Cell Histology Research Articles

Salvage Radiotherapy as a Bridge for Relapsed Secondary CNS Lymphoma.

Secondary CNS lymphoma (SCNSL) is a challenging clinical scenario observed in 2-5% of non-Hodgkin lymphoma patients, for which a standard of care has not been defined. We studied the indications for, and outcomes of SCNSL patients referred for radiotherapy (RT). We identified patients with aggressive B cell lymphoma who received brain RT for SCNSL between 1999-2023 at a tertiary cancer center. Patients were grouped and analyzed by RT indication. Overall survival (OS) was determined from RT start using the Kaplan-Meier method. OS analysis comparing patients who did and did not receive therapy after RT was landmarked at 60 days from start of RT to minimize immortal time bias. "SCNSL-directed therapy" is defined as systemic therapy for the treatment of SCNSL, as opposed to CNS prophylaxis. We identified 99 SCNSL patients treated with RT. To account for the heterogeneity of RT referrals, we focused on the most common indication: salvage of radiographic progression after SCNSL-directed systemic therapy (n = 58). Among this group, median age was 62 (interquartile range [IQR]: 48-69) and 86% had diffuse large B cell histology. At initial lymphoma diagnosis, 10% of patients had CNS involvement, 90% received Rituximab-based therapy, and 25% received prior CNS prophylaxis. For SCNSL directed therapy, 90% received methotrexate (MTX)-based regimen. Median time from initial SCNSL diagnosis to RT was 4.4 months (IQR 1.7-7.0), with a median of 2.0 lines of therapy prior to RT (IQR 1.0-3.0). 86% of patients were symptomatic at RT with median KPS of 70 (IQR: 60-80). RT targets included whole brain (86%) and partial brain (14%). 1 patient had craniospinal RT. Median RT dose was 30 Gy (IQR: 24-30) over 10 fractions. Median OS for the entire salvage cohort was 3.5 months (m). Landmark analysis 2m post RT showed that median OS differed when patients were stratified by receipt of further therapy: CAR-T (9.4m, n = 4), hematopoietic cell transplant (8.5m, n = 6), other systemic therapy (4.4m, n = 17), no systemic therapy (0.6m, n = 10) (p = 0.0004). 29% of patients who received further therapy after RT achieved long term survival. In our cohort, most SCNSL patients are referred for salvage RT, with a median OS of 3.5m. 86% of patients had neurologic symptoms after having failed a median of 2 lines of SCNSL-directed therapy; the clinical urgency of this scenario implies that without RT, patients may not have been suitable candidates for further therapy. However, among patients for whom RT was successfully used to bridge to additional therapy, 29% could achieve long-term survival. This study supports further investigation of RT as a combined modality strategy for relapsed/refractory SCNSL, including with emerging cellular therapies.

Partial-Length Treatment With Brachytherapy in Patients With Endometrial Cancer With High-Risk Features Is as Effective as Full-Length Vaginal Brachytherapy but With Reduced Toxicity

Full length vaginal (FLV) brachytherapy for patients with endometrial cancer and high-risk features should be considered as per ABS in order to reduce distal vaginal recurrence in patients with endometrial cancers (EC) with papillary serous/clear cell histologies, grade 3 or extensive lymphovascular invasion.We sought to investigate this patient population and report outcomes of treatment with high dose rate brachytherapy (HDR) in women treated to FLV versus partial length vaginal (PLV). With IRB approval, we identified patients with EC meeting ABS criteria of high-risk features treated withadjuvant HDR between 2004 - 2010. HDR doses were 21 Gy, in 3 fractions delivered to either FLV orPLV. Acute and late toxicities were evaluated using the RTOG scale and RTOG/EORTC grading, respectfully.Vaginal recurrences were assessed by physical examination and Pap smears. Statistical analyses were performed using the SPSS v. 23 software (IBM, Armonk, NY, USA). Of 240 patients treated with HDR, 121 were treated with FLV and 119 with PLV brachytherapy. With median follow up of 9.5 years (range, 8-11 years) for FLV and 8.5 years (range, 7-10 years) for PLV patients, 0% of these patients had vaginal recurrences, and 1.4% and 0.9% of proximal vaginal recurrences, respectively (p=0.54). All patients treated with FLV developed grade 3 mucositis of the lower vagina/introitus (p<0.0001) and had increased analgesics use compared to PLV (p<0.0001). 23% of patients treated with FLV developed grade 3 stenosis of lower vagina/introitus in contrast to 0% patients treated with PLV (p<0.0001). PLV brachytherapy is as effective as FLV in reducing local recurrence but causes significantly lower incidence of acute and late toxicities. The results of this study caution radiation oncologists regarding careful use of FLV in endometrial cancer patients with high-risk features.

Biological Role and Clinical Implications of MYOD1L122R Mutation in Rhabdomyosarcoma

Major progress in recent decades has furthered our clinical and biological understanding of rhabdomyosarcoma (RMS) with improved stratification for treatment based on risk factors. Clinical risk factors alone were used to stratify patients for treatment in the European Pediatric Soft Tissue Sarcoma Study Group (EpSSG) RMS 2005 protocol. The current EpSSG overarching study for children and adults with frontline and relapsed rhabdomyosarcoma (FaR-RMS NCT04625907) includes FOXO1 fusion gene status in place of histology as a risk factor. Additional molecular features of significance have recently been recognized, including the MYOD1L122R gene mutation. Here, we review biological information showing that MYOD1L122R blocks cell differentiation and has a MYC-like activity that enhances tumorigenesis and is linked to an aggressive cellular phenotype. MYOD1L122R mutations can be found together with mutations in other genes, such as PIK3CA, as potentially cooperating events. Using Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines, ten publications in the clinical literature involving 72 cases were reviewed. MYOD1L122R mutation in RMS can occur in both adults and children and is frequent in sclerosing/spindle cell histology, although it is also significantly reported in a subset of embryonal RMS. MYOD1L122R mutated tumors most frequently arise in the head and neck and extremities and are associated with poor outcome, raising the issue of how to use MYOD1L122R in risk stratification and how to treat these patients most effectively.

Radiotherapy for Secondary CNS Lymphoma: Patterns of Utilization and Clinical Outcomes

Introduction: Secondary CNS lymphoma (SCNSL) is a challenging clinical scenario observed in 2-5% of non-Hodgkin lymphoma patients, without well-defined standard of care. We sought to understand the indications and outcomes of patients with SCNSL referred for radiotherapy (RT) at a tertiary cancer center. Methods: We analyzed 93 consecutive patients with SCNSL of aggressive B cell histology who received brain RT at Memorial Sloan Kettering Cancer Center between 1999-2021. Overall survival (OS) was estimated using the Kaplan-Meier method. Time to intracranial progression was estimated for patients with brain imaging at least 30 days after treatment start using competing risk analysis with death as a competing risk. Outcomes were considered from the start of RT. Patients were further analyzed by treatment intent and by the presence of leptomeningeal disease (LMD), defined radiographically or by cerebrospinal fluid analysis. Results: Median age was 63 (interquartile range [IQR]: 51-70). 9% of patients had CNS involvement at initial diagnosis. Histologies included diffuse large B cell (76%), mantle cell (7%), post-transplant lymphoproliferative disorder (7%), Burkitt (5%), Richter's transformed CLL (4%), lymphoblastic lymphoma (1%). 55% of patients received R-CHOP and 12% received R-EPOCH as initial therapy, and 25% received CNS prophylaxis. 21 (23%) patients received RT as their initial SCNSL treatment. Of the 72 (77%) patients who received CNS-directed systemic therapy for SCNSL prior to brain RT, 88% received a methotrexate (MTX)-based regimen. Median time interval between MTX and RT was 32 days (IQR: 15 - 82). 86 (92%) of patients received whole brain (WBRT) and 7 (8%) received partial brain RT. Median RT dose was 30 Gy (IQR: 23.4-30). 3 patients received craniospinal irradiation (CSI). Median OS for the entire cohort was 2.9 months. Median OS differed by RT treatment intent as follows: consolidation after complete response to systemic therapy (61.7 months, n=8), RT bridging to hematopoietic cell transplantation (HCT) (not reached, n=7), RT bridging to CAR-T (5.7 months, n =3), RT salvage after progression or intolerance of systemic therapy (2.5 months, n = 54), RT as initial SCNSL treatment typically in an emergency or palliative setting (1.2 months, n=21) (p < 0.0001). Across the full cohort, median OS was longer for patients without evidence of LMD at the time of RT (6.3 vs. 1.7 months, p < 0.0001). Among patients with LMD at SCNSL diagnosis, those who cleared LMD by the time of RT had longer median OS than those with persistent LMD (8.3 vs. 2.2 months, p = 0.014). The estimated 3-year risk of intracranial failure after salvage RT was 35 ± 11% for patients without LMD and was not evaluable for patient with LMD at salvage as all patients' treatment failed or the patients died by 4 months. Conclusion: SCNSL remains a clinical challenge and patients referred for brain RT have overall poor prognosis. However, the subset of patients who receive RT as consolidation or bridge to HCT can achieve long term survival. The presence of LMD at RT is prognostic for inferior survival. For SCNSL patients without LMD, intracranial disease control after salvage RT is possible. Alternate treatment strategies including broader consideration of CSI for persistent LMD are warranted. Further identification of clinical, anatomic, and molecular subtypes of SCNSL that impact treatment response will help to clarify optimal treatment strategies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Abstract 775: Evaluation of Erb-B2 expression from archival tumor tissue in early stage epithelial ovarian carcinoma patient prognosis

Abstract In this retrospective study, Erb-B2 expression was assessed in 106 patients using Paraffin-Embedded Tissue (PET) to determine the prognostic value in Epithelial Ovarian Carcinoma (EOC) patients diagnosed at Saskatoon Cancer Center between the years 1983-1995. Relationship between Erb-B2 positivity and patient tumor characteristics was assessed for Overall Survival (OS) and Relapse-Free Survival (RFS) by Kaplan-Meier method. Based on the intensity of stain with a polyclonal antibody (A485, 1/400 dilution), cases with markedly intense cytoplasmic and membranous immunoreactivity were classified as Erb-B2 over-expressors. Erb-B2 was overexpressed in 13 (12%) cases. The mean age of the patients was 56 years (range 18-84 years). The patient population consisted of 64 Stage I and II, Early Stage (ES) and 36 cases with Stage III and IV Advanced Stage (AS). Data on histologic grade was available in 81 cases. 58 patients received platinum-containing regimen, 13 melphalan, and in 9 cases combination chemotherapy. Erb-B2 expression did not influence OS and RFS in these patients. Similarly, there was no significant relation to OS and RFS between Erb-B2 and age, grade, and patients with No Evidence of Disease (NED). Of the 64 cases with ES disease, 16 cases had Grade 2 tumor with Erb-B2 over-expression in 4 cases. Grade 2 tumors with Erb-B2 overexpression indicated significant relationship to OS and RFS, p=0.01, (median = 30 months, CI: 26 - 68) and p=0.05, (median = 15 months, CI: 13 - 68). Furthermore, in 21 cases with ES, Endometrioid histology and Erb-B2 expressors 6/21 (28%) experienced significantly shorter OS compared to non-expressors p=0.005, (median = 48 months, CI: 22 - 74) (mean = 134 months, CI: 124 - 144). In conclusion, Erb-B2 overexpression could be detected in only limited EOC cases in PET and appears to have no prognostic influence. Additionally, endometrioid carcinoma and grade 2, ES certainly reflect the importance of sub-group analysis. In ovarian carcinoma distribution of stage, histology, and grading of tumor cells are conventional markers for prognosis. Finally, Erb-B2 status detected by immunohistochemistry, in a subgroup of patients with high-degree of HER2 positivity/focal amplification may likely have better clinical outcome with anti-HER2 agents. Citation Format: Kavitha Advikolanu Rao, Anthony Magliocco, Andrew Maksymiuk. Evaluation of Erb-B2 expression from archival tumor tissue in early stage epithelial ovarian carcinoma patient prognosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 775.

Multicenter phase II study of abemaciclib and ramucirumab in metastatic esophageal/gastroesophageal junction carcinoma.

TPS4169 Background: Cyclin dependent kinases (CDKs) are serine/threonine kinases that are responsible for phosphorylating the intracellular proteins that coordinate cell-cycle progression. The Cancer Genome Atlas (TCGA) molecular analysis of esophageal/gastroesophageal junction (E/GEJ) carcinomas has highlighted significant dysregulation of CDKN2A, the gene coding for the tumor suppressor p16, through deletion, mutation or epigenetic silencing in up to 76% of cases and the associated significant upregulation of the CDK4/6-Cyclin D-axis (The Cancer Genome Atlas Research Network. Integrated genomic characterization of oesophageal carcinoma. Nature 2017). Preclinical experiments demonstrated that single agent abemaciclib has potent antitumor efficacy both in vitro and in vivo in esophageal adenocarcinoma with direct pathway inhibition (Kosovec J et al. CDK4/6 dual inhibitor abemaciclib demonstrates compelling preclinical activity against esophageal adenocarcinoma: a novel therapeutic options for a deadly disease. Oncotarget 2017 Nov 1;8[59]). Significant activity was seen in the OE33 (p=0.048) and FLO1 (p=0.043) esophageal cancer cell lines while western blot revealed downregulation of Cyclin D, phospho-pRb, E2F1, and Cyclin A2 (Kosovec J et al, 2017). In vivo experiments , utilizing the Levrat model of end-to-side esophagojejunostomy performed on Sprague-Dawley rats demonstrated that 78.9% of animals given abemaciclib demonstrated &gt;20% tumor volume decrease (placebo 0%). Post treatment comparison between placebo and abemaciclib of qRT-PCR gene expression demonstrated downregulation of pathway correlates, including CDK4 (p=0.023), CDK6 (p=0.068), E2F1 (p=0.005), Rb1 (p=0.067), Cyclin D (p=0.031), and Cyclin A (p=0.039) (Kosovec J et al, 2017). The JPBJ phase 1b study has previously demonstrated the safety of combining abemaciclib with ramucirumab in NSCLC. Methods: Subjects with previously treated metastatic esophageal or gastroesophageal junction adenocarcinomas are eligible for this study which is currently open and enrolling patients. Squamous cell and mixed histology with neuroendocrine features are excluded. A total of 30 patients will be enrolled across 3 sites at Baylor University Medical Center in Dallas, The Allegheny Health Network in Pittsburgh and at Johns Hopkins Hospital in Baltimore. The primary objective is to describe the safety profile of abemaciclib (150mg po bid) and ramucirumab (8mg/kg iv every 2 weeks) in this patient population as assessed according to Common Terminology Criteria for Adverse Events version 4.0. Secondary objectives are to assess response rate, progression free and overall survival. In addition, we will determine the rate of stable disease at 3 months post targeted therapy. Correlative studies investigating changes in the expression of selected serum/tissue genomic markers of interest will be performed. Clinical trial information: NCT04921904.

Interaction of race and socioeconomic status as risk modulators of treatment delay and cancer-specific mortality in uterine cancer.

5595 Background: The majority of studies of uterine cancer combine high and low-grade histologies and do not sample a diverse cohort of patients. In many studies race is treated as biologic construct, when it may be better thought of as a proxy for socioeconomic inequity and deprivation. Socioeconomic (SE) deprivation may play a significant role in the disease trajectory of women with uterine cancer. Methods: Data were drawn from the Metropolitan Detroit Cancer Surveillance System which covers a tri-county area of approximately 4 million people. We included non-Hispanic Black (NHB) and White (NHW) women diagnosed with uterine cancer between 2010 and 2018. Poorly differentiated and undifferentiated endometrioid, serous, clear cell, mixed, carcinosarcoma and mucinous histologies were considered high grade. Patients diagnosed by death certificate, or with unknown stage or histology were excluded. Socioeconomic status was assessed using the Yost Score, an area-level composite measure of socioeconomic deprivation derived from census-tract data at cancer diagnosis. Lower Yost quintile indicates higher deprivation. Competing risk analysis was used to determine risk of uterine cancer specific mortality (reported as subdistribution hazard ratio [SHR]) and to assess statistical interaction between race and Yost score. Results: A total of 4,840 patients were identified. Race conferred significant increased risk of cancer-specific mortality (SHR 2.11, p &lt; 0.0001). Race and Yost score interacted to increase risk of cancer-specific mortality in NHB women in the lowest Yost quintile (SHR 2.23, p &lt; 0.0001) compared to NHW and NHB women in the highest quintiles. The interaction between race and Yost score persisted only among women with low grade cancers (SHR 1.7, p = 0.04). Time from diagnosis to surgery increased as Yost score decreased. Women in the lowest Yost quintile had lower likelihood of receiving surgery within 6 weeks of diagnosis (OR 0.74, p = 0.001). This effect persisted among women with low grade cancer (NHB OR 0.75, p = 0.014; lowest Yost quintile OR 0.68, p &lt; 0.0001). An association between race, Yost score and delays in time to surgery was not seen among women with high grade cancers. Conclusions: Race and Yost score, an area-based measure of socioeconomic deprivation, are associated with increased cancer-specific mortality risk among women with low grade cancer. NHB race and high socioeconomic deprivation are associated with delayed primary surgery. The interaction between race and socioeconomic deprivation may underlie known disparities in uterine cancer survival, particularly in low grade disease where there is the greatest opportunity for timely curative surgery.

Characterization of the microbial resistome in a prospective trial of CBM588 in metastatic renal cell carcinoma (mRCC) offers mechanism for interplay between antibiotic (abx) use and immune checkpoint inhibitor (ICI) activity.

4510 Background: The negative association between ICI response and abx therapy is well defined (Derosa et al Cancer Discov 2021). Paradoxically, a retrospective assessment of the live bacterial product (LBP) CBM588 in patients (pts) with advanced lung cancer showed improved outcome with ICIs when the combination of CBM588 and abx (as compared to CBM588 alone) was employed (Tomita et al Cancer Immunol Res 2020). We postulated that the microbial resistome (genes encoding antimicrobial resistance) could shift in a manner with CBM588 therapy that facilitated ICI response. Methods: Pts with newly diagnosed mRCC with clear cell and/or sarcomatoid histology and intermediate/high risk disease per IMDC criteria were randomized to nivolumab/ipilimumab (nivo/ipi) or nivo/ipi/CBM588 in a 1:2 ratio. Stool samples were collected at baseline and week 12. Whole-metagenome sequencing was performed to analyze stool microbiome composition. Abx resistance genes (RGs) were inferred using publicly available database (McArthur et al. Antimicrob Agents Chemother 2013), and groups of abx RGs for various classes of abx were characterized. Wilcoxon signed-rank test was used for comparison of abx RG abundance between baseline and week 12 in each treatment arm and in responders (R) and non-responders (NR). Results: The study enrolled 30 pts, with the final analysis including 29 eligible pts (median age: 66 years, M:F 21:8, nivo/ipi: 19 pts, nivo/ipi/CBM588:10 pts). Objective response was 20% and 58% in nivo/ipi and nivo/ipi/CBM588 arms, respectively. The overall abundance of abx RGs remained unchanged between baseline and week 12 in pts receiving nivo/ipi alone. In contrast, a decrease in abx RGs was observed in pts receiving nivo/ipi with CBM588 arm from baseline to week 12 (p = 0.042 in Rs; p = 0.078 in NRs). More specifically, nivo/ipi/CBM588 treatment led to a significant reduction in fosfomycin RGs and nitroimidazole (e.g., metronidazole) RGs in both pts with R (p = 0.019 and 0.042, respectively) and NR (p = 0.031 and p = 0.031, respectively). A multitude of other clinically relevant abx RGs were downregulated in pts receiving CBM588, including those mediating resistance to glycopeptide (e.g., vancomycin) and lincosamide (e.g., clindamycin) abx. Conclusions: In the first interrogation of the resistome in mRCC, we demonstrate that CBM588 decreases abx RGs associated with multiple commonly used classes of abx. Abx clear commensals and increase pathogenic (abx resistant) bacteria in the gut. Based on our data, we formulate the hypothesis that combining abx with CBM588 may decrease potentially pathobionts and favor butyrogenic species, thereby improving CPI response. Clinical studies using CBM588 with abx priming may be warranted. Clinical trial information: NCT03829111.

Survival Outcomes After Cytoreductive Surgery with Hyperthermic Intraperitoneal Chemotherapy in Patients with Synchronous Versus Metachronous Onset of Peritoneal Metastases of Colorectal Carcinoma

BackgroundCytoreductive surgery (CRS) with hyperthermic intraperitoneal chemotherapy (HIPEC) is a treatment option for peritoneal metastases (PM) from colorectal carcinoma (CRC). Because of considerable morbidity, optimal patient selection is essential. This study was designed to determine the impact of the onset of PM (synchronous vs. metachronous) on survival outcomes after CRS-HIPEC.MethodsPatients undergoing CRS-HIPEC for colorectal PM in two academic centers in the Netherlands between 2010 and 2020 were eligible for inclusion. Patients were classified as synchronous (s-PM, i.e., diagnosis at time of presentation, staging, or primary surgery) or metachronous onset (m-PM, i.e., diagnosis during follow-up) of colorectal PM. Survival outcomes were compared between groups by Kaplan–Meier survival and Cox regression analyses.ResultsOf 390 included patients, 179 (45.9%) had synchronous onset of colorectal PM. These patients more often presented with higher TN-stage and poor differentiation/signet cell histology. Treatment with perioperative chemotherapy was more common in s-PM patients. m-PM patients experienced more serious postoperative complications (Clavien-Dindo ≥ III). There was no significant difference in disease-free survival (DFS) between s-PM (median 9 months, interquartile range [IQR] 5–15) and m-PM patients (median 8 months, IQR 5–17). Overall survival (OS) was significantly shorter for s-PM (median 28 months, IQR 11–48) versus m-PM patients (median 33 months, IQR 18–66, p = 0.049). Synchronous onset of PM was not independently associated with OS in a multivariable analysis.ConclusionsSynchronous onset of colorectal PM was associated with poor tumor characteristics and more advanced disease, but was not an independent predictor of survival outcomes after CRS-HIPEC.

Food Allergen Nitration Enhances Safety and Efficacy of Oral Immunotherapy in Food Allergy.

(1) Background: Posttranslational protein modifications have been demonstrated to change protein allergenicity. Previously, it was reported that pretreatment with highly nitrated food proteins induced a tolerogenic immune response in an experimental mouse model and in human immune cells. Here, we investigated a possible therapeutic effect of modified proteins and evaluated the safety of oral exposure to highly nitrated proteins in an experimental food allergy model. (2) Methods: BALB/c mice were orally sensitized towards ovalbumin (OVA) under gastric acid suppression. Thereafter, treatment via intragastric gavage with maximally nitrated OVA (nOVAmax) and OVA as a control was performed six times every 2 weeks. On the last day of experiments, all the treated mice were orally challenged with OVA. Systemic anaphylactic reaction was determined by measuring the core body temperature. Moreover, antibody levels, regulatory T cell numbers, cytokine levels and histology of antrum tissues were analyzed. (3) Results: After oral immunotherapy, OVA-specific IgE titers were decreased while IgG1 titers were significantly elevated in the mice receiving OVA. After oral challenge with OVA, nOVAmax-treated allergic animals showed no drop of the core body temperature, which was observed for OVA-allergic and OVA-treated allergic animals. Significantly fewer eosinophils and mast cells were found in the gastric mucosa of the allergic mice after nOVAmax treatment. (4) Conclusions: Oral immunotherapy with nOVAmax reduced allergic reactions upon allergen exposure and the number of allergen effector cells in the gastric mucosa. Thus, maximally nitrated allergens enabled an efficient and safe treatment for food allergy in our experimental model.

Nivolumab/ipilimumab with or without CBM588 in metastatic renal cell carcinoma: A randomized phase Ib study and the evolution of the functionality of microbial communities with treatment.

371 Background: The role of gut microbial composition as a determinant of clinical outcomes has been well established in several cancers, including metastatic renal cell carcinoma (mRCC) (Routy et al Science 2018). A growing body of evidence suggests that examining the metabolic function of microbial communities may provide a more insightful understanding of these associations (Helmink et al Nature Medicine 2019). Herein, we aimed to examine the effect of nivolumab/ipilimumab with or without CBM588 on clinical outcomes and gut microbiome functionality. Methods: Treatment naïve mRCC pts with clear cell and/or sarcomatoid histology and IMDC intermediate/high risk disease were enrolled and randomized into receiving nivolumab/ipilimumab or nivolumab/ipilimumab with CBM588 in 1:2 fashion. Whole metagenome sequencing was performed on stool samples collected at baseline and week 12. Generated MetaPhlan 3.0 data was run through HUMAnN 3.0 to identify differentially expressed metabolic pathways between two timepoints in each arm and with respect to treatment response. Results: A total of 30 pts were enrolled and randomized, and 29 pts were eligible for analysis as one patient was excluded as tumor tissue next-generation sequencing revealed genomic alterations pathognomonic for sarcoma after initiation of protocol-based therapy. Median age of the participants was 66 years, 21 pts (72%) were male, 10 pts (34%) had sarcomatoid features, and 29 pts (45%) had prior nephrectomy. Objective response was achieved in 58% and 20% of the pts in nivolumab/ipilimumab/CBM-588 and nivolumab/ipilimumab arm, respectively. Significant changes in 40 metabolic pathways (37 with upregulation and 3 with downregulation) in nivolumab/ipilimumab arm and 52 metabolic pathways (49 with downregulation and 3 with upregulation) in nivolumab/ipilimumab with CBM588 arm were identified. In detail, dTDP-β-L-rhamnose biosynthesis, L-lysine biosynthesis II and superpathway of pyrimidine ribonucleosides degradation pathways were found upregulated while O-antigen building blocks biosynthesis (E. coli) pathway was found downregulated after treatment with nivolumab/ipilimumab and CBM588 (p = 0.001, p = 0.007, p = 0.037, p = 0.005 respectively). Heatmaps detailing the dynamics of metabolic pathway expressions in regard to response in each arm will be presented. Conclusions: We observed an increase in the activity of the pathways associated with butyrate consumption and a resultant decrease in glycolytic dependence. Further, suppression of the pathogenic E. coli function was observed, suggesting a role for CBM588 in protection from pathogenic species. Our findings provide mechanistic evidence for the effect of the addition of CBM588 to nivolumab/ipilimumab on gut microbiome function and resultant improvement in clinical outcomes in mRCC. Clinical trial information: NCT03829111.

Avelumab plus axitinib in advanced renal cell carcinoma (aRCC): 12-month interim results from a real-world observational study in the United Kingdom.

301 Background: The combination therapy avelumab + axitinib (A + Ax) has demonstrated superior progression-free survival (PFS) and objective response rate (ORR) benefit across all International Metastatic RCC Database Consortium (IMDC) risk groups (favourable, intermediate, and poor) vs sunitinib in patients with previously untreated aRCC. A + Ax is now approved and funded in the UK; on 29 October 2019, the European Commission approved A + Ax for first-line treatment of adult patients with aRCC, and it has been funded by the Cancer Drugs Fund since 30 July 2020. Here we report 12-month real-world outcomes of A + Ax in patients with aRCC in the UK. Methods: Patients ≥18 years with diagnosed aRCC who initiated A + Ax treatment outside of a clinical trial on or after 1 August 2019 via an early access to medicines scheme were recruited at 4 UK sites. Patient informed consent was obtained. Primary endpoints of interest include overall survival (OS), best response, PFS and ORR at 12 months. Descriptive statistics are provided. Recruitment is ongoing until Jan 2022 and follow-up until July 2023. Results: This analysis includes 36 patients with a minimum 12 month follow up. Median age at baseline was 66.2 (range, 39.8-84.1) years. 78% were male, 69% were White, and 6% were Asian/Asian British; ethnicities of 25% of patients were not recorded. Patient IMDC risk statuses were: 39% favourable, 42% intermediate, 17% poor, and 3% unknown; 89% had an ECOG PS of 0 or 1. Median time between aRCC diagnosis and A + Ax initiation was 1.8 months. Most patients had clear cell (72%) or other (25%) histology, had undergone nephrectomy (61%), and had 1 or 2 sites of metastatic disease (69%). The most common metastatic sites were lung (50%), bone (36%), and lymph node (25%). The 12 month OS rate was 86% (95% CI 74.8-97.4), and ORR was 53% (95% CI 36.5-69.1) with 6% achieving a complete response and 47% achieving partial response. Median duration of follow up and PFS was 12 months. Best responses within 12 months of observation were: complete response = 2 (6%), partial response = 17 (47%), stable disease = 14 (39%), and progressive disease = 3 (8%). At the time of the first and second infusions, 39% of patients received Ax at the starting dose of 5 mg twice daily, 47% had a dose escalation, and 14% had a dose reduction. Infusion-related reactions (IRRs) were recorded in 9 patients (25%) in the first 12 months, with 10 events total. All 10 IRRs required the use of high-dose corticosteroids. Median time from A + Ax initiation to IRR was 1.3 months. A + Ax was discontinued in 1 patient (3%) due to toxicity and 4 patients (11%) due to disease progression. Conclusions: In this real-world study of A + Ax treatment in the UK, A + Ax displayed comparable clinical responses to previously published data. OS, ORR, and best response figures were in line with published randomised controlled trials, which is encouraging in a nontrial population.

Abstract ES11-3: Optimizing immunotherapy efficacy in the clinic through biomarkers: Advances in single cell and spatial histology analyse

Abstract Single cell technologies and spatial analyses are emerging as powerful tools to investigate tumor heterogeneity, cell state of key effector or suppressing immune cells and detailed distribution of cells within the tumor microenvironment. Their application will undoubtedly provide further understanding of the effects of cancer immunotherapy on the tumor microenvironment, which is particularly important for the largely non-immunogenic breast carcinomas. However, analyses of clinical samples require well-coordinated, multidisciplinary teams and specific expertise. Moreover, large scale analyses have major technical challenges and is still relatively costly. We will discuss the promise and hurdles of single cell and spatial analyses on the road to find biomarker for breast cancer immunotherapy and avenues for novel immunomodulatory treatments for breast cancer patients. Citation Format: M Kok. Optimizing immunotherapy efficacy in the clinic through biomarkers: Advances in single cell and spatial histology analyse [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr ES11-3.

Abstract 463: Features consistent with a small-cell neuroendocrine like (SC/NE-L) transformation can be readily detected and quantified in circulating tumor cells (CTCs)

Abstract Background: The increasing availability and earlier use of life prolonging drugs targeting the androgen receptor signaling axis (ARSI) has resulted in an increase in the frequency of late state tumors with “small cell/neuroendocrine like (SC/NE-L) phenotypes” similar to small-cell lung cancer (SCLC). These tumors are typically heterogenous wherein the small-cell component represents only a portion of CTC's present. Definitive pathologic criteria to diagnose small-cell neuroendocrine transformation are lacking, and the eligibility criteria across trials in this space inconsistent, limiting the ability to relate outcomes between studies. We hypothesize that an analytically validated assay for a rigorously defined “small-cell CTC” phenotype would clarify the identification and diagnosis of SC/NE-L tumors. Methods: Using the WHO guidelines for small-cell diagnosis in tissue as reference, we defined an equivalent set of single-cell criteria for defining a CTC with small cell or neuroendocrine like (SC/NE) histology that include: a circulating CD45- cell with a high nuclear to cytoplasm area ratio, small size, high circularity, lack of detectable nucleoli, salt and pepper chromatin, SC/NE or tumor-related protein expression (DLL3, CD56, CK etc.), and other features associated with lineage plasticity (i.e., CK speckling). Clinical feasibility of feature extraction was explored on CTCs detected using the Epic Sciences platform in the blood of patients with small-cell lung and late state prostate cancers. Results: Using the Epic Sciences platform, morphologic features associated with small-cell or neuroendocrine transformation can be readily identified and quantified automatically in CTCs in a liquid biopsy from patients with small-cell lung and prostate cancers. Conclusions: Identifying small-cell to neuroendocrine like (SC/NE-L) CTC subtypes may aid in establishing that lineage transformation to a SC/NE-L phenotype has occurred where invasive single site tissue biopsies may not be informative or feasible. Citation Format: Howard I. Scher, Adam Jendrisak, Cole Gilbertson, Audrey Gill, Ethan Barnett, Anuradha Gopalan, Niamh Keegan, Samir Zaidi, Hannah Benoliel, Carbone Emily, Eva Wang, James Lu, Alisa Tubbs, Amanda Anderson, Joshua Jones, Joseph D. Schonhoft, Rick Wenstrup. Features consistent with a small-cell neuroendocrine like (SC/NE-L) transformation can be readily detected and quantified in circulating tumor cells (CTCs) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 463.

TIME‐TO‐RESPONSE FOR PATIENTS WITH RELAPSED/REFRACTORY AGGRESSIVE B CELL NON‐HODGKIN LYMPHOMA TREATED WITH POLATUZUMAB‐BASED THERAPY

Introduction: Knowledge of time-to-response after initiation of therapy (TTR) may influence treatment recommendations made to patients (pts) with relapsed/refractory (R/R) aggressive B cell non-Hodgkin lymphoma (aBNHL) given the potential for rapid disease growth. If reported in clinical trials, TTR is dependent upon the timing of protocol-specified imaging response assessments (IRA) and does not account for clinical response to therapy (Rc), which may precede radiographic response (Rr). Here we report TTR and other response-related outcomes in pts with R/R aBNHL treated with polatuzumab (pola). Methods: Pts analyzed were those with non-Burkitt R/R aBNHL diagnosed on most recent tissue biopsy and first treated with pola at the University of Pennsylvania from 7/2019 to 12/2020. Rc was defined as reduction in lymph node size on examination, resolution of disease-related symptoms, normalization of lactate dehydrogenase (LDH) previously >2x upper limit of normal or disease response on imaging performed for indication other than IRA. Rr was assessed per Revised Response Criteria for Malignant Lymphoma. Timing of clinical/laboratory assessments and IRA were at the discretion of the treating clinician. Data were censored on 3/1/2021. Results: Fifty pts were included in this analysis. Baseline clinicopathologic and treatment characteristics at time of pola initiation are listed in Table 1. Nineteen pts (38%) were treated with the intent to bridge to cellular therapies (CT). Median number of cycles of pola received was 3. Rc was achieved by 18 pts (36%), Rr by 16 pts (32%) and the composite outcome of Rc or Rr (overall response, [Ro]) by 24 pts (48%). IRA was not performed for 9 pts: 7 due to proceeding to planned CT (5 with Rc) and 2 due to death not attributed to aBNHL with ongoing Rc. The median TTRc, TTRr and earliest of TTRc or TTRr (TTRe) were 21 d (range 7-41 d), 64 d (range 17-137 d) and 22 d (7-67 d), respectively. Of 12 pts who achieved Rc and had subsequent IRA performed, 8 achieved Rr. Twelve pts stopped tx in remission: 7 pts due to proceeding to planned CT, 3 due to toxicity (gastrointestinal in 2 pts and neuropathy in 1 pt) and 2 pts due to choice. Excluding the 7 pts proceeding to planned CT in remission, the estimated proportion of pts with ongoing Rc and Rr at 180 d were 53% and 51%, respectively. Of characteristics listed in Table 1, non-high grade B cell histology and receipt of prior CD19-directed chimeric antigen receptor-modified T cell therapy were significantly associated with achievement of Ro; additionally, age ≤60 years, largest tumor diameter ≤7.5 cm and disease refractory to most recent prior therapy were significantly associated with shorter TTRe. Conclusions: For R/R aBHL pts treated with pola at our institution, frequently with the intention to bridge to CT, 48% achieved Rc or Rr with median TTR of 22 d. These findings may further inform use of pola in the standard-of-care and investigational settings. Keywords: Aggressive B-cell non-Hodgkin lymphoma Conflicts of interests pertinent to the abstract M. E Hughes Consultant or advisory role: AstraZeneca, Genzyme, Janssen, AbbVie, Karyopharm Research funding: Acerta S. D Nasta Consultant or advisory role: Morphosys, Merck Research funding: Roche/Genentech, Millennium/Takeda, Pharmacyclics, ATARA, Forty Seven J. N Gerson Consultant or advisory role: Pharmacyclics, Genentech, AbbVie Research funding: Loxo J. Svoboda Consultant or advisory role: Seattle Genetics, Bristol-Myers Squibb, Pharmacyclics, Imbrium Therapeutics, Genmab, Adaptive Biotechnologies, ADC Therapeutics, Atara Biotherapeutics Research funding: Celgene, Seattle Genetics, Pharmacyclics, Merck, Bristol-Myers Squibb, Incyte Educational grants: Imbrium Therapeutics E. A Chong Consultant or advisory role: Novartis, Tessa Therapeutics, Bristol-Myers Squibb, Kite/Gilead S. J Schuster Consultant or advisory role: Celgene, Nordic Nanovector, Novartis, AbbVie, Acerta, Alimera Sciences, BeiGene, Juno Therapeutics, Loxo Oncology, Tessa Therapeutics, Genetech/Roche, Research funding: Novartis, Pharmacyclics, Adaptive Biotechnologies, Merck, Genetech/Roche, Celgene, Juno Therapeutics, AbbVie, Incyte, TG Therapeutics, DTRM, S. K Barta Consultant or advisory role: Monsanto Honoraria: Atara, Seattle Genetics, Janssen, Pfizer, Acrotech Research funding: Seattle Genetics, D. J Landsburg Consultant or advisory role: Morphosys, Karyoparhm, Celgene Research funding: Takeda, Curis, Triphase Other remuneration: Karyopharm

Immune checkpoint inhibitors (ICI) in advanced sarcomatoid renal cell carcinoma (sRCC): A multicenter study.

4568 Background: Advanced sRCC is an aggressive disease with limited responsiveness to chemotherapy and VEGF-targeted therapies. Subgroup analyses from randomized trials showed improved outcomes with ICI, though sample sizes were relatively small. Methods: We conducted a multi-institutional retrospective analysis of consecutive patients (pts) who had RCC with any sarcomatoid component and received systemic therapy for advanced disease. The pts were classified into ICI+ and ICI- groups (gp) based on whether they had received ICI in any treatment line. Overall survival (OS) was measured from the initiation of first systemic therapy. Time to ICI failure (TIF) was defined as the interval from initiation of ICI to subsequent therapy or death. Survival distributions were estimated using the Kaplan-Meier method. Association between covariates and survival was analyzed using multivariate Cox regression. Two-tailed P &lt; 0.05 was considered statistically significant. Results: 203 pts from 6 US academic cancer centers met the inclusion criteria – 155 in ICI+ gp and 48 in ICI- gp. Overall, 137 (67%) pts were male and 181 (89%) were white; median age at mRCC diagnosis was 59.7 (IQR 52.4-67.7) years; 129 (63%) pts presented de novo with distant metastases, 154 (76%) had clear cell (CC) histology, and 182 (90%) had intermediate/poor risk by IMDC criteria. ICI+ had a higher proportion of purely CC tumors (81% vs 64%, P =.02); other demographic and clinical features were similar between the two gps. After a median follow-up of 48.1 (95% CI 40.7-55.5) months (mos), median OS and response rates were significantly higher in the ICI+ gp (Table). OS benefit, compared to ICI-, was maintained in pts who received ICI in ≥ second line (39.6 vs 7.6 mos, HR 0.33, 95% CI 0.22-0.51, log-rank P &lt;.001). TIF was comparable between pts treated with ICI upfront vs in ≥ second line (6.0 vs 5.3 mos, HR 1.27, 95% CI 0.87-1.85, P =.21). On multivariate analysis, ICI- (HR 2.50, 95% CI 1.61-3.88, P &lt;.001), non-CC histology (HR 3.14, 95% CI 1.98-5.00, P &lt;.001) and sarcomatoid component ≥20% (HR 1.92, 95% CI 1.28-2.90, P =.002) were predictive of all-cause mortality. Among pts with non-CC or mixed histology (n=45), ICI+ had higher OS (18.0 vs 5.5 mos, HR 0.20, 95% CI 0.09-0.44, P &lt;.001) and ORR (44% vs 12%, P =.03), compared to ICI-. Conclusions: Treatment with ICI led to markedly higher survival and response rates in advanced sRCC. OS benefit was maintained with ICI in the second line and beyond. Significant benefit was also noted among pts with non-CC or mixed histology sRCC.[Table: see text]

Effective Removal of Crystal Violet Dye Using Neoteric Magnetic Nanostructures Based on Functionalized Poly(Benzofuran-co-Arylacetic Acid): Investigation of the Adsorption Behaviour and Reusability.

Synthetic dyes represent a significant class of contaminants released in the environment. Crystal violet is a triarylmethane dye used in several fields such as printing inks, the textile or paper industries, as well as in cell histology. Coating magnetic nanoparticles with functionalized polymers has been proved to improve their efficiency, offering unique properties for applications in wastewater treatment. The current paper focuses on preparing and characterising magnetic core-shell nanoparticles coated with poly(benzofuran-co-arylacetic acid) functionalized with folic acid as an organic shell. The new polymer-based magnetic nanostructures were applied for crystal violet extraction from aqueous solutions. The nanostructures were structurally and morphologically investigated by Fourier-transform infrared (FTIR) spectroscopy and transmission electron microscopy (TEM). While thermal and magnetic properties of the magnetic nanostructures were determined by thermogravimetric analysis (TGA) and magnetization measurements (VSM). At the same time, crystal violet concentrations were determined by UV-VIS spectroscopy. The influence of initial dye concentration and contact time on the removal efficiency has been studied to achieve the optimum adsorption conditions. The dye adsorbent neoteric magnetic nanostructure was easily desorbed and reused, the adsorption capacity decreasing from 100% to 97.63% in the first five cycles, reaching a minimum of 88.74% after the 10th recycling step.

Genomic landscape of variant urinary tumor histologies.

467 Background: The genetics of urothelial carcinoma (UC), the most common histology of urinary tract (UT) tumors, is well characterized; much less is known about the genomic features of rare histologic variants of UT tumors. We aim to compare the genomic alterations (GA) of UT tumors with adenocarcinoma (AD), small cell (SC), squamous cell (SQ), or plasmacytoid (PC) histologies, to UC tumors. Methods: We identified patients with pure variant (AD, SC, SQ, PC) or UC histology with genetic characterization through the GENIE registry. Patient tumor genomic data were captured by Memorial Sloan Kettering Cancer Center (MSK)-IMPACT and Dana-Farber Cancer Institute (DFCI)-Oncopanel NGS initiatives. Tumors with mixed histology were excluded. We limited our analysis to genes tested &gt;1000 times (N=211). Mutation frequencies and copy number variants (CNVs), collectively called GAs, were determined for AD, SC, SQ, PC, and UC, and were compared using the Fisher’s Exact test and Kruskall Wallis test. Nominal p values were obtained, and FDR correction was employed (q &lt; 0.1). Results: We identified 1199 patients with available genomic data who met the inclusion criteria. Histologic distribution was: 32 AD, 13 SC, 15 SQ, 11 PC, and 1128 UC tumors. The median age was 68 years and 77% of patients were male. Statistically significant differences in genetic alterations by subtype are shown in the table below. ARID1A and KDM6A GAs were higher in UC; PC and SC; CDH1 GAs higher in PC; RB1 and TP53 GAs higher in SC; SMAD4 GAs higher in AD; and NFE2L2 GAs higher in SQ. Conclusions: Variant UT histologies exhibit a distinct pattern of alterations compared to UC, consistent with their divergent clinical behavior. This suggests different biological origins for these variant histologies and possibly different therapeutic vulnerabilities. Exploring the GAs of these UT tumors in larger datasets is warranted. [Table: see text]

P53 immunohistochemical analysis of fusion-positive uterine sarcomas.

Uterine sarcomas can be grouped into tumours with pathognomonic genetic fusions such as low-grade endometrial stromal sarcoma (LGESS), high-grade endometrial stromal sarcoma (HGESS), and inflammatory myofibroblastic tumour (IMT), and tumours lacking genetic fusions such as leiomyosarcoma (LMS) and undifferentiated uterine sarcoma (UUS). Members of the latter group frequently harbour TP53 mutations. The aim of this study was to evaluate TP53 mutations by the use of immunohistochemistry in fusion-positive uterine sarcomas. We performed p53 immunohistochemical staining on 124 uterine sarcomas harbouring genetic fusions and 38 fusion-negative LMSs and UUSs. These included 41 HGESSs with YWHAE, BCOR and BCORL1 fusions/rearrangements, 13 IMTs with ALK fusion, 12 sarcomas with NTRK1/3 fusion, three sarcomas with PDGFB fusion, and 55 LGESSs with JAZF1, SUZ12 and PHF1 fusions/rearrangements. All HGESSs, LGESSs, IMTs and sarcomas with PDGFB fusion showed wild-type p53 expression. Among NTRK1/3-positive sarcomas, a TPR-NTRK1-positive sarcoma with nuclear pleomorphism showed mutation-type p53 expression. The remaining 11 NTRK1/3-positive sarcomas showed wild-type p53 expression, except for the subclonal p53 mutation-type staining in a minor pleomorphic focus of an NTRK3-positive sarcoma. Twenty-one of 27 (78%) LMSs and six of nine (67%) UUSs showed mutation-type p53 expression. p53 immunohistochemistry may be considered in the initial work-up of a uterine sarcoma, as mutation-type staining would make a fusion-positive sarcoma very unlikely. Mutation-type p53 expression, however, can be seen in a small subset of NTRK1/3-positive sarcomas showing pleomorphic round/ovoid cell histology, which may represent a mechanism of progression in these tumours.

Effects of Oxygen on Renal Function and Oxidative Stress During Hypothermic Machine Perfusion in an Experimental Porcine Model of Kidney Donation after Cardiac Death

The efficacy and safety of using high concentrations of oxygen during hypothermic machine perfusion (HMP) have not been fully elucidated to date. This study investigated the impact of administering high concentrations of oxygen on renal function during HMP in a porcine donation after circulatory death (DCD), as well as the metabolic and biochemical effects of this method. A randomized nonblinded cohort study was established in a porcine transplant (KT) model mimicking Maastricht type III DCD under oxygen-supplemented HMP (Ox-HMP) compared to non-supplemented (nOx-HMP) (LifePort® kidney transporter) conditions. The primary endpoint was evolution of renal function post-KT, whereas secondary endpoints included changes in perfusion dynamics, miRNA expression and cellular lesion measured by LDH and lactate levels in perfusate, lipid peroxidation in kidney biopsies, ATP generation, epithelial mesenchymal transition (EMT) and oxidative gene expression in cell cultures and histology evaluation. ATP generation and oxidative stress, as measured by lipid peroxidation, increased simultaneously after warm ischemia in the Ox-HMP group. Ox-HMP did not exhibit a significant effect on kidney function or animal survival. A significant increase in lipid peroxidation was observed in the Ox-HMP group. This resulted in a greater expression of the genes responsible for producing superoxide dismutase 1 (SOD-1) and catalase oxygenation enzymes, although only SOD-1 showed statistical significance. Respiratory chain dysfunction was maintained in the Ox-HMP group with a non-significant decrease in ATP production, increased proton leakage, and a decrease in respiratory reserve. Regarding epithelial-mesenchymal transition, an upward trend in the expression of vimentin, fibronectin, and collagen genes was observed only in the Ox-HMP group. Finally, the expression levels of miR-101 and miR-126, related to characteristic functions of the tubular epithelium, were significantly modified (miRNA levels expressed as DCT. A brief bubble Ox-HMP treatment did not show a clear positive effect on renal function and oxidative stress markers. The role and safety of adding oxygen during HMP still need to be elucidated. Currently, this Ox-HMP method cannot be considered standard practice.