Selda Aydin

Molecular Response to Cetuximab and Efficacy of Preoperative Cetuximab-Based Chemoradiation in Rectal Cancer

PURPOSE To characterize the molecular pathways activated or inhibited by cetuximab when combined with chemoradiotherapy (CRT) in rectal cancer and to identify molecular profiles and biomarkers that might improve patient selection for such treatments. PATIENTS AND METHODS Forty-one patients with rectal cancer (T3-4 and/or N+) received preoperative radiotherapy (1.8 Gy, 5 days/wk, 45 Gy) in combination with capecitabine and cetuximab (400 mg/m2 as initial dose 1 week before CRT followed by 250 mg/m2 /wk for 5 weeks). Biopsies and plasma samples were taken before treatment, after cetuximab but before CRT, and at the time of surgery. Proteomics and microarrays were used to monitor the molecular response to cetuximab and to identify profiles and biomarkers to predict treatment efficacy. RESULTS Cetuximab on its own downregulated genes involved in proliferation and invasion and upregulated inflammatory gene expression, with 16 genes being significantly influenced in microarray analysis. The decrease in proliferation was confirmed by immunohistochemistry for Ki67 (P = .01) and was accompanied by an increase in transforming growth factor-alpha in plasma samples (P < .001). Disease-free survival (DFS) was better in patients if epidermal growth factor receptor expression was upregulated in the tumor after the initial cetuximab dose (P = .02) and when fibro-inflammatory changes were present in the surgical specimen (P = .03). Microarray and proteomic profiles were predictive of DFS. CONCLUSION Our study showed that a single dose of cetuximab has a significant impact on the expression of genes involved in tumor proliferation and inflammation. We identified potential biomarkers that might predict response to cetuximab-based CRT.

SDHB/SDHA immunohistochemistry in pheochromocytomas and paragangliomas: A multicenter interobserver variation analysis using virtual microscopy: A Multinational Study of the European Network for the Study of Adrenal Tumors (ENS@T)

Despite the established role of SDHB/SDHA immunohistochemistry as a valuable tool to identify patients at risk for familial succinate dehydrogenase-related pheochromocytoma/paraganglioma syndromes, the reproducibility of the assessment methods has not as yet been determined. The aim of this study was to investigate interobserver variability among seven expert endocrine pathologists using a web-based virtual microscopy approach in a large multicenter pheochromocytoma/paraganglioma cohort (n=351): (1) 73 SDH mutated, (2) 105 non-SDH mutated, (3) 128 samples without identified SDH-x mutations, and (4) 45 with incomplete SDH molecular genetic analysis. Substantial agreement among all the reviewers was observed either with a two-tiered classification (SDHB κ=0.7338; SDHA κ=0.6707) or a three-tiered classification approach (SDHB κ=0.6543; SDHA κ=0.7516). Consensus was achieved in 315 cases (89.74%) for SDHB immunohistochemistry and in 348 cases (99.15%) for SDHA immunohistochemistry. Among the concordant cases, 62 of 69 (~90%) SDHB-/C-/D-/AF2-mutated cases displayed SDHB immunonegativity and SDHA immunopositivity, 3 of 4 (75%) with SDHA mutations showed loss of SDHA/SDHB protein expression, whereas 98 of 105 (93%) non-SDH-x-mutated counterparts demonstrated retention of SDHA/SDHB protein expression. Two SDHD-mutated extra-adrenal paragangliomas were scored as SDHB immunopositive, whereas 9 of 128 (7%) tumors without identified SDH-x mutations, 6 of 37 (~16%) VHL-mutated, as well as 1 of 21 (~5%) NF1-mutated tumors were evaluated as SDHB immunonegative. Although 14 out of those 16 SDHB-immunonegative cases were nonmetastatic, an overall significant correlation between SDHB immunonegativity and malignancy was observed (P=0.00019). We conclude that SDHB/SDHA immunohistochemistry is a reliable tool to identify patients with SDH-x mutations with an additional value in the assessment of genetic variants of unknown significance. If SDH molecular genetic analysis fails to detect a mutation in SDHB-immunonegative tumor, SDHC promoter methylation and/or VHL/NF1 testing with the use of targeted next-generation sequencing is advisable.

Repeat kidney biopsies fail to detect differences between azathioprine and mycophenolate mofetil maintenance therapy for lupus nephritis: data from the MAINTAIN Nephritis Trial

BACKGROUND In the MAINTAIN Nephritis Trial, azathioprine (AZA) and mycophenolate mofetil (MMF) were compared as maintenance immunosuppressive treatment of proliferative lupus nephritis (LN) after a short-course of intravenous cyclophosphamide. Here, we compare the pathological findings on repeat kidney biopsies between the two groups. METHODS Per protocol, repeat renal biopsies were obtained in 30 patients (16 AZA and 14 MMF) at 2 years (±6 months). Baseline and follow-up biopsies were graded according to the International Society of Nephrology/Renal Pathological Society (ISN/RPS) classification. The activity and chronicity indices (AI, CI) were calculated using two different semiquantitative scoring systems (Morel-Maroger and National Institutes of Health). Statistics were performed by non-parametric tests. RESULTS The clinical characteristics of the 30 re-biopsied patients only marginally differ from the entire MAINTAIN cohort (105 patients). Clinical baseline and follow-up characteristics of AZA- and MMF-treated re-biopsied patients did not differ. Time (SD) to repeat renal biopsy was 25.0 (2.0) and 26.5 (3.3) months in AZA and MMF patients, respectively. More patients had normal renal biopsies or Classes I/II/V LN at follow-up compared to baseline and conversely, less patients had Class IV LN at follow-up. In both groups, the AI statistically decreased at follow-up compared to baseline, while the CI slightly, but significantly, increased. No differences could be detected between the groups. CONCLUSION Centralized pathological analyses, including ISN/RPS classification and comparisons of AI/CI, failed to find differences between MMF and AZA at 2 years, a result well in line with the absence of difference in long-term clinical outcome reported elsewhere.

Serum soluble interleukin 7 receptor is strongly associated with lupus nephritis in patients with systemic lupus erythematosus

Background The soluble form of the interleukin 7 receptor (sIL-7R) is produced by fibroblasts after stimulation with proinflammatory cytokines. Increased sIL-7R serum and synovial fluid levels were recently demonstrated in patients with rheumatoid arthritis. Objectives To investigate whether sIL-7R production is dysregulated in systemic lupus erythematosus (SLE), and whether this correlates with disease activity. Methods Serum and urine sIL-7R concentrations were measured by ELISA, and sIL-7R quantitative PCR (qPCR) studies were performed in peripheral blood mononuclear cells (PBMCs). IL-7R, tumour necrosis factor α (TNFα), IL-1β and IL-17 immunostainings were performed on kidney sections. Results sIL-7R concentrations were significantly higher in SLE sera than in controls, and correlated with SLE Disease Activity Index (SLEDAI) scores. Accordingly, serum sIL-7R levels were strongly raised in patients with nephritis. Moreover in patients with lupus nephritis, serum sIL-7R decreased upon treatment. sIL-7R gene expression in PBMCs was similar in patients with lupus nephritis and controls. By contrast, abundant perivascular IL-7R expression was seen in SLE kidney biopsy specimens, which was associated with expression of TNFα in the surrounding tissue. Conclusions Our data indicate that sIL-7R is a marker of SLE disease activity, especially nephritis. In contrast to conventional disease activity markers, sIL-7R is not produced by immune cells, but might instead reflect activation of tissue cells in the target organ.

What is the best way to predict disease-free survival after preoperative radiochemotherapy for rectal cancer patients: tumor regression grading, nodal status, or circumferential resection margin invasion?

is an important issue. The excellent article by Rodel et al 1 suggested that patients with complete (tumor regression grading [TRG] 4) and intermediate pathologic response (TRG 2 3) had an improved disease-free survival (DFS) after preoperative RCT. We would like to make a few comments regarding this interesting analysis. In this series, although TRG seemed promising in the univariate analysis, the most important independent prognostic factor for DFS was the pathological (T) category and the nodal (N) status in the multivariate model. So, it is possible that, in the clinical routine, the only relevant practical parameter for DFS remains the postoperative TN stadification. It could be also speculated that pathologic TN staging after surgery not only reflects the initial staging of the tumor, but alsothetumorresponsetothepreoperativetreatmentassuggestedby some correlations in the Rodel article. 1 Itcouldalsobeinterestingtoclarifyifclearanceofthecircumferential resection margin (defined according to standard guidelines as 1or2mmbetweenthetumorandthesurgicalmargin 2,3 )wasalsoa

Prognosis of proliferative lupus nephritis subsets in the Louvain Lupus Nephritis inception Cohort

Objective The objective of this paper is to evaluate whether the different International Society of Nephrology/Renal Pathology Society (ISN/RPS) classes of proliferative lupus nephritis (LN) have a distinct baseline presentation, short-term response to immunosuppression (IS) and long-term prognosis. Methods Ninety-eight patients with new onset (first renal biopsy) ISN/RPS proliferative LN (Class III: n = 24; IV-S: n = 23; IV-G: n = 51) were diagnosed at our institution between 1995 and 2012 (Louvain Lupus Nephritis inception Cohort). Their baseline renal parameters, primary response to IS at one year, survival and long-term renal outcome (mean follow-up: 77 months) were compared. Results At baseline, serum creatinine and 24-hour proteinuria were higher in Class IV-G, as was activity index on renal biopsy in Class IV-S and IV-G compared to III. Upon treatment, renal parameters improved with the same kinetics and to the same extent in the three pathological classes. On repeat renal biopsies (n = 43), activity indices dropped similarly. Poor outcomes (death, end-stage renal disease, renal impairment defined by an eGFR <60 ml/min/1.73 m2) did not statistically differ between groups, although there was a trend toward more renal impairment at follow-up in Class IV-G compared to IV-S and III. Finally, the presence of even mild chronic lesions on baseline biopsy was clearly predictive of late renal outcome. Conclusion Subsetting proliferative LN into Class III, IV-S and IV-G provides less clinically discriminant prognostic information than baseline chronicity index.

Primary leptomeningeal melanoma is part of the BAP1-related cancer syndrome.

In this case report, a 40-year-old woman, with a recent history of seizures, was diagnosed with a supratentorial brain mass. Surgical exploration revealed a heavily pigmented, 5-cm mass adhered to the leptomeninges. Pathological analysis and immunohistochemistry confirmed a malignant melanoma. Due to the absence of a cutaneous or ocular melanoma, a diagnosis of primary leptomeningeal melanoma was made. A few months after this diagnosis, the patient developed a papillary thyroid carcinoma. One year later, the melanoma relapsed. Excision with complementary radiotherapy was performed. Four months after this operation, the patient relapsed again, leading to a progressive, strictly intracranial, multifocal diffusion of the tumor. The patient died from intracranial hypertension related to tumor compression 26 months after the initial surgery. Familial anamnesis revealed a medical history of The BRCA1-associated Protein 1 (BAP1) gene encodes a ubiquitin hydrolase that plays a role in several key cellular processes, including transcriptional regulation, growth, DNA damage response, and chromatin dynamics [1]. Heterozygous germline BAP1 mutations were initially associated with a higher risk of mesothelioma and uveal melanoma, as well as multiple atypical cutaneous melanocytic tumors. However, recent reports have extended their clinical phenotype to others tumors such as clear cell renal cancer, lung adenocarcinoma, meningioma, paraganglioma, and multiple basal cell carcinomas [1, 4].

Unambiguous Detection of Multiple TP53 Gene Mutations in AAN-Associated Urothelial Cancer in Belgium Using Laser Capture Microdissection

In the Balkan and Taiwan, the relationship between exposure to aristolochic acid and risk of urothelial neoplasms was inferred from the A>T genetic hallmark in TP53 gene from malignant cells. This study aimed to characterize the TP53 mutational spectrum in urothelial cancers consecutive to Aristolochic Acid Nephropathy in Belgium. Serial frozen tumor sections from female patients (n = 5) exposed to aristolochic acid during weight-loss regimen were alternatively used either for p53 immunostaining or laser microdissection. Tissue areas with at least 60% p53-positive nuclei were selected for microdissecting sections according to p53-positive matching areas. All areas appeared to be carcinoma in situ. After DNA extraction, mutations in the TP53 hot spot region (exons 5–8) were identified using nested-PCR and sequencing. False-negative controls consisted in microdissecting fresh-frozen tumor tissues both from a patient with a Li-Fraumeni syndrome who carried a p53 constitutional mutation, and from KRas mutated adenocarcinomas. To rule out false-positive results potentially generated by microdissection and nested-PCR, a phenacetin-associated urothelial carcinoma and normal fresh ureteral tissues (n = 4) were processed with high laser power. No unexpected results being identified, molecular analysis was pursued on malignant tissues, showing at least one mutation in all (six different mutations in two) patients, with 13/16 exonic (nonsense, 2; missense, 11) and 3/16 intronic (one splice site) mutations. They were distributed as transitions (n = 7) or transversions (n = 9), with an equal prevalence of A>T and G>T (3/16 each). While current results are in line with A>T prevalence previously reported in Balkan and Taiwan studies, they also demonstrate that multiple mutations in the TP53 hot spot region and a high frequency of G>T transversion appear as a complementary signature reflecting the toxicity of a cumulative dose of aristolochic acid ingested over a short period of time.

Complement activation and effect of eculizumab in scleroderma renal crisis.

Background:Scleroderma renal crisis (SRC) is a life-threatening complication of systemic sclerosis characterized by abrupt onset of hypertension, thrombotic microangiopathy, and kidney injury. The mechanisms of the disease remain ill-defined, but a growing body of evidence suggests that activation of the complement system may be involved. Methods:Here, we report the case of a patient presenting with severe SRC and strong evidence of complement activation, both in serum and in the kidney, in the absence of genetic defect of the complement system. Results:Immunofluorescence studies on kidney biopsy showed significant deposits of C1q and C4d in the endothelium of renal arterioles, pointing toward activation of the classical pathway. Because of the dramatic clinical and histological severity, and the lack of response to early treatment with angiotensin-converting enzyme inhibitors, calcium channel blockers and plasma exchange, the patient was treated with the specific C5 blocker eculizumab.Contrarily to conventional treatment, eculizumab efficiently blocked C5b-9 deposition ex vivo and maintained hematological remission. Unfortunately, the patient died from heart failure a few weeks later. Postmortem examination of the heart showed diffuse patchy interstitial fibrosis, the typical lesion of systemic sclerosis-related cardiomyopathy, but normal coronary arteries and myocardial microvasculature. Conclusion:SRC may lead to complement system activation through the classical pathway. Early administration of C5 inhibitor eculizumab may have therapeutic potential in patients with life-threatening SRC refractory to conventional treatment using angiotensin-converting enzyme inhibitors.

Gitelman syndrome and glomerular proteinuria: a link between loss of sodium-chloride cotransporter and podocyte dysfunction?

We report on a 27-year-old patient presenting with chronic hypokalaemia, inappropriate kaliuresis, hypomagnesaemia and alkalosis, associated with moderate proteinuria. Genetic analysis evidenced a homozygous mutation (p.Arg399Cys) in the SLC12A3 gene coding for the sodium-chloride cotransporter (NCC), confirming the diagnosis of Gitelman syndrome. Further genetic testing did not show any mutation in NPHS2. A renal biopsy was performed in view of the unusual association with proteinuria. Light microscopy showed hypertrophy of the juxtaglomerular apparatus and discrete mesangial thickening. In addition to possible focal segmental glomerular sclerosis lesions, electron microscopy showed extensive segments of variably thickened glomerular basement membrane (GBM), contrasting with segments of regular GBM of low range thickness, and effacement of podocyte foot processes. Of interest, alterations of the GBM were also observed in a Slc12a3 knock-out mouse model for Gitelman syndrome. These data suggest that the association between Gitelman syndrome and secondary changes of the GBM is probably not coincidental. Possible mechanisms include angiotensin II- or renin-induced podocyte lesions, as well as chronic hypokalaemia.