Irina A. Lubensky

Cyclophosphamide-induced cystitis and bladder cancer in patients with Wegener granulomatosis.

Wegener granulomatosis is a necrotizing granulomatous vasculitis that typically involves the upper and lower respiratory tract and the kidneys. Standard therapy for Wegener granulomatosis includes the daily administration of low-dose oral cyclophosphamide and corticosteroid therapy [1, 2]. This therapeutic regimen has dramatically improved the survival of patients with this otherwise fatal disease: More than 90% of patients treated with cyclophosphamide and corticosteroid therapy improve markedly, and 75% achieve complete remission of disease [1, 3]. However, extended follow-up of patients with Wegener granulomatosis indicates that relapse of disease is common, and repeated and prolonged courses of cyclophosphamide can be associated with serious long-term toxicities, including bone marrow suppression, infertility, hemorrhagic cystitis, and the development of cancer [3]. Hemorrhagic cystitis and bladder cancer are well-recognized complications of cyclophosphamide therapy for both malignant [4] and nonmalignant diseases [3, 5-7]. However, the relations among total cyclophosphamide dose, the development of cystitis, and the occurrence of bladder cancer have not been well defined. In this report, we describe the incidence, clinical manifestations, and natural history of cyclophosphamide-mediated urotoxicity in a cohort of patients with Wegener granulomatosis. We identify risk factors associated with the development of cyclophosphamide-induced bladder cancer and discuss recommendations for surveillance. Methods Patients From 1967 to 1993, 145 patients with Wegener granulomatosis were treated with cyclophosphamide at the Warren G. Magnuson Clinical Center of the National Institutes of Health (NIH). The clinical features of the underlying disease in all but 3 of these patients have been reported previously [3]; the clinical and demographic characteristics of these patients are summarized in Table 1. Individual patients were followed for 0.5 to 27 years (median, 8.5 years), for a total of 1333 patient-years. Table 1. Demographic and Clinical Characteristics of 145 Patients with Wegener Granulomatosis Who Were Treated with Cyclophosphamide* Treatment Protocol We used the standard cyclophosphamide treatment regimen, which has been described previously [1, 3]. Therapy consisted of 1) oral cyclophosphamide, 2 mg/kg of body weight per day and 2) prednisone, 1 mg/kg of body weight per day. If patients improved substantially after the first month of treatment, the prednisone dose was gradually tapered to an alternate-day regimen, and prednisone therapy was eventually discontinued. Cyclophosphamide therapy was continued for at least 1 year after patients achieved complete remission. The cyclophosphamide dose was then tapered by 25-mg decrements of therapy every 2 to 3 months until discontinuation of therapy or until disease recurrence required an increase in dose. The cyclophosphamide dose was adjusted as needed to keep the absolute neutrophil count higher than 1.0 106/L. If substantial toxicity required the permanent discontinuation of cyclophosphamide therapy and if signs of active vasculitis were present, azathioprine, chlorambucil, or (after 1990) low-dose weekly methotrexate therapy was allowed. Eight patients with fulminant disease initially received intravenous cyclophosphamide at daily doses of 3 to 5 mg/kg. When their disease stabilized, these patients were switched to the standard oral cyclophosphamide regimen. Fourteen patients received monthly pulses of intravenous cyclophosphamide, 1 g/m2 body surface area; 13 of these 14 patients also received one or more courses of the standard oral cyclophosphamide regimen. Thus, 144 of the 145 patients received oral cyclophosphamide therapy for some period of time. Patients were evaluated at the NIH every 1 to 3 months. Those who achieved remission of disease and maintained it for 1 year were subsequently seen every 6 months. For each patient, urinalysis was done at every visit, and a cytologic examination of urine was done every 6 to 12 months. All patients received cyclophosphamide as part of clinical research protocols approved by the National Institute of Allergy and Infectious Diseases (NI-AID) Institutional Review Board, the NIAID Clinical Director, and the director of the NIH Clinical Center. All patients gave written informed consent. Urine Cytology Cytologic examination of urine was done at each evaluation. Sediments obtained from voided urine specimens were fixed in Saccomanno solution (Lerner Laboratories, Pittsburgh, Pennsylvania), immobilized on membrane filters (Millipore, Chicago, Illinois) or by cytospin, placed in 95% ethanol, and stained with Papanicolaou stain. Urine samples obtained as much as 6 months before each cystoscopic examination were reviewed retrospectively and were correlated with subsequent bladder biopsy specimens. Cellular cytologic features were placed in the following categories according to the following criteria [8-10]: 1. Negative: no important epithelial abnormalities. 2. Atypia: some nuclear abnormalities in epithelial cells, but the changes could not be definitely placed in categories 3, 4, or 5. 3. Therapeutic or viral: cytologic changes consistent with polyomavirus infection or cyclophosphamide toxicity. In the absence of diagnostic inclusions, the two types of changes are indistinguishable, and we therefore grouped them together. The nuclear enlargement and hyperchromasia associated with polyomavirus or chemotherapeutic effect should be distinguished from high-grade dysplasia or carcinoma. 4. Dysplasia or possible low-grade transitional-cell carcinoma: a few cells in a voided urine sample, either singly or in clusters, that have slightly enlarged, irregular nuclei with increased granularity in chromatin distribution and small or absent nucleoli. 5. Transitional-cell carcinoma: high-grade lesions meeting unequivocal criteria of malignancy. Definition of Terms Nonglomerular hematuria was defined as microscopic or gross hematuria not associated with the presence of erythrocyte casts or declining renal function. Glomerulonephritis causing hematuria associated with erythrocyte casts (glomerular hematuria) occurred at least once in 116 of the 145 patients (Table 1). If hematuria persisted after the treatment of glomerulonephritis and the disappearance of erythrocyte casts, or if hematuria not associated with the presence of erythrocyte casts ever developed, patients were considered to have nonglomerular hematuria and had cystoscopy (see below). Cyclophosphamide-induced cystitis was defined as nonglomerular hematuria associated with characteristic cystoscopic bladder changes. These changes included patchy areas of neovascularity and telangiectasia manifested as an increased number of tortuous, thin-walled veins and small areas of hemorrhage in or under the bladder epithelium. The mucosa between the hypervascular areas may appear normal or pale with decreased vascularity. Cystoscopy Cystoscopy was done to evaluate nonglomerular hematuria (microscopic or gross) in patients receiving cyclophosphamide. Only the results of cystoscopies done at the NIH Clinical Center by members of the Urologic Oncology Section of the National Cancer Institute are included in this report. Most of these cystoscopies were done by two of the authors; all cystoscopy results were reviewed by these two authors. Random biopsies were done only if the results of cytologic examination of urine suggested malignancy. Patients having cystoscopy had intravenous pyelography or retrograde pyelography, or both, at least once to evaluate the upper urinary tract. Statistical Analysis The frequencies of clinical findings were compared by using the Fisher exact test for association with bladder cancer and nonglomerular hematuria; adjustments were made for multiple comparisons of microscopic and gross hematuria with bladder cancer using the modified Bonferroni method [11]. The effects of fixed covariates (sex, history of smoking, age at onset of Wegener granulomatosis disease, and duration of disease before first cyclophosphamide treatment) and time-varying covariates (microscopic hematuria, gross hematuria, total cyclophosphamide dose, and duration of cyclophosphamide therapy) on the development of bladder cancer were examined using Cox proportional-hazards regression analysis [12, 13]. Medians and other percentiles for variables dependent on follow-up time were estimated by using the Kaplan-Meier method [14]. The cumulative distributions determined by the Kaplan-Meier method were compared with the log-rank test. Risk estimates for the development of bladder cancer in patients with Wegener granulomatosis who were treated with cyclophosphamide were determined by comparing observed rates with the expected rates for the United States population, which were obtained from the Surveillance, Epidemiology, and End Results (SEER) Cancer Statistics Review, 1973-1991 [15]. Results Hematuria Seventy-three of 145 patients treated with cyclophosphamide (50%) developed nonglomerular hematuria; the median time to development for all patients was 37 months of receipt of cyclophosphamide (95% CI, 32 to 55 months), and the median dose before development was 124 g (CI, 82 to 149 g) (Table 2 and Figure 1). Forty-one patients (56%) presented with microscopic hematuria; 32 (44%) presented with gross hematuria. Twenty-eight patients had more than one recurrent episode of nonglomerular hematuria (microscopic or gross), sometimes years after cyclophosphamide therapy had been discontinued. Eighteen of the 73 patients developed hematuria after cyclophosphamide therapy was discontinued. Manifestations of active vasculitis necessitated that cyclophosphamide therapy be continued in 26 of the 55 patients who developed nonglomerular hematuria while receiving this therapy. Table 2. Clinical Characteristics of the 73 Patients Treated with Cyclophosphamide Who Developed Nonglomerular Hematuria Figure 1. Cumulative r

Trisomy 7-harbouring non-random duplication of the mutant MET allele in hereditary papillary renal carcinomas

The gene defect for hereditary papillary renal carcinoma (HPRC) has recently been mapped to chromosome 7q, and germline mutations of MET (also known as c-met) at 7q31 have been detected in patients with HPRC (ref. 2). Tumours from these patients commonly show trisomy of chromosome 7 when analysed by cytogenetic studies and comparative genomic hybridization (CGH). However, the relationship between trisomy 7 and MET germline mutations is not clear. We studied 16 renal tumours from two patients with documented germline mutations in exon 16 of MET. Flourescent in situ hybridization (FISH) analysis showed trisomy 7 in all tumours. To determine whether the chromosome bearing the mutant or wild-type MET gene was duplicated, we performed duplex PCR and phosphoimage densitometry using polymorphic microsatellite markers D7S1801 and D7S1822, which were linked to the disease gene locus, and D1S1646 as an internal control. We determined the parental origin of chromosome alleles by genotyping parental DNA. In all 16 tumours there was an increased signal intensity (2:1 ratio) of the microsatellite allele from the chromosome bearing the mutant MET compared with the allele from the chromosome bearing the wild-type MET. Our study demonstrates a non-random duplication of the chromosome bearing the mutated MET in HPRC and implicates this event in tumorigenesis.

Multiple Neuroendocrine Tumors of the Pancreas in von Hippel-Lindau Disease Patients : Histopathological and Molecular Genetic Analysis

Although pancreatic neuroendocrine tumors (NETs) in von Hippel-Lindau (VHL) disease have been reported, their pathological features have not been characterized. In addition, it is unknown whether alterations of the VHL gene are responsible for pancreatic NET development. To evaluate NETs in VHL patients, we performed histopathological analysis of 30 pancreatic tumors in 14 patients. In addition, DNA from NETs and normal pancreatic tissue from 6 patients with documented germ-line VHL gene mutations was studied for allelic deletions of the second copy of the VHL gene by fluorescence in situ hybridization and polymerase chain reaction-based single-strand conformational polymorphism analysis. Morphologically, the tumors were characterized by solid, trabecular, and/or glandular architecture and prominent stromal collagen bands. Sixty percent of the tumors revealed at least focally clear-cell cytology. All tumors were positive for panendocrine immunohistochemistry markers (chromogranin A and/or synaptophysin); 35% of NETs demonstrated focal positivity for pancreatic polypeptide, somatostatin, insulin, and/or glucagon; and no immunostaining for pancreatic and gastrointestinal hormones was observed in 65% of tumors. Dense core neurosecretory granules were evident by electron microscopic examination, and the clear cells additionally revealed abundant intracytoplasmic lipid. All NETs that were subjected to genetic analysis showed allelic loss of the second copy of the VHL gene. We conclude that multiple, nonfunctional pancreatic NETs occur in VHL patients. Stromal collagen bands and clear-cell morphology are important histological features of VHL-associated NETs. The presence of allelic deletions of the VHL gene in pancreatic NETs provides direct molecular evidence for a role of the gene in their tumorigenesis and establishes NET as an independent tumor type of VHL disease.

Hereditary and Sporadic Papillary Renal Carcinomas with c-met Mutations Share a Distinct Morphological Phenotype

Germline mutations of c-met oncogene at 7q31 have been detected in patients with hereditary papillary renal cell carcinoma. In addition, c-met mutations were shown to play a role in 13% of patients with papillary renal cell carcinoma and no family history of renal tumors. The histopathology of papillary renal cell carcinoma with c-met mutations has not been previously described. We analyzed the histopathology of 103 bilateral archival papillary renal cell carcinomas and 4 metastases in 29 patients from 6 hereditary papillary renal cell carcinoma families with germline c-met mutations and 6 papillary renal cell carcinomas with c-met mutations from 5 patients with no family history of renal tumors. Twenty-five sporadic renal tumors with prominent papillary architecture and without somatic c-met mutations were evaluated for comparison. All papillary renal cell carcinomas with c-met mutations were 75 to 100% papillary/tubulopapillary in architecture and showed chromophil basophilic, papillary renal cell carcinoma type 1 histology. Fuhrman nuclear grade 1-2 was seen in tumors from 23 patients, and nuclear grade 3 was observed focally in 8 patients. Seventeen patients had multiple papillary adenomas and microscopic papillary lesions in the surrounding renal parenchyma. Clear cells with intracytoplasmic lipid and glycogen were focally present in tumors of 94% papillary renal cell carcinoma patients. Clear cells of papillary renal cell carcinoma had small basophilic nuclei, and clear cell areas lacked a fine vascular network characteristic of conventional (clear) cell renal cell carcinoma. We conclude that papillary renal cell carcinoma patients with c-met mutations develop multiple, bilateral, papillary macroscopic and microscopic renal lesions. Renal tumors with c-met genotype show a distinctive papillary renal cell carcinoma type 1 phenotype and are genetically and histologically different from renal tumors seen in other hereditary renal syndromes and most sporadic renal tumors with papillary architecture. Although all hereditary and sporadic papillary renal cell carcinomas with c-met mutations share papillary renal cell carcinoma type 1 histology, not all type 1 sporadic papillary renal cell carcinomas harbor c-met mutations.

Hereditary papillary renal cell carcinoma: clinical studies in 10 families.

We recently described a 3-generation family with members affected with papillary renal cell carcinoma, an uncommon histological type of renal cell carcinoma. Possibly family 150 is an isolated occurrence, a reflection of some as yet unknown environmental factor. Alternatively, family 150 may represent a distinct class of inherited cancer. To distinguish between these 2 possibilities we sought additional families with papillary renal cell carcinoma and we identified 9 with members affected with papillary renal cell carcinoma. There were 29 affected male and 12 affected female subjects (ratio 2.41:1), including affected members of family 150. Papillary renal cell carcinomas were often detected incidentally in asymptomatic individuals or during screening of asymptomatic members of renal cell carcinoma families. The penetrance, the proportion of obligate gene carriers that showed clinical evidence of the disease, was reduced. The median survival of affected individuals was 52 years. The results support the concept that the predisposition to develop papillary renal cell carcinomas may be inherited and that hereditary papillary renal cell carcinoma constitutes a distinct class of inherited cancer.

HISTOPATHOLOGY AND MOLECULAR GENETICS OF RENAL TUMORS: TOWARD UNIFICATION OF A CLASSIFICATION SYSTEM

PURPOSE We characterize the genetic abnormalities associated with pathological subtypes of renal tumors, which may help diagnosis or prognostication. MATERIALS AND METHODS A comprehensive literature review of genetic abnormalities associated with different renal tumor subtypes was performed. RESULTS Studies of sporadic and hereditary forms suggest that abnormalities in the von Hippel-Lindau and met genes are the earliest changes in conventional (clear cell) and papillary basophilic renal cancers, respectively. Renal oncocytoma and chromophobe carcinoma have common genetic abnormalities, suggesting a relationship. A similar finding has been observed between papillary adenoma and papillary basophilic renal cancer. CONCLUSIONS These findings suggest that molecular diagnostic testing will help determine histopathological diagnosis, identify tumor types with similar genetic abnormalities suggesting a common origin and indicate potential prognostic markers for future study.

Prevalence of microscopic lesions in grossly normal renal parenchyma from patients with von Hippel-Lindau disease, sporadic renal cell carcinoma and no renal disease: clinical implications.

PURPOSE We sought to describe the earliest renal lesions in patients with von Hippel-Lindau disease to gain insight into the genesis of renal neoplasms in this condition. MATERIALS AND METHODS Grossly normal renal parenchyma from von Hippel-Lindau disease patients was examined microscopically and compared to findings in similar tissues obtained from patients with sporadic renal cancer and from autopsy subjects with no renal disease. RESULTS Microscopic renal cystic and solid neoplasms containing only clear cell cytological features were found in patients with von Hippel-Lindau disease. Benign cysts with clear cell cytological features were found only in patients with von Hippel-Lindau disease. Benign cysts lined by cuboidal cells with eosinophilic cytoplasm, similar to renal tubular cells, were present only in patients with renal cancer. The extrapolated number of clear cell lesions in an average kidney with von Hippel-Lindau disease was estimated as 1,100 cysts (benign or atypical) with clear cell lining and 600 clear cell neoplasms. CONCLUSIONS These findings support the hypothesis that abnormalities in the von Hippel-Lindau gene in a kidney results in the cytological cell type of clear cell renal cancer as the initial product of cellular transformation.

The multiple endocrine neoplasia type I gene locus is involved in the pathogenesis of type II gastric carcinoids

BACKGROUND & AIMS Both gastrin and genetic factors were suggested to underlie the pathogenesis of multiple gastric enterochromaffin-like (ECL) cell carcinoids. To assess the role of genetic alterations in carcinoid tumorigenesis, loss of heterozygosity (LOH) at the locus of the multiple endocrine neoplasia type 1 (MEN-1) gene was studied in gastric carcinoids of patients with MEN-1 and chronic atrophic type A gastritis (A-CAG), as well as in sporadically arising intestinal carcinoids. METHODS DNA extracted from archival tissue sections of 35 carcinoid tumors was assessed for LOH with eight polymorphic markers on chromosome 11q13. A combined tumor and family study was performed in 1 patient with MEN-1-Zollinger-Ellison syndrome (ZES). RESULTS LOH at 11q13 loci was detected in 15 of 20 (75%) MEN-1-ZES carcinoids, and each ECL-cell carcinoid with LOH showed deletion of the wild-type allele. Only 1 of 6 A-CAG carcinoids displayed LOH at the MEN-1 gene locus, and none of the 9 intestinal and rectal carcinoids showed 11q13 LOH. CONCLUSIONS Gastric ECL-cell carcinoid is an independent tumor type of MEN-1 that shares a common developmental mechanism (via inactivation of the MEN-1 gene) with enteropancreatic and parathyroid MEN-1 tumors. Further analysis of sporadic and A-CAG carcinoids is needed to elucidate genetic factors involved in their tumorigenesis.

FAMILIAL RENAL ONCOCYTOMA: CLINICOPATHOLOGICAL STUDY OF 5 FAMILIES

PURPOSE We analyzed familial renal oncocytoma to provide a foundation for studies aimed at defining genes involved in the pathogenesis of renal oncocytoma. MATERIALS AND METHODS We describe 5 families with multiple members affected with renal oncocytoma. Tumors were analyzed pathologically, and affected and nonaffected members were screened clinically and genetically. RESULTS We identified 12 affected male and 3 affected female (ratio 4:1) individuals in the 5 families. In affected family members renal oncocytomas were often multiple and bilateral. No metastatic disease was observed. Most renal oncocytomas were detected incidentally in asymptomatic individuals or during screening of asymptomatic members of renal oncocytoma families. One identical twin pair was affected with bilateral multiple renal oncocytomas. CONCLUSIONS Renal oncocytoma may be inherited in some families.

Characterization of the Renal Pathology of a Familial Form of Renal Cell Carcinoma Associated With Von Hippel-Lindau Disease: Clinical and Molecular Genetic Implications

We examined 12 patients with von Hippel-Lindau disease and renal cell carcinoma to characterize the pathological findings after renal surgery (total 161 lesions). Tissue from 15 partial nephrectomies and 1 radical nephrectomy was obtained for examination. Pathological evaluation identified 116 cystic lesions, 25 of which (21%) were malignant, and 45 solid lesions, 41 of which (91%) were renal cell carcinoma. Each kidney had a mean of 7.8 cystic and 3.0 solid lesions. Of 66 malignant lesions 35 (53%) were comprised of cells with only clear cell cytological features and 30 (45.5%) contained predominantly clear cells with scattered granular cells. One malignant lesion (1.5%) contained sarcomatoid renal cell carcinoma with clear and granular cells. Microscopic invasion of the pseudocapsule was common (13 of 25 clinically cystic tumors [52%] and 31 of 41 clinically solid lesions [76%]) but no tumor extended through the pseudocapsule. All lesions without a pseudocapsule were well circumscribed. Our study establishes the clear cell cytological features as the primary finding observed in patients with von Hippel-Lindau disease. Many cystic lesions contained renal cancer, demonstrating the need for removal of all cystic lesions when this can be safely performed. The well circumscribed nature of the tumors lends them to parenchymal sparing procedures.