address the high risk of recurrence for patients with residual disease. Omission of breast surgery following neoadjuvant chemotherapy requires further development of imaging and biopsy techniques to accurately assess the extent of residual disease before clinical application.

Microplastics combined with tetracycline in soils facilitate the formation of antibiotic resistance in the Enchytraeus crypticus microbiome

Growing evidence suggests that microplastics can adsorb antibiotics and may consequently exacerbate effects on the health of exposed organisms. Our current understanding of the combined effects of microplastics and antibiotics on antibiotic resistance genes (ARGs) in soil invertebrates is limited. This study aimed to investigate changes in the microbiome and ARGs in Enchytraeus crypticus following exposure to a soil environment that contained both microplastics and antibiotics. Tetracycline (TC), polyamide (PA) and polyvinyl chloride (PVC) were used to construct microcosms of polluted soil environments (TC, PA, PVC, PA+TC, PVC+TC). The differences in microbiomes and ARGs were determined by bacterial 16S rRNA gene amplicon sequencing and high throughput quantitative PCR. The results show that compared with the Control or microplastics alone treatments, TC was significantly accumulated in E.crypticus when exposed to TC alone or in combination with microplastics (P<0.05), but there were no significant differences about TC accumulation between TC, PA+TC, and PVC+TC treated E.crypticus (P>0.05). Microplastics and TC significantly disturbed the microbial community, and decreased the microbial alpha diversity of E.crypticus (P<0.05). However, there were no significant differences between TC, microplastics and their combined exposure treatments, and no toxic synergies on the diversity of E.crypticus microbiome between tetracycline and microplastics in soil environment. All the treatments increased the diversity of ARGs in E.crypticus (39-49 ARGs vs. 25 ARGs of control). In particular, treatments combining PVC and TC or PA and TC exposure resulted in greater ARGs abundance than the treatments when E.crypticus was exposed to PVC, PA or TC alone. These results add to our understanding of the combined effects of microplastics and antibiotics on the ARGs and microbiome of soil invertebrates.

Real-time PCR for identification of five species of Cryptolestes based on COI barcode region

Cryptolestes(Coleoptera: Laemophloeidae) are economically important pests, which damage stored products in granaries and mills. Because it is difficult to distinguish Cryptolestes species morphologically, we develop here a high sensitivity real-time PCR method for distinguishing five species of Cryptolestes (C. ferrugineus, C. pusillus, C. turcicus, C. pusilloides and C. capensis). The lower limit of DNA concentration required for species identification is 0.1 ng/μl for C. ferruginues and C. pusilloides, and 0.01 ng/μl for C. pusillus, C. turcicus, and C. capensis. This method successfully determined species assignments for previously unidentified Cryptolestes spp. Collected from the field and will facilitate Cryptolestes identification in the field of plant quarantine and stored product protection.

SAT-429 CMV-ASSOCIATED CONGENITAL NEPHROTIC SYNDROME: CORRELATION OF CLINICAL, HISTOPATHOLOGICAL AND GENETIC FINDINGS

Congenital nephrotic syndrome (cNS), defined by onset during the first three months of life, is a rare clinical disorder with traditionally poor renal outcome. It is caused by homozygous or compound heterozygous mutations of a growing number of genes affecting podocyte and/or glomerular basement membrane integrity, with or without syndromic features. Other potential etiologies include antenatal or perinatal infections, or (other) systemic disorders. Case description and systematic review of the literature in PubMed using the key words "CMV", "congenital” or "infant", and "nephrotic” or "glomerulonephritis". This report describes an ex-preterm baby who had been referred at two weeks corrected gestational age for suspected surgical abdomen in view of progressive abdominal distention, scrotal edema and vomiting. The patient was found to have full-blown nephrotic syndrome with urine protein-to-creatinine ratio of > 6 g/g, hypoalbuminemia <10 g/L, hypercholesterolemia and anemia, and elevated liver transaminases. He had syndromic features. Diagnostic workup revealed an active CMV infection, based on CMV-specific IgM and positive CMV PCR from blood and urine. Routine pregnancy screen for intrauterine infections had been negative. Subsequent maternal diagnostic revealed high maternal anti-CMV IgG (IgM negative) and positive CMV PCR. The infant’s kidney biopsy showed focal mesangial proliferation and sclerosing glomerulonephritis with cellular or fibro-cellular crescents in less than 5% of glomeruli, while global or segmental sclerosis associated with older fibrous crescents was noted in 10% of glomeruli, in addition to interstitial fibrosis and tubular atrophy. Routine immune fluorescence was negative. Electron microscopy revealed diffuse podocyte foot process effacement, but no immune complexes or endothelial tubule-reticular inclusions. Infiltrating (interstitial) lymphocytes stained positive for CD3 and CD20, respectively. Results of the genetic mutation screen are pending. After 6 weeks of treatment with valganciclovir, the CMV PCR has become negative, but the infant continues to demonstrate severe proteinuria and hypoalbuminemia. Review of the available literature reveals an important overlap between cNS due to known monogenic causes and CMV infection. Although the number of defined cases in the literature is scarce, we noted divergent histopathological features in CMV-associated cNS, dependent on the presence or absence of cognate genetic mutations as shown in the Table. CMV is a rare cause of nephrotic syndrome presenting soon after birth. Screening for relevant mutations is essential since congenital infections can complicate or mask an underlying genetic etiology leading to the failure of CMV-directed antiviral therapy and poor renal outcome.

MHCI and FOXP3 predicting outcome to neoadjuvant atezolizumab in sequential urothelial cancer tissue.

541 Background: Biomarker analysis predicting outcome with neoadjuvant atezolizumab in muscle invasive bladder cancer (MIBC) was performed. This includes previously unreported findings with FOXP3 and MHC1. Methods: Sequential tissue obtained from 95 patients in the ABACUSstudy (NCT02662309) which investigated two cycles of atezolizumab prior to cystectomy in MIBC patients. Multiplex immunohistochemistry staining, Foundation One analysis and RNA sequencing was performed on paired pre- and post- treatment samples. CD8+ T cells, fibroblast activation protein (FAP), granzyme – B (GZMB), FOXP3, MHC1, PD-L1 expressions were analyzed and correlated with outcome. Results: After a median follow up of 13.1 months, 31% had a pathological complete response (pCR) and 18% have relapsed. 40% were PD-L1 positive at baseline and 73%, 19% and 8%had inflamed, excluded and desert phenotype respectively. CD8/GZMB and CD8/MHC1 expression prior to therapy was significantly associated with response in inflamed tumors. CD8, PDL1, GZMB and MHC1 expression all increased with treatment in this group of patients. Immune desert phenotype at baseline, showed low and static expression of immune biomarker. TGFb correlated with relapse in excluded phenotypes. Treatment was associated with increase in cell cycle genes and FAP, both of which were associated with relapse. FOXP3 expression correlated with CD8 and increased with therapy in responding tumors. Combining FAP (high) to CD8 (low) and MHC1 (low) increased the positive predictive value for relapse. TMB did not correlate with outcome or increase with therapy. FGF DNA alterations were associated with response. Conclusions: MIBCs have high T effector immune expression, which may account for high pCR rates. Biomarkers at baseline and after treatment can predict outcome. Assessment of multiple biomarker within algorithms improves accuracy in predicting outcome. FOXP3 expression correlates with activated T cell expression, potentially accounting for the counterintuitive findings. Clinical trial information: NCT02662309.

Abstract P1-20-02: Axillary conversion rates according to breast cancer subtype among patients with pathological complete remission (pCR) in the breast after primary systemic therapy (PST) - A transSENTINA subproject

Abstract Background: Modern chemotherapy carries a high potential of converting patients with a clinically positive axilla (cN+) prior to PST to a clinically (ycN0) or even pathologically (ypN0) negative axilla after PST. Patients achieving a pathological complete remission (pCR) in the breast after PST carry a favorable prognosis including a high probability of axillary conversion. Among patients with highly chemosensitive tumors (i.e. high pCR rate), axillary staging may be adjusted accordingly. Methods: In a retrospective study we analyzed the relation between breast pCR, nodal pCR and the intrinsic subtype in clinically node-positive patients who convert to ycN0. Arm C of the SENTINA trial included patients converting from cN+ to ycN0 through PST as part of a prospective study. Patients were categorized into luminal (HR+/HER2-), triple negative (TN; HR- / HER2-), triple positive (TP (HR+ / HER2+), and HER2 pos ER neg HER2 positive (HR- / HER2+) subgroups. Results: Overall 416 of 596 patients were included (exclusions due to missing parameters) with 167 luminal, 119 TN, 77 TP and 56 HER2 pos ER neg HER2 positive tumors. Among all patients, pathological axillary conversion occurred in 154 (80.21%) and 91 (32.97%) cases among patients with and without pCR, respectively (odds ratio (OR) 0.23, p-value &amp;lt; .001). Among all subgroups, there was a significant increase in the probability of axillary conversion in case of pCR. Axillary conversion rates for patients with and without pCR were, respectively: • TN 56 (84.85%) and 24 (45.28%)• TP 33 (73.33%) and 12 (37.50%) • Luminal 18 (66.67%) and 34 (24.29%) • HER2 pos ER neg 33 (91.67%) and 9 (45.00%) We identified patient subgroups, among whom conversion rates were &amp;gt; 90%, i.e. among patients with (i) TN breast cancer and age at diagnosis &amp;lt; 50 years (91.89%) and (ii) HER2 pos ER negitive breast cancer and grade 3 disease (94.74%) or age &amp;gt; 50 years (94.44%). Conclusion: Our analysis demonstrates that among patients with pCR, probabilities of axillary conversion were significantly higher among patients with pCR compared to patients without pCR. However, results differed according to biological breast cancer subtypes, with more aggressive subgroups presenting with higher probability of axillary conversion. We identified subgroups of patients among whom the rate of axillary residual disease after PST was lower compared to the false-negative rate of sentinel lymph node biopsy. Provided independent validation of our results, these subgroups may benefit from de-escalation of axillary stagingsurgery. Citation Format: Cornelia Kolberg-Liedtke, Hans-Christian Kolberg, Maja Krajewska, Ingo Bauerfeind, Tanja Fehm, Barbara Fleige, Gisela Helms, Annette Lebeau, Annette Staebler, Sibylle Loibl, Michael Untch, Thorsten Kühn. Axillary conversion rates according to breast cancer subtype among patients with pathological complete remission (pCR) in the breast after primary systemic therapy (PST) - A transSENTINA subproject [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-20-02.

Abstract P2-16-19: Neoadjuvant weekly paclitaxel (wP) and carboplatin (Cb) with trastuzumab (T) and pertuzumab (P) in HER2-positive breast cancer (H+BC): A Brown University oncology group (BrUOG) study

Abstract In H+BC, neoadjuvant chemotherapy (NACT) with dual HER2-targeted therapy achieves high pathologic complete response (pCR) rates, but the most common anthracycline-free regimen, every 3-week docetaxel and Cb with T and P (TCHP), causes significant hematologic and non-hematologic toxicities. We conducted a pilot study to assess the efficacy and toxicity of a weekly anthracycline-free regimen, and to assess whether patients (pts) with a suboptimal response to this regimen benefit from switching to doxorubicin/cyclophosphamide (AC). Methods Pts with clinical stage II-III H+BC receive wP 80 mg/m2 and Cb AUC 2 weekly with every 3-week T and P (wPCbTP). Investigators may split the week 1 loading dose of P (840 mg) into two weekly doses, and hold the 2nd dose for early onset toxicity, particularly diarrhea. Pts are restaged after 12 weeks with ultrasound or MRI. Responding pts receive another 6 weeks of wPCbTP, while nonresponders are switched to AC x 4, followed by surgery. Study participation is complete after a 3-month post-op visit. Dose modifications and post-op therapy are at investigator discretion. Results Enrollment (30 pts) is complete. Pathologic findings are available for 23 pts; the other 7 are due to undergo surgery by 10/2019. No pts have required switching to AC for suboptimal response at week 12; 2 have been switched for toxicity (1 each for diarrhea and recurrent cytopenias). pCR (T0/isN0) rates are tabulated below: Pt grouppCR% (95% CI)All18/2378 (56-92) %ER(-)10/1191 (59-100) %ER(+)8/1267 (35-90) %Operable (Stage IIA-IIIA)14/1782 (57-96) %Inoperable (Stage IIIB-C)4/667 (22-96) % In 23 pts, grade 3/4 toxicities observed include neutropenia (10/2, but no febrile neutropenia), anemia (3/0), thrombocytopenia (1/0), diarrhea (4/1), and 5 cases of grade 2 (but no grade ≥3) peripheral neuropathy. One pt each has required hospitalization for recurrent urinary tract, port site or breast infections while not neutropenic. wP has been dose-reduced for neuropathy after week 12 in 8 pts. Two pts discontinued Cb after hypersensitivity reactions in week 10. Treatment was discontinued during weeks 12-18 in 8 pts. P was held during weeks 1-6 for diarrhea in 5 pts and discontinued before week 12 in 3 pts; splitting the loading dose of P has not substantially reduced the incidence or severity of diarrhea. Conclusions Treatment with wPCbTP is associated with a high pCR rate (78%), comparable to those reported with TCHP in multicenter phase II-III trials, and is generally well tolerated, with fewer grade ≥3 toxicities, though diarrhea remains a concern. In some pts, cumulative toxicities make it difficult to continue treatment beyond week 12. Thus far, we have failed to identify a subset of pts in whom to test the efficacy of switching to AC after a suboptimal response to wPCbTP. This regimen appears to be worth exploring further, though we might consider switching to a less toxic regimen, or taking pts with an excellent response to surgery, after week 12. Supported by LifeCycle and the Lura Cook Hull Trust Citation Format: Mary Lorraine Lopresti, Jessica J. Bian, Bachir J Sakr, Rochelle S Strenger, Robert D. Legare, Mary Anne Fenton, Sabrina M Witherby, Don S Dizon, Sonali V Pandya, Ashley R Stuckey, David A Edmondson, Jennifer S Gass, Christine M Emmick, Theresa A Graves, Marlene Cutitar, Adam J Olszewski, William M Sikov. Neoadjuvant weekly paclitaxel (wP) and carboplatin (Cb) with trastuzumab (T) and pertuzumab (P) in HER2-positive breast cancer (H+BC): A Brown University oncology group (BrUOG) study [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P2-16-19.

Abstract P1-18-28: Myocet or carboplatin as part of chemotherapy in HER2-positive operable breast cancer treated with trastuzumab and pertuzumab in the neoadjuvant setting: Efficacy data and cardiotoxicity in 60 patients diagnosed from 2016-2019 at a single institution

Abstract Objectives: To evaluate the efficacy and cardiotoxicity profile of liposomal anthracycline or Carboplatin as part of neoadjuvant therapy added to taxanes and dual blockade in HER2-positive operable breast cancer patients. Methods: A total of 60 patients diagnosed from August 2016 to May 2019 were included in the study. The treatment consisted of a sequential regimen of taxanes and non- pegilated liposomal anthracycline (Myocet) plus trastuzumab and pertuzumab, or taxanes in combination with carboplatin plus trastuzumab and pertuzumab. The clinical and pathologic responses were evaluated and correlated with clinical and biological factors. Pathologic complete response was defined as the total absence of invasive tumor in both breast and axillary nodes (ypT0/is ypN0).The cardiotoxicity profile was also analyzed. Results: The median age was 52(26-77) years and 5%, 62% and 33% of patients had stage I, II and III breast cancer, respectively, while 7% had inflammatory breast cancer at diagnosis. Hormone receptor (HR) status was negative in 48%, and 65% and 50% had grade III and ki-67 value up to 35%, respectively breast cancer. 65% of patients received weekly paclitaxelx12-trastuzumab-pertuzumab followed by Myocet-cyclophosphamide with -trastuzumab x 4 cycles or this sequence but beginning with the anthracycline, 23% of patients the same regimen but with pertuzumab concurrently with all chemotherapy treatment. The remaining 12% received docetaxel-carboplatin x6 cycles plus trastuzumab and pertuzumab. All treatment was administered previous surgery. The clinical complete response rate, was 57% and 63%, as assessed using ultrasound and MRI, respectively, and this allowed a high rate of conservative surgery (60%). The pathologic complete response (pCR) rate was 52%, and it was higher in HR-negative (61%) than in HR-positive (39%), in grade 3 (71%) than in grade 2(29%) tumors. There were not statistical significant differences in pCR between different chemotherapy treatments. Five patients showed a 10% decrease in left ventricular ejection fraction (LVEF) to below 50% at the end of neoadjuvant treatment. Asymptomatic drops in 2 patients and with heart failure symptoms in 3 of them. Myocet was part of chemotherapy in all of them. All patients recovered after discontinuation of adjuvant trastuzumab and heart medication. Two of these patients did not complete all preplanned adjuvant trastuzumab doses. Conclusion: A sequential regimen of taxanes and non-pegilated liposomal anthracycline or carboplatin plus trastuzumab and pertuzumab was effective, with high pCR rates and a good cardiotoxicity profile. Citation Format: Silvia Antolin, Cristina Reboredo, Rocío Lesta, Aurea Molina, Joaquin Mosquera, Patricia Cordeiro, Eva Pérez, Lourdes Calvo. Myocet or carboplatin as part of chemotherapy in HER2-positive operable breast cancer treated with trastuzumab and pertuzumab in the neoadjuvant setting: Efficacy data and cardiotoxicity in 60 patients diagnosed from 2016-2019 at a single institution [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-18-28.

Abstract P2-10-10: Lower pathologic complete response rate with dual-HER2 blockage and chemotherapy in African Americans and ethnic minorities

Abstract Introduction: Racial disparities in breast cancer outcomes are well documented with age-adjusted mortality among African American (AA) women 40% higher than Caucasian women. Some studies have demonstrated poor response to neoadjuvant chemotherapy in AA with triple negative breast cancer but data regarding pathologic complete response rate (pCR) in HER2 positive disease are lacking. TRYPHAENA trial demonstrated a high pCR rate after treatment with dual HER2 blockage along with chemotherapy. Due to underrepresentation of African Americans and minorities in the trials leading to the approval of dual HER2 blockage (Gianni et al; Lancet Oncology;13: 25-32) in early stage breast cancer, data is lacking on the efficacy and tolerability of this treatment in this subgroup of patients. Methods: After IRB approval, pharmacy records were utilized to identify patients treated with dual HER2 blockade in neoadjuvant setting at our institution. Individual patient charts were reviewed to identify AA patients and other ethnic minorities. We also obtained details of the patient age, clinical stage, pathological stage, ER/PR status, treatment received, side effects leading to dose reductions if any. pCR was defined as absence of invasive or noninvasive cancer in the breast and lymph nodes (ypT0N0). Results:50 patients with a median age of 53 years met our inclusion criteria. 86% patients self-reported as AA and 14% were other minorities as recorded in EMR. 90% patients were treated with Taxotere, Carboplatin, Trastuzumab and Pertuzumab (TCHP) and 10% received Adriamycin, Cyclophosphamide, Paclitaxel, Trastuzumab and Pertuzumab (AC-THP) chemotherapy. Clinical staging was: Stage I- 4 %; Stage II- 84 %, and Stage III- 12 %. 46% of patients had hormone receptor (HR) positive tumors and 54% patients were HR negative. 30% (15/50) of patients required dose reduction or omission for diarrhea, neuropathy, or cytopenia. 18%(9/50) patients had doses omitted. The overall observed pCR rate (ypT0N0) was 42%(21/50). As expected, the pCR rate was higher in patients with HR negative/HER2 positive breast cancer compared to HR positive/HER2 positive breast cancer. 44% (12/27) vs. 39% (9/23), respectively.Conclusion: Dual HER2 blockade was associated with a lower pCR rate (42%) in AA and other minorities compared to rate of 52% as reported by TRYPHAENA trial (n=77). The treatment was well tolerated but higher percent of patients required a dose reduction or omission (Tiwari et al; Breast Cancer Res Treat; 158:189-193).The most common side effects were diarrhea, neuropathy and cytopenias. Further prospective studies are needed to elucidate the role of biologic differences in chemosensitivity and balancing the variables of increased number of dose reductions and effect of socioeconomic differences in an underserved minority population. Citation Format: Shruti R Tiwari, Prasun Mishra, Asma Dilawari, Christopher M Gallagher, Ami Chitalia, Patricia Wehner, Marc E Boisvert. Lower pathologic complete response rate with dual-HER2 blockage and chemotherapy in African Americans and ethnic minorities [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P2-10-10.

Abstract P1-10-11: Pathological complete response after carboplatin-based neoadjuvant chemotherapy in triple negative breast cancer: The importance of stromal tumor infiltrating lymphocytes as a predictive biomarker

Abstract Background: Lymphocyte predominant breast cancers (LPBC) account for 5-30% of TNBCs; 65-80% of TNBCs have low/moderate levels of sTILs and 15-20% have no lymphocyte infiltration. TNBCs with high stromal tumor infiltrating lymphocytes (sTILs) have higher rates of pathological complete response (pCR) with anthracycline-taxane (AT) neoadjuvant chemotherapy (NACT), but the effect of carboplatin (Cb) is unclear. In GeparSixto, Cb increased pCR rates in LPBC but there was no interaction between LPBC and Cb in TNBCs. This study examines the predictive role of sTILs in TNBCs, focusing on the association between sTILs and pCR with and without Cb based NACT. Methods: Patients diagnosed with TNBC and treated with NACT in University Hospital Galway, Ireland from 2004-2016 were identified. Patients were treated with a carboplatin-anthracycline-taxane regimen (Cb-AT) (n=30) or an AT regimen (n=54). As per the International TILs Working Group, sTILs were scored on digitized slides of full-face tumor sections from diagnostic needle core biopsies. sTILs were categorized as 0-10%; 11-25%; 26-49%; ≥50% (LPBC). Odds ratio (OR) was calculated using non-pCR as the baseline. pCR was defined as ypT0/isN0. Results: Eighty-eight (n=88) cases with sufficient sTILs for analysis were identified. The median sTIL count was 12.5% (range 0-75%): 50% of cases (n=44) had sTILs 0-10%; 25% (n=22) had sTILs 11-25%; 14% (n=12) had sTILs 26-49% and 11% (n=10) had sTILs ≥50%. Cases with sTILs &amp;gt;10% had higher rates of pCR compared to those with sTILs ≤10% (52% vs 29%; p=0.021). In cases treated with an AT regimen, tumors with sTILs &amp;gt;10% had higher pCR rates than those with sTILs ≤10% (46% vs 12%; p=0.005). In cases treated with a Cb-AT regimen, there was no difference in pCR rates between sTILs &amp;gt;10% tumors and sTILs ≤10% tumors (60% vs 53%; p=0.500). In this cohort, pCR rates were higher in LPBC compared to non-LPBCs (80% vs 36%; p=0.010). In cases treated with AT, pCR rates were higher in LPBC than non-LPBC (83% vs 23%; p=0.007). However, in cases treated with Cb-AT, there were no differences in pCR rates between LPBC and non-LPBC (67% vs 56%; p=0.603). In LPBC, the choice of chemotherapy did not affect pCR rates: 67% vs 83% for Cb-AT and AT regimens respectively (p=0.583). In non-LPBC, pCR rates increased with Cb: 56% vs 23% for Cb-AT and AT regimens respectively (p=0.005). By univariate analysis, there was a significant association between sTILs and pCR. The association was greatest in LPBC (OR 0.14; p=0.016) and remained significant in those treated with AT (OR 0.06; p=0.014) but not in cases treated with Cb-AT (OR 0.63; p=0.714). In a multivariable model, with tumor grade and Cb use as co-variables, LPBC was independently associated with pCR (OR0.14; p=0.030). In cases treated with AT, sTILs were associated with pCR (OR 0.18, p=0.019 for sTILs &amp;gt;10%; OR 0.08, p=0.029 for LPBC). In cases treated with Cb-AT, sTILs were not associated with pCR (p=0.372 and p=0.622 for sTILs &amp;gt;10% and LPBC respectively). Conclusions: In TNBC, sTILs are predictive of response to NACT: as sTILs increase, pCR rates increase. The likelihood of a pCR increased by 86% for LPBC vs non-LPBC. The effect of sTILs on pCR was most notable in LPBC treated with AT based NACT where the odds of a pCR increased by 94%. In cases treated with Cb-AT, there was no association between sTILs and pCR. This study suggests that tumors with high sTILs are more chemosensitive than tumors with low sTILs. High sTIL tumors had high pCR rates with AT based NACT but the addition of Cb did not increase pCR rates. Cb-AT NACT could be reserved for patients with low sTIL tumors, in order to increase the rates of pCR in that subgroup, who had low rates of pCR in this study. In the future, sTILs could be used as a predictive biomarker to guide chemotherapy selection. Citation Format: Elaine M Walsh, Mark O'Loughlin, Aliaa Shalaby, Mark Webber, Michael J Kerin, Sharon A Glynn, Grace Callagy, Maccon M Keane. Pathological complete response after carboplatin-based neoadjuvant chemotherapy in triple negative breast cancer: The importance of stromal tumor infiltrating lymphocytes as a predictive biomarker [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-10-11.

Detection of rice blast pathogen- Expedition from Petri plate to PCR tube - a mini review

Rice blast caused by Magnaporthe oryzae (anamorph: Pyricularia oryzae) is a serious disease of rice threatening its production worldwide. the pathogen has high genomic plasticity, wide host range and rapid rate of evolution resulting in fungicidal resistance and breakdown of resistant varieties. though integrated disease management modules have been adopted for the management of the pathogen, the disease continues to be a challenge to plant pathologists. The pathogen affects all the above-ground parts of the plant including seeds. Seeds are implicated as important overwintering source of inoculum of the pathogen and may serve as an important source of primary inoculum during the rice season. Further, the spread of pathogen to previously un- invaded areas has been found associated with the movement of the infected seeds. therefore, use of pathogen-free healthy seeds should be an integral part of the rice blast management module, for quarantine purposes. Further, with the appearance of wheat blast as a global threat to wheat production and invasion of rice blast pathogen to previously uninvaded areas, accurate detection of M. oryzae strains is of paramount importance. Conventionally used seed testing assays are time-consuming, labor-intensive and have low sensitivity and specificity. Advances in the detection strategies of rice blast pathogen from blotter test to high throughput real time PCR is remarkable. But still, commercial application of these technologies is lacking. the review focuses on the advances in detection strategies of M. oryaze and their utility as commercial protocols.

Rapid bacterial detection and antibiotic susceptibility testing in whole blood using one-step, high throughput blood digital PCR.

Sepsis due to antimicrobial resistant pathogens is a major health problem worldwide. The inability to rapidly detect and thus treat bacteria with appropriate agents in the early stages of infections leads to excess morbidity, mortality, and healthcare costs. Here we report a rapid diagnostic platform that integrates a novel one-step blood droplet digital PCR assay and a high throughput 3D particle counter system with potential to perform bacterial identification and antibiotic susceptibility profiling directly from whole blood specimens, without requiring culture and sample processing steps. Using CTX-M-9 family ESBLs as a model system, we demonstrated that our technology can simultaneously achieve unprecedented high sensitivity (10 CFU per ml) and rapid sample-to-answer assay time (one hour). In head-to-head studies, by contrast, real time PCR and BioRad ddPCR only exhibited a limit of detection of 1000 CFU per ml and 50-100 CFU per ml, respectively. In a blinded test inoculating clinical isolates into whole blood, we demonstrated 100% sensitivity and specificity in identifying pathogens carrying a particular resistance gene. We further demonstrated that our technology can be broadly applicable for targeted detection of a wide range of antibiotic resistant genes found in both Gram-positive (vanA, nuc, and mecA) and Gram-negative bacteria, including ESBLs (blaCTX-M-1 and blaCTX-M-2 families) and CREs (blaOXA-48 and blaKPC), as well as bacterial speciation (E. coli and Klebsiella spp.) and pan-bacterial detection, without requiring blood culture or sample processing. Our rapid diagnostic technology holds great potential in directing early, appropriate therapy and improved antibiotic stewardship in combating bloodstream infections and antibiotic resistance.