Association between pathologic response and survival after neoadjuvant therapy in lung cancer.

Neoadjuvant immunotherapy plus chemotherapy improves event-free survival (EFS) and pathologic complete response (0% residual viable tumor (RVT) in primary tumor (PT) and lymph nodes (LNs)), and is approved for treatment of resectable lung cancer. Pathologic response assessment after neoadjuvant therapy is the potential analog to radiographic response for advanced disease. However, %RVT thresholds beyond pathologic complete response and major pathologic response (≤10% RVT) have not been explored. Pathologic response was prospectively assessed in the randomized, phase 3 CheckMate 816 trial (NCT02998528), which evaluated neoadjuvant nivolumab (anti-programmed death protein 1) plus chemotherapy in patients with resectable lung cancer. RVT, regression and necrosis were quantified (0-100%) in PT and LNs using a pan-tumor scoring system and tested for association with EFS in a prespecified exploratory analysis. Regardless of LN involvement, EFS improved with 0% versus >0% RVT-PT (hazard ratio = 0.18). RVT-PT predicted EFS for nivolumab plus chemotherapy (area under the curve = 0.74); 2-year EFS rates were 90%, 60%, 57% and 39% for patients with 0-5%, >5-30%, >30-80% and >80% RVT, respectively. Each 1% RVT associated with a 0.017 hazard ratio increase for EFS. Combining pathologic response from PT and LNs helped differentiate outcomes. When compared with radiographic response and circulating tumor DNA clearance, %RVT best approximated EFS. These findings support pathologic response as an emerging survival surrogate. Further assessment of the full spectrum of %RVT in lung cancer and other tumor types is warranted. ClinicalTrials.gov registration: NCT02998528 .

Improved bladder cancer antitumor efficacy with a recombinant BCG that releases a STING agonist.

Despite the introduction of several new agents for the treatment of bladder cancer (BC), intravesical BCG remains a first line agent for the management of non-muscle invasive bladder cancer. In this study we evaluated the antitumor efficacy in animal models of BC of a recombinant BCG known as BCG-disA-OE that releases the small molecule STING agonist c-di-AMP. We found that compared to wild-type BCG (BCG-WT), in both the orthotopic, carcinogen-induced rat MNU model and the heterotopic syngeneic mouse MB-49 model BCG-disA-OE afforded improved antitumor efficacy. A mouse safety evaluation further revealed that BCG-disA-OE proliferated to lesser degree than BCG-WT in BALB/c mice and displayed reduced lethality in SCID mice. To probe the mechanisms that may underlie these effects, we found that BCG-disA-OE was more potent than BCG-WT in eliciting IFN-ß release by exposed macrophages, in reprogramming myeloid cell subsets towards an M1-like proinflammatory phenotypes, inducing epigenetic activation marks in proinflammatory cytokine promoters, and in shifting monocyte metabolomic profiles towards glycolysis. Many of the parameters elevated in cells exposed to BCG-disA-OE are associated with BCG-mediated trained innate immunity suggesting that STING agonist overexpression may enhance trained immunity. These results indicate that modifying BCG to release high levels of proinflammatory PAMP molecules such as the STING agonist c-di-AMP can enhance antitumor efficacy in bladder cancer.

A Phase 1 Trial of Durvalumab in Combination with Bacillus Calmette-Guerin (BCG) or External Beam Radiation Therapy in Patients with BCG-unresponsive Non-muscle-Invasive Bladder Cancer: The Hoosier Cancer Research Network GU16-243 ADAPT-BLADDER Study.

BACKGROUND: Novel treatments and trial designs remain a high priority for bacillus Calmette-Guerin (BCG)-unresponsive non-muscle-invasive bladder cancer (NMIBC) patients. OBJECTIVE: To evaluate the safety and preliminary efficacy of anti-PD-L1 directed therapy with durvalumab (D), durvalumab plus BCG (D + BCG), and durvalumab plus external beam radiation therapy (D + EBRT). DESIGN, SETTING, AND PARTICIPANTS: A multicenter phase 1 trial was conducted at community and academic sites. INTERVENTION: Patients received 1120 mg of D intravenously every 3 wk for eight cycles. D + BCG patients also received full-dose intravesical BCG weekly for 6 wk with BCG maintenance recommended. D + EBRT patients received concurrent EBRT (6 Gy × 3 in cycle 1 only). OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Post-treatment cystoscopy and urine cytology were performed at 3 and 6 -mo, with bladder biopsies required at the 6-mo evaluation. The recommended phase 2 dose (RP2D) for each regimen was the primary endpoint. Secondary endpoints included toxicity profiles and complete response (CR) rates. RESULTS AND LIMITATIONS: Twenty-eight patients were treated in the D (n = 3), D + BCG (n = 13), and D + EBRT (n = 12) cohorts. Full-dose D, full-dose BCG, and 6 Gy fractions × 3 were determined as the RP2Ds. One patient (4%) experienced a grade 3 dose limiting toxicity event of autoimmune hepatitis. The 3-mo CR occurred in 64% of all patients and in 33%, 85%, and 50% within the D, D + BCG, and D + EBRT cohorts, respectively. Twelve-month CRs were achieved in 46% of all patients and in 73% of D + BCG and 33% of D + EBRT patients. CONCLUSIONS: D combined with intravesical BCG or EBRT proved feasible and safe in BCG-unresponsive NMIBC patients. Encouraging preliminary efficacy justifies further study of combination therapy approaches. PATIENT SUMMARY: Durvalumab combination therapy can be safely administered to non-muscle-invasive bladder cancer patients with the goal of increasing durable response rates.

Integrative genomics identifies DSCR1 (RCAN1) as a novel NFAT-dependent mediator of phenotypic modulation in vascular smooth muscle cells.

Vascular smooth muscle cells (SMCs) display remarkable phenotypic plasticity in response to environmental cues. The nuclear factor of activated T-cells (NFAT) family of transcription factors plays a critical role in vascular pathology. However, known functional NFAT gene targets in vascular SMCs are currently limited. Publicly available whole-genome expression array data sets were analyzed to identify differentially expressed genes in human, mouse and rat SMCs. Comparison between vehicle and phenotypic modulatory stimuli identified 63 species-conserved, upregulated genes. Integration of the 63 upregulated genes with an in silico NFAT-ome (a species-conserved list of gene promoters containing at least one NFAT binding site) identified 18 putative NFAT-dependent genes. Further intersection of these 18 potential NFAT target genes with a mouse in vivo vascular injury microarray identified four putative NFAT-dependent, injury-responsive genes. In vitro validations substantiated the NFAT-dependent role of Cyclooxygenase 2 (COX2/PTGS2) in SMC phenotypic modulation and uncovered Down Syndrome Candidate Region 1 (DSCR1/RCAN1) as a novel NFAT target gene in SMCs. We show that induction of DSCR1 inhibits calcineurin/NFAT signaling through a negative feedback mechanism; DSCR1 overexpression attenuates NFAT transcriptional activity and COX2 protein expression, whereas knockdown of endogenous DSCR1 enhances NFAT transcriptional activity. Our integrative genomics approach illustrates how the combination of publicly available gene expression arrays, computational databases and empirical research methods can answer specific questions in any cell type for a transcriptional network of interest. Herein, we report DSCR1 as a novel NFAT-dependent, injury-inducible, early gene that may serve to negatively regulate SMC phenotypic switching.

Re-engineered BCG overexpressing cyclic di-AMP augments trained immunity and exhibits improved efficacy against bladder cancer.

In addition to its role as a TB vaccine, BCG has been shown to elicit heterologous protection against many other pathogens including viruses through a process termed trained immunity. Despite its potential as a broadly protective vaccine, little has been done to determine if BCG-mediated trained immunity levels can be optimized. Here we re-engineer BCG to express high levels of c-di-AMP, a PAMP recognized by stimulator of interferon genes (STING). We find that BCG overexpressing c-di-AMP elicits more potent signatures of trained immunity including higher pro-inflammatory cytokine responses, greater myeloid cell reprogramming toward inflammatory and activated states, and enhances epigenetic and metabolomic changes. In a model of bladder cancer, we also show that re-engineered BCG induces trained immunity and improved functionality. These results indicate that trained immunity levels and antitumor efficacy may be increased by modifying BCG to express higher levels of key PAMP molecules.

Combined Next-generation Sequencing and Flow Cytometry Analysis for an Anti-PD-L1 Partial Responder over Time: An Exploration of Mechanisms of PD-L1 Activity and Resistance in Bladder Cancer.

Anti-PD-L1/PD-1 immunotherapy has improved survival for certain patients with metastatic urothelial carcinoma. However, the mechanisms of resistance to these agents have not been fully elucidated. We report the first combined analysis using RNA sequencing, whole-exome sequencing (WES), and flow cytometry of multiple tumor specimens over a 5-yr period for a patient undergoing anti-PD-L1 therapy. Initial sensitivity to anti-PD-L1 immunotherapy was associated with conversion to a basal molecular subtype and a rising tumor mutational burden. We found that as the tumor became more resistant to anti-PD-L1, the proportion of regulatory T cells and CD8+ T cells expressing alternative immune checkpoints including CTLA-4, TIM-3, and LAG-3 increased. This suggests that alternative immune checkpoint upregulation may be one form of anti-PD-L1 resistance in urothelial carcinoma. These data support the concept of combined immune checkpoint blockade for urothelial carcinoma, a concept that is being evaluated in prospective clinical trials. PATIENT SUMMARY: In this study we characterized how a patient with metastatic urothelial cancer became resistant to anti-PD-L1 immunotherapy. By tracking changes in protein and gene expression over time, we found that as urothelial carcinoma becomes resistant to PD-L1 blockade, additional immune checkpoints may be upregulated. These data support the concept of combined checkpoint blockade for urothelial carcinoma.

Pathology Residency Program Special Expertise Tracks Meet the Needs of an Evolving Field.

Pathologists who enter the workforce must have a diverse skill set beyond that of clinical diagnostics alone. Anticipating this need, the Johns Hopkins Pathology Residency Program developed Special Expertise Tracks to enhance training in relevant subspecialty domains. Using a combination of discussions and surveys, we assessed: (1) our current resident curriculum; (2) perceived curricular strengths and needs; (3) resident career preferences and ultimate career paths; (4) perceived barriers to implementing an advanced elective curriculum; and (5) available departmental/institutional resources. Additionally, we utilized the Accreditation Council for Graduate Medical Education Pathology Milestones as a curricular guide. Six professional residency training Special Expertise Tracks were established: Education, Physician-Scientist Research, Informatics, Quality Improvement/Quality Assurance/Value-Based Care, Health Policy/Hospital Management and Global Health. After implementation in 2017, the Education track has had 4 residents complete the curriculum successfully; the Physician-Scientist Research track has had 2 residents and the Informatics and Global Health tracks have each had one resident successfully complete their respective curricula. Currently, 5 residents are pursuing the Education track, one is pursuing the Physician-Scientist Research track, one is pursuing the Informatics track, and 2 residents are pursuing the Global Health track. Five residents have completed long-term projects including developing several e-learning modules, an online free digital cytopathology atlas, peer-reviewed articles, book chapters, and books. The Johns Hopkins Pathology Resident Special Expertise Track program provides pathology residents an opportunity to gain meaningful experience and additional skills tailored to their individual career interests.

Pathologic response in patients receiving neoadjuvant chemotherapy for muscle-invasive bladder cancer: Is therapeutic effect owing to chemotherapy or TURBT?

PURPOSE: We estimated the proportion of patients who received neoadjuvant chemotherapy for muscle-invasive bladder cancer whose tumors were downstaged by transurethral resection. MATERIALS AND METHODS: We identified patients with cT2 N0 urothelial carcinoma who underwent cystectomy at our institution from 2005 to 2014-overall, 139 underwent transurethral resection without chemotherapy, and 146 underwent transurethral resection with chemotherapy. Pathologic response was defined as

Correlation of 99mTc-sestamibi uptake in renal masses with mitochondrial content and multi-drug resistance pump expression.

BACKGROUND: 99mTc-sestamibi single-photon emission computed tomography/computed tomography (SPECT/CT) has recently been explored for the characterization of indeterminate renal masses. As judged by increased intra-tumoral radiotracer uptake, we have previously reported the excellent diagnostic performance characteristics of this test for identifying benign/indolent oncocytomas and hybrid oncocytic/chromophobe tumors (HOCTs). In this study, we investigated potential molecular mechanisms underlying the discriminatory ability of 99mTc-sestamibi SPECT/CT for renal masses. Fifty renal masses imaged with 99mTc-sestamibi SPECT/CT prior to surgical resection were evaluated by immunohistochemistry for mitochondrial content and expression of the multi-drug resistance pump 1 (MDR1/P-gp). Immunohistochemical staining was scored semi-quantitatively, and results were compared across renal tumor histologies and correlated with 99mTc-sestamibi uptake. RESULTS: In total, 6/6 (100%) and 2/2 (100%) HOCTs demonstrated strong mitochondrial content staining combined with low MDR1 staining. Clear cell renal cell carcinoma showed an opposite pattern with the majority having low mitochondrial (14/26, 54%) and high MDR1 staining (18/26, 69%). Other tumor types were more variable in staining pattern, although the staining pattern reliably predicted 99mTc-sestamibi uptake in almost all tumors except chromophobe renal cell carcinoma. CONCLUSIONS: Our findings confirm that renal tumors with high mitochondrial content and relatively low MDR pump expression activity accumulate 99mTc-sestamibi and allow for the accurate diagnosis of the benign/indolent tumor class that includes oncocytomas and HOCTs. For masses in which MDR activity outweighs the presence of mitochondria, the tumors appear cold on 99mTc-sestamibi SPECT/CT, allowing for high confidence in the diagnosis of renal cell carcinoma.

Intravesical BCG Induces CD4+ T-Cell Expansion in an Immune Competent Model of Bladder Cancer.

Intravesical bacillus Calmette-Guérin (BCG) immunotherapy is the standard of care in treating non-muscle-invasive bladder cancer, yet its mechanism of action remains elusive. Both innate and adaptive immune responses have been implicated in BCG activity. Although prior research has indirectly demonstrated the importance of T cells and shown a rise in CD4+ T cells in bladder tissue after BCG, T-cell subpopulations have not been fully characterized. We investigated the relationship between effector and regulatory T cells in an immune competent, clinically relevant rodent model of bladder cancer. Our data demonstrate that cancer progression in the N-methyl-N-nitrosourea (MNU) rat model of bladder cancer was characterized by a decline in the CD8/FoxP3 ratio, consistent with decreased adaptive immunity. In contrast, treatment with intravesical BCG led to a large, transient rise in the CD4+ T-cell population in the urothelium and was both more effective and immunogenic compared with intravesical chemotherapy. Whole-transcriptome expression profiling of posttreatment intravesical CD4+ and CD8+ T cells revealed minimal differences in gene expression after BCG treatment. Together, our results suggest that although BCG induces T-cell recruitment to the bladder, the T-cell phenotype does not markedly change, implying that combining T-cell-activating agents with BCG might improve clinical activity. Cancer Immunol Res; 5(7); 594-603. ©2017 AACR.

Defining the Added Value of 99mTc-MIBI SPECT/CT to Conventional Cross-Sectional Imaging in the Characterization of Enhancing Solid Renal Masses.

PURPOSE: This study investigates whether the addition of preoperative Tc-MIBI SPECT/CT can increase the degree of diagnostic confidence in the differentiation of benign from malignant enhancing renal masses. PATIENTS AND METHODS: Patients were recruited as part of an institutional review board-approved prospective clinical trial. Forty-eight patients with clinical stage T1 solid renal masses who underwent a Tc-MIBI SPECT/CT before partial or radical nephrectomy were evaluated. Conventional CT and MRI, which were approximately performed within 8 weeks before Tc-MIBI, were retrospectively retrieved. Based on a 5-point scale (1 = definitely benign, 5 = definitely malignant), 2 blinded readers recorded their degree of confidence for each lesion using conventional imaging before and after reviewing the Tc-MIBI uptake ratios. Surgical pathology was considered as the reference standard. RESULTS: Additional review of Tc-MIBI SPECT/CT uptake ratios increased diagnostic confidence in the differentiation of solid renal masses in 14/48 lesions (29.2%). In 9 lesions, the addition of Tc-MIBI changed the initial confidence levels of malignancy toward benign diagnosis. Postsurgical pathology confirmed the diagnosis of benign histology (oncocytoma/hybrid oncocytic-chromophobe tumors) in 7 and chromophobe renal cell carcinoma (behave as indolent) in 2 of these lesions. Tc-MIBI increased the confidence level toward malignancy in 5 cases; all were confirmed as RCC on surgical pathology. The area under the receiver operative characteristic curve was 0.60 for conventional imaging alone and 0.85 after reviewing Tc-MIBI (P for difference = 0.03). CONCLUSIONS: Preoperative Tc-MIBI SPECT/CT enhances the performance of conventional imaging, improving the characterization of benign histologies and lowering the possibility of misclassification.

Accuracy of urethral frozen section during radical cystectomy for bladder cancer.

OBJECTIVE: Our objective was to determine the accuracy of urethral frozen section (FS) by analyzing our clinical experience. MATERIALS AND METHODS: A total of 298 patients undergoing radical cystectomy for bladder cancer with benign or malignant urethral FS were identified between 2000 and 2012. Urethral FS were compared with rereviewed FS to calculate the positive and negative predictive values of the FS. To assess the ability of the positive FS to be cleared with further sampling/resection, FS were then compared with the final urethral margin. The cases of positive urethral FS were then specifically analyzed to assess rates of urethral recurrence and survival. RESULTS: All negative FS were confirmed to be negative on FS rereview and on final pathology, resulting in a NPV of 100%. Urethral FS were positive in 28 (8.7%) patients, of whom 2 (7%) were negative on FS rereview, yielding a positive predictive value of 93%. Both false positives were because of contamination of detached cancer from the bladder being present in the FS. After additional sampling/resection, the final margin was negative in 13 (46%) patients. CONCLUSIONS: A negative urethral FS reliably identifies individuals for whom urethrectomy is unnecessary and provides robust information for decision-making regarding the safety of orthotopic reconstruction. Nearly half of the patients with a positive FS were ultimately determined to have a negative final margin. Accordingly, we recommend that surgeons and pathologists discuss positive FS findings at the time of surgery and consider whether additional tissue should be analyzed in real time.

DNA microarray analysis of neonatal mouse lung connects regulation of KDR with dexamethasone-induced inhibition of alveolar formation.

Treatment of newborn mice with dexamethasone (Dex) inhibits the subdivision of lung saccules to form alveoli; treatment with all-trans retinoic acid (RA) prevents this inhibition of septation. To better understand the early molecular signals responsible for the effects of Dex and RA, Affymetrix gene profiling was done on RNA isolated from 4-day-old mice after treatment with 1) diluent, 2) RA (1 mg/kg), 3) Dex (0.7 microg/pup), or 4) RA + Dex. Each sample was assayed in duplicate on U74Av2 GeneChips. Data were analyzed with Affymetrix suite 5.0, corrected for saturation, and evaluated with GeneSpring 5.1 software. Stringent filtering of data by the global error model and condition-to-condition comparisons was used to identify 46 genes demonstrating significantly different expression between the lungs of Dex- and RA + Dex-treated mice. A query of the gene ontology database revealed that the major biological processes affected by treatment with Dex and RA were cell growth/maintenance and cellular communication. On the basis of microarray data analysis, we hypothesize that Dex-induced inhibition of septation is associated with a block in angiogenesis due to downregulation of the kinase domain receptor (KDR), also known as VEGF receptor-2 and fetal liver kinase, and that the downregulation of KDR is prevented by treatment with RA.