Serial Biopsy Specimens Research Articles

Molecular profiling of serial liquid biopsy specimens utilizing cell free DNA (cfDNA) and circulating tumor cells (CTCs) in TBCRC041: A phase II study of alisertib in endocrine resistant metastatic breast cancer (MBC).

1037 Background: TBCRC041, a randomized phase 2 trial of alisertib (A) ± fulvestrant (F) in women with HR+/HER2- MBC demonstrated promising clinical activity for alisertib (1). Interrogation of baseline (PreC1) and end of cycle 1 (EOC1) cfDNA and CTCs may identify biomarkers associated with response to A±F. Methods: Plasma cfDNA was sequenced using the Guardant INFINITY platform, which includes genomic and methylated tumor fraction (mTF) analysis. Pathogenic variants were determined using publicly available databases and Mayo Clinic annotation pipelines. CTCs were identified as nucleated, EpCAM+/ cytokeratin+/ CD45- cells and assessed for ER/HER2 staining (RareCyte, Seattle). PFS was defined as the time from randomization to progression or death. EOC1-PFS differs from PFS as time starts at C2D1. Stratified log-rank tests and conditional Cox modeling were used to assess whether PFS or EOC1-PFS differed by mutations in ESR1 or PIK3CA, pre-CTC ≥ 5, or mTF>1%. Results: Of the 91 eligible pts, 84 progressed, and 73 were deceased. In the 86 pts with PreC1-cfDNA results, variants were identified in ESR1 (42; 48.8%), PIK3CA (36; 41.9%), AKT1 (7; 8.1%), PTEN(7; 8.1%), BRCA1 (4; 4.7%), and BRCA2 (7; 8.1%). Pts with a PIK3CAmut had significantly decreased PFS (HR 1.8; 95%CI: 1.1 -2.9, p=0.014) but not by ESR1mut status (p=0.583). PreC1 and EOC1-mTF were determined for 78 and 76 pts, respectively. PreC1-mTF was 0-1% in 21; 1.1-10.0% in 27; and 10.1-100% in 30. mTF levels remained between 0-1% in 17 pts fell to 0-1% in 11 pts, and was 0-1% in 5 pts without a preC1-mTF determination. PFS did not differ with respect to PreC1-mTF (p=0.210), but EOC1-PFS was worse in pts with mTF >1% (HR 3.0; 95%CI: 1.6 -5.2, p<0.001). PreC1 and EOC1-CTC were determined for 78 and 76 pts. 36 (46.2%) pts had a PreC1 CTC count ≥ 5. EOC1-CTC levels remained at (n=17) or increased to (n=1) ≥ 5 and was ≥ 5 in 2 pts without a preC1-CTC determination. EOC1-PFS was not significantly different for EOC1-CTC ≥ 5 (p=0.065), but was significantly decreased in pts with PreC1-CTC≥5 (HR=1.8; 95%CI: 1.1-3.0; p=0.018). Broader cfDNA NGS panel and CTC-derived ER/HER2 staining results will be presented at the meeting. Conclusions: Among patients receiving A±F, PFS varied based on the PIK3CAmutation status. cfDNA and CTC assessment PreC1 and EOC1 provided complementary information. Further validation in larger datasets is needed. 1. JAMA Oncol 2023;9:815. Clinical trial information: NCT02860000 .

Combining amino acid PET and MRI imaging increases accuracy to define malignant areas in adult glioma

Accurate determination of the extent and grade of adult-type diffuse gliomas is critical to patient management. In clinical practice, contrast-enhancing areas of diffuse gliomas in magnetic resonance imaging (MRI) sequences are usually used to target biopsy, surgery, and radiation therapy, but there can be discrepancies between these areas and the actual tumor extent. Here we show that adding 18F-fluoro-ethyl-tyrosine positron emission tomography (FET-PET) to MRI sequences accurately locates the most malignant areas of contrast-enhancing gliomas, potentially impacting subsequent management and outcomes. We present a prospective analysis of over 300 serial biopsy specimens from 23 patients with contrast-enhancing adult-type diffuse gliomas using a hybrid PET-MRI scanner to compare T2-weighted and contrast-enhancing MRI images with FET-PET. In all cases, we observe and confirm high FET uptake in early PET acquisitions (5–15 min after 18F-FET administration) outside areas of contrast enhancement on MRI, indicative of high-grade glioma. In 30% cases, inclusion of FET-positive sites changes the biopsy result to a higher tumor grade.

Histological Analysis of Serial Renal Biopsy Specimens from Children with Immunoglobulin A Nephropathy Not Treated with Immunosuppressants.

Although pediatric immunoglobulin A nephropathy (IgAN) is considered to have a good prognosis, few studies have investigated histological changes over time in IgAN. Serial renal biopsies were performed during the course of the disease and histological changes were observed in patients who did not receive immunosuppressive treatment. To our knowledge, this is the first report of two or more histological evaluations of renal biopsies from patients with pediatric IgAN who did not receive immunosuppressive drugs. Forty-two patients with biopsy-proven IgAN who did not receive immunosuppressive agents and underwent serial renal biopsies were followed in our hospital between 1990 and 2003. This retrospective study evaluated findings from renal biopsy specimens and medical records. Analysis of histological findings showed that 19 of 42 patients improved and 16 showed exacerbation of mesangial proliferation. Seven patients showed no obvious histological changes. Of the improved cases, 11 showed spreading of chronic lesions, and there was a significant difference between patients with and without segmental glomerular sclerosis or adhesion at the first biopsy. Of the exacerbated cases, only 5 of 16 patients showed strong active lesions at the first renal biopsy. Histological changes were investigated in pediatric IgAN patients not receiving immunosuppressive treatment. The results suggest that, even if mesangial hypercellularity improves, chronic lesions may spread during the natural history of the disease. Predicting histological changes by using findings from renal biopsies performed early after onset is difficult; therefore, patients should be carefully followed.

Abstract P4-09-10: Spatial protein and RNA expression in tumor, immune and stromal cells in BRCA1/2 mutated metastatic breast cancer

Abstract Background: Spatial transcriptomics and proteomics are cutting-edge techniques that allow for quantification of RNA and protein expression within separate cell populations or regions of interest in a tissue biopsy specimen. We aimed to independently characterize the tumor cells and immune infiltrate in metastatic site biopsy specimens from patients with somatic or germline BRCA1/2 mutations. Methods: We performed spatial transcriptomics and proteomics using the Nanostring GeoMx Digital Spatial Profiler (DSP) on 9 formalin-fixed paraffin-embedded (FFPE) tissue biopsy specimens from 5 patients with metastatic breast cancer and a known somatic or germline BRCA1/2 mutation. Each patient had 1-3 separate metastatic site tissue biopsy specimens. Cell populations of interest were delineated with fluorescently labeled antibodies to pan-cytokeratin on tumor cells, CD45 of immune cells, and a nuclear stain. On each sample, 4-5 regions of interest (ROIs) were selected near the interface of the metastatic tumor deposit and surrounding normal tissue. Each ROI was segmented into 3 areas of interest (AOIs): pan-cytokeratin-positive tumor cells, CD45-positive immune cells, and the remaining stromal cells negative for both pan-cytokeratin and CD45. Data was background corrected and normalized with internal negative and positive internal controls. Relative abundance of immune cell subtypes was derived from CD45+ cell gene expression data using the TIMER algorithm. Results: The expression of 57 proteins and 84 RNAs was quantified in each of the 396 AOIs. Separate analysis of CD45+ cells, pan-cytokeratin cells, and stromal cell populations confirmed expected differential expression of immune, tumor and stromal markers, respectively. Protein expression of the estrogen receptor (ER) and progesterone receptor (PR) on pan-cytokeratin-positive tumor cells using the DSP correlated with pathologist reviewed immunohistochemistry (IHC) for ER and PR. Significant differential expression was found within each cell population between hormone receptor (HR) positive and negative cohorts, including enrichment for S100B in HR negative samples. Comparison will be shown for expression of 14 overlapping genes/proteins. Immune cell compartment gene expression deconvolution showed heterogeneity in the relative proportion of infiltrating immune cell subtypes between samples and also between ROIs within a sample. Changes in gene and protein expression from a patient with 3 serial biopsy specimens can be tracked with substantial heterogeneity across distinct AOIs within individual specimens and across metastatic sites.. Conclusions: Spatial transcriptomics and proteomics is feasible for concurrent, relative quantification of protein and gene expression in tumor, immune, and stromal cells in FFPE tissue biopsies from patients with metastatic breast cancer harboring germline or somatic BRCA1 or BRCA2 mutations. Heterogeneity within individual samples and across serial time points offers opportunity for methods development in the analyses and interpretation of spatial transcriptomic/proteomic data. Citation Format: Katharine A. Collier, Katherine Miller, Zaibo Li, Jesse Westfall, David Tallman, Mark Vater, Gabriel Tinoco, Elaine Mardis, Daniel Stover. Spatial protein and RNA expression in tumor, immune and stromal cells in BRCA1/2 mutated metastatic breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-09-10.

CD44 as a pathological marker for the early detection of calcineurin inhibitor-induced nephrotoxicity post kidney transplantation.

Long-term calcineurin inhibitor (CNI) administration causes irreversible nephrotoxicity. Therefore, early CNI-induced nephrotoxicity detection is necessary for patients who will need long-term CNI administration. There is no pathological indicator for early CNI-induced nephrotoxicity. Here, serial protocol kidney biopsy specimens from five kidney-transplant patients with severe CNI-induced nephrotoxicity were examined. We observed that the increase in CD44 expression in glomerular parietal epithelial cells (PECs) preceded the chronic pathological changes of CNI-induced nephrotoxicity such as tubular atrophy/interstitial fibrosis, arterial hyaline thickening, and focal segmental glomerulosclerosis (FSGS). This result suggests that CD44-positive PECs have pivotal roles in FSGS development in human CNI-induced nephrotoxicity as well as rodent models. CD44 could be useful as a pathological marker for early CNI-induced nephrotoxicity detection post kidney transplantation.

Breast implant-associated anaplastic large cell lymphoma: clinical follow-up and analysis of sequential pathologic specimens of untreated patients shows persistent or progressive disease

Breast implant-associated anaplastic large cell lymphoma (ALCL) is a distinctive type of T-cell lymphoma that arises around textured-surface breast implants. In a subset of patients, this disease can involve surrounding tissues, spread to regional lymph nodes, and rarely metastasize to distant sites. The aim of this study was to assess sequential pathologic specimens from patients with breast implant-associated ALCL to better understand the natural history of early-stage disease. To achieve this goal, we searched our files for patients who had breast implant-associated ALCL and who had undergone earlier surgical intervention with assessment of biopsy or cytologic specimens. We then focused on the patient subset in whom a definitive diagnosis was not established, and patients did not receive current standard-of-care therapy at that time. We identified a study group of ten patients with breast implant-associated ALCL in whom pathologic specimens were collected 0.5 to 4 years before a definitive diagnosis was established. A comparison of these serial biopsy specimens showed persistent disease without change in pathologic stage in three patients, progression in five patients, and persistence versus progression in two patients. Eventually, six patients underwent implant removal with complete capsulectomy and four underwent partial capsulectomy. Seven patients also received chemotherapy because of invasive disease, three of whom also received radiation therapy, two brentuximab vedotin after chemotherapy failure, and one allogeneic stem cell transplant. Eight patients achieved complete remission and two had partial remission after definitive therapy. At time of last follow-up, six patients were alive without disease, one had evidence of disease, one died of disease, and two patients died of unrelated cancers. In summary, this analysis of sequential specimens from patients with breast implant-associated ALCL suggests these neoplasms persist or progress over time if not treated with standard-of-care therapy.

Abstract 5593: Inhibition of A2AR induces anti-tumor immunity alone and in combination with anti-PD-L1 in preclinical and clinical studies

Abstract Adenosine signaling via A2A receptor (A2AR) on immune cells suppresses anti-tumor immunity and limits the efficacy of immunotherapy, chemotherapy, CAR-T, and vaccines. CPI-444 is a potent and selective oral A2AR antagonist. Daily treatment of mice with CPI-444 led to dose-dependent inhibition of tumor growth in multiple syngeneic tumor models. Combining CPI-444 with anti-PD-L1 treatment synergistically eliminated tumors in up to 90% of treated mice, including restoration of immune responses in models that are poorly responsive to anti-PD-1 or anti-PD-L1 monotherapy. We have initiated a Phase 1/1b clinical trial to examine safety, tolerability, biomarkers, and preliminary efficacy of CPI-444 as a single agent and in combination with the anti-PD-L1 antibody, atezolizumab, in patients with non-small cell lung, melanoma, renal, triple negative breast, and other (bladder, prostate, head and neck, colorectal) tumors. Step 1 of the trial focused on determining the optimal dose and schedule for CPI-444; Step 2 is currently evaluating the efficacy of optimal CPI-444 dosing alone and with atezolizumab. In 48 patients treated in Step 1, CPI-444 was well tolerated with 1 Grade 3 or 4 treatment related adverse events. Preliminary evidence of clinical activity was observed in patients treated with single agent CPI-444, including patients who previously failed anti-PD-1 therapy. A pCREB-based pharmacodyamic assay showed that 100 mg, BID dose of CPI-444 resulted in a complete, sustained inhibition of A2AR on circulating immune cells. CPI-444 treatment alone or in combination with atezolizumab resulted in increased frequency of circulating CD8+PD-1+ cells and memory/effector subsets of CD4+ and CD8+ T cells. Substantial changes in TCR repertoire (Morisita Index <0.9) were observed in both anti-PD-1 native and refractory patients and correlated to patient responses. CD73 expression, CD8+ infiltration, TREG distribution, and gene expression signatures are currently being evaluated in serial tumor biopsy specimens and will be presented. In total, this shows that CPI-444 is well tolerated in cancer patients, exhibits functional inhibition of adenosine signaling, and treatment is associated with activation of anti-tumor immunity and clinical activity. Citation Format: Stephen Willingham, Andrew Hotson, Po Ho, Carmen Choy, Kim Walter, Erik Yusko, Sharon Benzeno, Ginna Laport, Richard Miller, Ian McCaffery. Inhibition of A2AR induces anti-tumor immunity alone and in combination with anti-PD-L1 in preclinical and clinical studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5593. doi:10.1158/1538-7445.AM2017-5593

Effect of Macroanatomic Bone Type and Estrogen Loss on Osteocyte Lacunar Properties in Healthy Adult Women.

This is the first study to examine clinical human bone specimens by three-dimensional imaging to characterize osteocyte lacunar properties as a function of macroanatomic bone type and estrogen loss. We applied laboratory-based instrumentation [3D X-ray microscope (3DXRM), MicroXCT-200; Carl Zeiss/Xradia, Inc.] that reaches the same resolution as synchrotron microscopy. We used serial transiliac bone biopsy specimens to examine the effect of macroanatomic bone type and estrogen status on osteocyte lacunar properties. These properties include lacunar size (volume, axes lengths of the ellipsoidal lacunar voids), distribution (density, average near-neighbor lacunar distance), and shape factors (sphericity ratio, average eigenvalues, degree of equancy, elongation, and flatness) in both cortical and trabecular bone tissue. The lacunar properties (volume, surface area, density, near-neighbor distance, etc.) and the shape factors (E1, L1, L2, degree of equancy, degree of elongation) were different between cortical and trabecular bone regardless of estrogen status. In cortical bone and trabecular nodes, the lacunar void volume and surface area were either smaller or tended to be smaller in postmenopausal as compared to premenopausal women. The void volume-to-bone volume ratio of cortical bone showed declining trends with estrogen loss. While there were differences between trabecular and cortical bone tissue, the lacunar void sphericity ratio for trabecular struts shows decreasing trends in postmenopausal women. These data suggest that using 3DXRM can provide new insight into osteocyte lacunar properties in transiliac bone biopsies from patients with various skeletal disease/conditions and pharmaceutical treatments.

Hepatic Infiltrates in Operational Tolerant Patients After Liver Transplantation Show Enrichment of Regulatory T Cells Before Proinflammatory Genes Are Downregulated.

Immunosuppression can be discontinued from selected and stable patients after liver transplantation resulting in spontaneous operational tolerance (SOT), although the underlying mechanisms remain elusive. Thus, we analyzed serial liver biopsy specimens from adult liver recipients enrolled in a prospective multicenter immunosuppression withdrawal trial that used immunophenotyping and transcriptional profiling. Liver specimens were collected before the initiation of weaning, at the time of rejection, or at 1 and 3 years after complete drug discontinuation. Unexpectedly, the tolerated grafts developed portal tract expansion with increased T cell infiltration after immunosuppression withdrawal. This was associated with transient and preferential accumulation of CD4(+) FOXP3(+) cells and a trend toward upregulation of immune activation and regulatory genes, without signs of rejection. At the same time, no markers of endothelial damage or activation were noted. Portal infiltrates persisted at 3 years but were characterized by decreased expression of genes associated with chronic immunological damage. Further, SOT was not associated with a progressive liver fibrosis up to 5 years. These data suggest that SOT involves several mechanisms: a long-lasting local immune cell persistence with a transient regulatory T cells accumulation followed by a downregulation of immune-activated genes over years. These results have important implications for designs and follow-up of weaning trials.

Abstract P5-13-04: A phase II neoadjuvant trial of MK-2206, an AKT inhibitor, in combination with anastrozole for clinical stage 2 or 3 PIK3CA mutant estrogen receptor positive HER2 negative (ER+HER2-) breast cancer (BC)

Abstract Background Activating mutations in PIK3CA occur in approximately 40% ER+BC. MK-2206 (M), a pan-AKT inhibitor, induced apoptosis of ER+ BC under estrogen deprivation in preclinical studies. We conducted this neoadjuvant trial to determine the pathologic complete response (pCR) rate of M plus anastrozole (A) for PIK3CA mutant (Mut) ER+ BC. Methods This single arm open label study of M+A used a 2-stage Simon phase II design (stage 1, n=16; stage 2, n=13, alpha=0.10, power=0.90) to test whether pCR rate <1% (based on historical data with A alone), against the alternative that pCR rate ≥15% in PIK3CA Mut ER+ BC. At least 1 pCR in stage 1 was required to proceed to stage 2. Eligible patients (pts) with clinical stage II or III ER+HER2- BC were pre-registered and proceeded to a research tumor biopsy for PIK3CA sequencing, followed by treatment with daily A monotherapy for 28 days (cycle 0). Pts with PIK3CA Mut BC were subsequently registered, underwent a second biopsy, and started M (150mg PO weekly) with daily A on cycle 1 day 1 (C1D1) for a maximum of four 28-day cycles followed by surgery. Goserelin was added for premenopausal pts. A tumor biopsy on C1D17, 17 days post the start of M, was performed. Those with C1D17 Ki67 >10% discontinued study treatment. pCR was defined as no invasive cancer in the breast and the lymph nodes. Tumor specimens collected at all timepoints are being analyzed for markers of proliferation, apoptosis, and PI3K pathway activity, gene expression microarray, intrinsic subtypes, and next generation sequencing of 83 genes. Results Of the 51 pts pre-registered, 35 pts did not register due to no PIK3CA mutation (n=22), inadequate specimen for testing (n=6), physician/pt decision (n=7). The remaining 16 pts (median age: 58, range: 40-77 years) received combination therapy. Three pts did not complete 4 cycles due to C1D17 Ki67 >10% (n=2) and intolerability (grade (Gr) 4 transaminase elevation in C1, n=1). Other severe toxicities possibly related to M included Gr 3 rash (25%) and pruritus (12.5%). Of the 13 pts completed study therapy and underwent surgery, all had residual disease in the breast and 7 also had positive nodes. Table 1 summarized changes in Ki67 during treatment. ComparisonsnAbsolute changes in Ki67 median (range)Wilcoxon signed rank p-valueC1D1 relative to pre-registration11-17.0% (-49.8 to 4.1%)0.0020C1D17 relative to pre-registration14-16.4% (-51.4 to 4.1%)0.0004C1D17 relative to C1D112-1.5% (-18.6 to 15.8%)0.9697C1D1, biopsy post 28 days of A alone; C1D17 biopsy post 17 days on combination therapy Although Ki67 levels post A monotherapy (C1D1) or M+A (C1D17) were significantly lower than that of pre-registration samples, Ki67 did not differ between C1D17 and C1D1 samples. Other correlative studies are ongoing and results will be presented. Conclusion Despite the small sample size, biomarker analysis on serial biopsy specimens demonstrated that M+A is unlikely to be more effective than A alone in PIK3CA Mut ER+ BC. This trial demonstrated the feasibility of genomic sequencing for pt selection and the value of a small, well-designed proof-of-principle neoadjuvant trial for the evaluation of targeted agents. Citation Format: Ma CX, Suman VJ, Goetz M, Northfelt D, Burkard M, Ademuyiwa F, Naughton M, Margenthaler J, Aft R, Gray R, Tavaarwerk A, Wilke L, Haddad T, Moynihan T, Loprinzi C, Hieken T, Hoog J, Guo Z, Han J, Vij K, Mardis E, Sanati S, Al-Kateb H, Doyle L, Erlichman C, Ellis MJ. A phase II neoadjuvant trial of MK-2206, an AKT inhibitor, in combination with anastrozole for clinical stage 2 or 3 PIK3CA mutant estrogen receptor positive HER2 negative (ER+HER2-) breast cancer (BC). [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-13-04.

Proteinuria rebound in IgA nephropathy associated with obesity-related glomerulopathy.

IgA nephropathy (IgAN), the most prevalent primary chronic glomerulonephritis worldwide, has three major risk factors: hypertension, proteinuria >1g/day, and severe renal lesions. Obesity also portends a poor prognosis. A Japanese boy with IgAN showed nephrotic syndrome at presentation. Pathological features resembled those of membranoproliferative glomerulonephritis (MPGN), although IgA deposition differed from MPGN and IgAN. Combination therapy improved renal lesions, but rebound deterioration of proteinuria occurred in this patient, who had marked obesity and hypertension. Serial kidney biopsy specimens were compatible with obesity-related glomerulopathy (ORG). Rebound proteinuria was apparently attributable to ORG rather than relapse and flaring up of IgAN.

Chronic Immune-Mediated Reaction Syndrome as the Cause of Late Graft Mortality in Living-Donor Liver Transplantation for Primary Biliary Cirrhosis

IntroductionFew studies to date have investigated the causes of late graft mortality after living-donor liver transplantation (LDLT) for primary biliary cirrhosis (PBC). Patients and MethodsFifty-five LDLTs for PBC were retrospectively reviewed. Factors prognostic of graft survival after LDLT were investigated, and histologic findings in patients with late graft loss were assessed. ResultsThe 1-, 5-, and 10-year cumulative graft survival rates were 85.1%, 82.5%, and 66.9%, respectively. Multivariate Cox regression analysis found that male donor and ≥4 HLA mismatches were independently associated with poor graft survival. Among the 13 grafts lost, 5 were lost >1 year after LDLT, including 1 each due to chronic rejection, veno-occlusive disease, and obliterative portal venopathy, and 2 to other causes. Pathologic reviews of the serial biopsy specimens and explanted grafts from these 5 patients, with graft rejections from “chronic immune-mediated reaction syndrome,” showed reciprocal changes over time. No patient died of recurrent PBC. ConclusionsMale donor and ≥4 HLA mismatches were independent factors associated with poor graft survival. Late graft mortality after LDLT for PBC in some patients was due to chronic immune-mediated reaction syndrome, including chronic rejection, veno-occlusive disease, and obliterative portal venopathy, but not to recurrent PBC.

Proteinuria Impairs Podocyte Regeneration by Sequestering Retinoic Acid

In CKD, the risk of kidney failure and death depends on the severity of proteinuria, which correlates with the extent of podocyte loss and glomerular scarring. We investigated whether proteinuria contributes directly to progressive glomerulosclerosis through the suppression of podocyte regeneration and found that individual components of proteinuria exert distinct effects on renal progenitor survival and differentiation toward a podocyte lineage. In particular, albumin prevented podocyte differentiation from human renal progenitors in vitro by sequestering retinoic acid, thus impairing retinoic acid response element (RARE)-mediated transcription of podocyte-specific genes. In mice with Adriamycin nephropathy, a model of human FSGS, blocking endogenous retinoic acid synthesis increased proteinuria and exacerbated glomerulosclerosis. This effect was related to a reduction in podocyte number, as validated through genetic podocyte labeling in NPHS2.Cre;mT/mG transgenic mice. In RARE-lacZ transgenic mice, albuminuria reduced retinoic acid bioavailability and impaired RARE activation in renal progenitors, inhibiting their differentiation into podocytes. Treatment with retinoic acid restored RARE activity and induced the expression of podocyte markers in renal progenitors, decreasing proteinuria and increasing podocyte number, as demonstrated in serial biopsy specimens. These results suggest that albumin loss through the damaged filtration barrier impairs podocyte regeneration by sequestering retinoic acid and promotes the generation of FSGS lesions. Our findings may explain why reducing proteinuria delays CKD progression and provide a biologic rationale for the clinical use of pharmacologic modulators to induce regression of glomerular diseases.

Predictive Role of Midtreatment Changes in Survivin, GSTP1, and Topoisomerase 2α Expressions for Pathologic Complete Response to Neoadjuvant Chemotherapy in Patients With Locally Advanced Breast Cancer

The primary aim of this study is to investigate the effect of change in the expression levels of survivin, glutathione-S-transferase P1 (GSTP1), and topoisomerase 2α (TOP2A) on the response to antracyclin-based and taxane-based neoadjuvant chemotherapy. This study included 32 locally advanced breast cancer patients. Tumoral expressions of survivin, TOP2A, and GSTP1 in serial biopsy specimens obtained before treatment, after sequential 4 cycles of doxorubicin+cyclophosphomide, and 4 cycles of docetaxel were analyzed by real-time polymerase chain reaction. Survivin expressions were additionally analyzed in serial blood samples. The pathologic complete response (pCR) rate and the overall response rate (clinical complete and partial) were 28% (n=9) and 91% (n=29), respectively. There were no statistically significant correlations between serial TOP2A expression levels and response. There was a nonsignificant trend toward an improved response rate with decreased survivin expression. A significant decrease in the GSTP1 expression level throughout treatment (P=0.014), which was also shown to be significantly correlated with a pCR (P=0.0001), was seen. Downregulation of GSTP1 after 4 cycles of anthracycline-based combination was independently associated with improved progression-free survival (P=0.01). Downregulation of GSTP1 is a significant predictor of pCR and improved progression-free survival during anthracycline-based and taxane-based neoadjuvant chemotherapy in patients with locally advanced breast cancer.

Abstract 2436: Fibroblast growth factor receptor blockade in prostate cancer bone metastases.

Abstract Castrate-resistant progression and bone metastases (BM) are hallmarks of advanced prostate cancer (PCa) for which there is no definitive cure. We previously reported that human PCa BM highly express fibroblast growth factor (FGF)-9 (J Clin Invest. 2008;118:2697). We now discovered that human PCa xenografts growing in the bone of mice induce bone cells to express components of the FGF/FGF receptor (FGFR) complex, Fgfr1, Fgfr2 and Fgf-2, as assessed by mouse-specific RT-PCR in bone bearing MDA PCa 118b tumors and in the contralateral bone (P<0.001). Similar results were found in 2 other human PCa xenografts. We then tested antitumor activity of dovitinib (Novartis), a receptor tyrosine kinase inhibitor (TKI) active against FGFR1-3 (IC50 <40 nM) in MDA PCa 118b bone tumors. We found that treated mice had significantly smaller tumors (P=0.016) than the controls had as assessed by magnetic resonance imaging. By human- and mouse-specific RT-PCR analyses we found that 7 days’ treatment reduces FGFR1 expression in tumor-bearing bones (P<0.01). Based on this evidence, a clinical study of dovitinib in men with castrate-resistant PCa and BM was initiated (Identifier: NCT00831792). In this study, serial bone marrow biopsy specimens are collected before and 8 weeks after treatment. To date, 29 of 40 patients have been enrolled. Preliminary results demonstrate tumor regression in BM and soft tissue metastases and progression-free survival of >20 weeks in a subset of patients (∼25%). Dovitinib inhibits vascular endothelial growth factor receptors (VEGFRs) 1-3, with IC50s (∼10 nM) similar to those of FGFR1-3. To assess the relative antitumor activity of FGFR blockade (with respect to VEGFR), we tested a TKI, AZD4547 (AstraZeneca), which inhibits FGFR1-3 (IC50s <3 nM) but not VEGFRs. We found that AZD4547 had antitumor activity against MDA PCa 118b bone tumors and reduces the volume of normal bones and of FGFR1 expression in tumor-associated bone cells (P<0.001). Accordingly, we observed a decrease in the blood levels of bone-specific alkaline phosphatase of men enrolled in the dovitinib trial, and this reduction was associated with the time men remained on treatment (P=0.035) (eg, those who benefited from treatment). No association was found between PSA blood levels or urinary N-telopeptides and response to therapy. Finally, we found that PCa cells and/or osteoblasts express FGFR1 in a fraction of human PCa BM (assessed by immunohistochemistry). We conclude that dovitinib therapy benefits a subset (∼25%) of men with PCa BM and that FGFR blockade contributes to this antitumor effect, at least partially, by blockade of FGFR-mediated PCa-bone cross talk. Ongoing studies focused on further understanding the mechanism of this antitumor activity will help identify likely responders and elucidate FGF signaling interaction with other candidate targets (eg, AR, c-Met, IGF, Src, VEGF). Citation Format: Xinhai Wan, Paul Corn, Jun Yang, Eleni Efstathiou, Elsa M. Li Ning Tapia, Fen Wang, Wallace McKeehan, Christopher Logothetis, Patricia Troncoso, Nora M. Navone. Fibroblast growth factor receptor blockade in prostate cancer bone metastases. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2436. doi:10.1158/1538-7445.AM2013-2436

Mucosal Plasma Cell Barrier Disruption During Intestine Transplant Rejection

Intestinal allograft mucosa undergoes repopulation with host immunocytes. However, critical changes within key immunocyte subsets are not known. To explain acute cellular rejection after intestine transplantation (ITx) on the basis of altered mucosal immunocytes, rejecting and rejection-free ITx allografts (n=17) were compared with genome-wide expression arrays. Cells identified by cell/lineage-specific genes were evaluated by immunohistochemistry. The corresponding phenotype and donor-specific alloreactivity were characterized in peripheral blood. Time-dependent changes in candidate cell(s) were evaluated in biopsies from an independent cohort of 12 children with ITx. Among 107 differentially expressed genes, three B-cell lineage-specific genes, CCR10, STAP1, and IGLL1, were down-regulated during ITx rejection and were selected for and achieved technical quantitative reverse transcription polymerase chain reaction replication. Down-regulation of the immunoglobulin (Ig)A+ plasma cell-specific CCR10 gene correlated with decreased mature mucosal CD138+ plasma cell numbers in corresponding biopsy specimens (r=0.761, P=0.006) and inversely correlated with enhanced alloreactivity of CD154+ T-cytotoxic memory cells (r=-0.56, P=0.031), which predict acute cellular rejection with high sensitivity. An independent cohort of serial biopsy specimens from 12 ITx recipients (1) confirmed relative CD138+ plasma cell depletion during rejection (P=0.042) and (2) showed increased IgG+-to-IgA+ cell ratios within 4 hr of reperfusion in rejection-prone allografts (P=0.037) and during ITx rejection (P=0.025), compared with rejection-free allografts. No differences existed late after ITx. Increased peripheral IgG+ CD27+ CD19+ memory B cells (P=0.004) were seen during ITx rejection in archived peripheral blood lymphocyte from test and replication cohorts. Protracted depletion of the mucosal CD138+ plasma cell barrier and early mucosal infiltration with memory IgG+ cells characterize the rejection-prone intestine allograft. Mucosal IgA+ plasma cell barrier reconstitution may augur resolution of ITx rejection.

Effect of omega-3 polyunsaturated fatty acids to reverse biopsy-proven parenteral nutrition-associated liver disease in adults

Parenteral nutrition-associated liver disease (PNALD) complicates the treatment of patients with short bowel syndrome (SBS). Some studies have shown omega-3 polyunsaturated fatty acids (ω-3 PUFAs) have favorable effects in treating PNALD in children. We, therefore, investigate the effects of ω-3 PUFAs supplemented intravenous lipid emulsion (ILE) on PNALD in adults. We performed an open-labeled study of a ω-3 PUFAs based ILE in 15 adults with SBS who developed cholestasis while receiving soybean oil-based ILE. Liver biopsies were performed before and after the initiation of ω-3 PUFAs to confirm the presence and improvement of cholestasis. Blood samples were collected every week for analysis of fatty acid composition and liver function. 12 of the 15 patients had their direct bilirubin normalized within 4 weeks and our data demonstrated a significant decrease in both DB (P ≤ 0.001) and TB (P ≤ 0.001) after 4 weeks compared to baseline. There were ameliorations of liver function and fatty acid patterns in all patients and serial liver biopsy specimens showed progressive histologic improvement. Parenteral ω-3 PUFAs supplemented fat emulsions are safe and effective in the treatment of PNALD in adults.

Predictors of Acute Rejection After Lung Transplantation

Acute rejection (AR) after lung transplantation (LTx) impacts survival and quality of life. The objective of this study, therefore, was to identify risk factors for AR after LTx, focusing on donor- and recipient-specific factors, operative variables, and immunologic issues, including pretransplant panel-reactive antibody (PRA) levels, and donor-recipient human leukocyte antigen (HLA) mismatch. From March 1996 to November 2007, 481 adults undergoing LTx had 3237 serial transbronchial biopsy specimens that were evaluated for perivascular rejection (grade A0 to A4). Longitudinal analysis was used to characterize the prevalence of rejection grade and influence of donor, recipient, technical, and immunologic variables. AR was highest (54%≥A1) in the first 2 months after LTx, decreased at 6 months (16%≥A1), then remained steady. Prevalence of AR at any time was dominated by donor-specific factors of young age (p<0.0001), blunt trauma (p=0.008), and nonblack race (p=0.012) and by recipient class II PRA exceeding 10% (p=0.005). AR within 2 months was associated with HLA mismatch at the DR locus (p=0.0006) and use of non-O blood-group donors (p=0.008). AR at 4 years and longer after LTx was associated with HLA mismatch at the B locus (p=0.01). Only a few recipient and operative factors were identified for AR after LTx. Moderately sensitized recipients identified by class II PRA exceeding 10% and those with HLA mismatches at the B and DR loci appear to be more susceptible to AR; however, such immunologic variations appear to be well controlled with current donor selection and immunosuppression protocols. The impact of donor-specific variables on AR is surprisingly strong and warrants closer inspection.

Histopathologic Study of Hidradenitis Suppurativa Following Long-Pulsed 1064-nm Nd:YAG Laser Treatment

To assess clinical and histopathologic changes occurring after long-pulsed 1064-nm Nd:YAG laser treatment of hidradenitis suppurativa (HS). Prospective, controlled clinical and histologic study of patients with Hurley stage II HS disease. Outpatient dermatology department at Henry Ford Hospital, Detroit, Michigan. Nineteen patients with Fitzpatrick skin types II to VI with Hurley stage II HS lesions of the axilla and groin. Interventions Two monthly laser sessions were performed using the long-pulsed 1064-nm Nd:YAG laser. Main Outcome Measure Clinical response was scored using the modified Sartorius scale for HS reflecting Lesion Area and Severity Index (LASI). Histologic changes were examined before treatment and 1 week, 1 month, and 2 months after treatment. The percentage change in HS severity after 2 sessions of laser treatment was -31.6 over all anatomic sites (P < .005), -24.4 for the axillary site (P = .008), and -36.8 for the inguinal site (P = .001). Histologic changes corresponded to clinical response. Findings from serial biopsy specimens showed increased inflammation at 1 week after treatment and decreased inflammation with resulting fibrosis and scarring at 1 month and 2 months after treatment. The long-pulsed 1064-nm Nd:YAG laser is a novel effective treatment option for HS. Our histopathologic data suggest that HS is primarily a follicular disorder. The Nd:YAG laser penetrates for selective photothermolysis of the follicular unit and destruction of organized inflammatory lesions in the superficial to mid dermis. Our study offers insight into the pathogenesis of HS and the mechanism of the Nd:YAG laser in treatment of patients with this chronic, debilitating disease.

CDNA Microarray Analysis of Serially Sampled Cervical Cancer Specimens From Patients Treated With Thermochemoradiotherapy

To elucidate changes in gene expression after treatment with regional thermochemoradiotherapy in locally advanced squamous cell cervical cancer. Tru-Cut biopsy specimens were serially collected from 16 patients. Microarray gene expression levels before and 24 h after the first and second trimodality treatment sessions were compared. Pathway and network analyses were conducted by use of Ingenuity Pathways Analysis (IPA; Ingenuity Systems, Redwood City, CA). Single gene expressions were analyzed by quantitative real-time reverse transcription-polymerase chain reaction. We detected 53 annotated genes that were differentially expressed after trimodality treatment. Central in the three top networks detected by IPA were interferon alfa, interferon beta, and interferon gamma receptor; nuclear factor kappaB; and tumor necrosis factor, respectively. These genes encode proteins that are important in regulation cell signaling, proliferation, gene expression, and immune stimulation. Biological processes over-represented among the 53 genes were fibrosis, tumorigenesis, and immune response. Microarrays showed minor changes in gene expression after thermochemoradiotherapy in locally advanced cervical cancer. We detected 53 differentially expressed genes, mainly involved in fibrosis, tumorigenesis, and immune response. A limitation with the use of serial biopsy specimens was low quality of ribonucleic acid from tumors that respond to highly effective therapy. Another "key limitation" is timing of the post-treatment biopsy, because 24 h may be too late to adequately assess the impact of hyperthermia on gene expression.