Case 1 -

Wilms Tumor D. Ashley Hill, M.D. Mihaela Onciu M.D.

Case History: A five-year-old female presented to her pediatrician with hematuria after being struck in the flank. Physical examination identified a left-sided abdominal mass. Radiographic studies showed a large left renal mass. A left radical nephrectomy was performed. Peri-aortic lymph nodes were sampled. Gross Examination: The lymph nodes were examined and submitted for processing prior to examining the kidney specimen. Grossly the left kidney weighed 341 grams and measured 8.9 x 7.0 x 6.0 cm. An irregular tumor thrombus protruded from the renal vein. External photographs were taken. No evidence of capsular rupture by tumor was noted. The surgeon was contacted and he reported that the tumor thrombus was identified in the proximal renal vein at the time of operation and the level of transection was distal to (closer to inferior vena cava) the thrombus. The irregular contour of the thrombus and lack of attachment to the vein wall was consistent with the surgeon's impression. Ureteral and vascular margins were identified and sampled. The external surface of the kidney was inked. A probe was placed through the ureteral lumen into the pelvis, which had been distorted by the mass, in order to select the best plane of section to demonstrate the tumor relationship to the anatomic landmarks of the kidney. The kidney was bivalved and showed a 7.2 x 6.4 x 5.9 cm pink-tan, gelatinous mass with areas of hemorrhage and necrosis. The tumor was well-circumscribed, encapsulated and appeared to push focally beyond the border of the renal capsule. The tumor protruded into the renal sinus fat and extended into the renal vein. There was yellow-tan firm ill-defined tissue consistent with nephrogenic rests adjacent to the tumor. The adrenal gland was unremarkable. Photographs of the cut surface were taken. Several 1 cm3 pieces of viable tumor were taken off the bulging surface of the tumor, snap frozen and stored at -70oC. Pieces of normal kidney and areas grossly resembling nephrogenic rests were also frozen. Using a clean knife, parallel sections were made through the kidney. The entire specimen was placed in a large container of formalin for overnight fixation. The next morning, an 8 x 10 digital print of the cut surface of the tumor was used to document the location of the sections submitted for histologic examination (A diagram or photocopy of the cut surface would be equally acceptable). Rinsing the scalpel blade after each cut and changing the blade often, sections were taken to demonstrate the relationship of the tumor to the renal capsule, renal vein, renal sinus and surgical margins. Samples of the discolored area resembling nephrogenic rests and normal kidney were taken. The renal hilar fat was searched for lymph nodes. Microscopic Examination: Sections showed a Wilms tumor composed predominantly of blastemal elements with small areas containing epithelial structures and stroma. The tumor was well-circumscribed and separated from the surrounding kidney by a pseudocapsule. Tumor was seen protruding out of the kidney into the perirenal fat but did not involve the inked surgical margin. The tumor extended into the renal vein but was not attached to the vessel wall at the renal vein margin. One of the peri-aortic lymph nodes showed metastatic Wilms tumor. On high power, many of the tumor cells showed marked nuclear enlargement and hyperchromatism. These cells were identified in most of the sections including the metastatic focus in the lymph node. Scattered large, abnormal mitotic figures including tripolar and tetrapolar forms were seen. Intralobar nephrogenic rests characterized by unencapsulated areas of persistent embryonal tubular and stromal elements with infiltrative borders were present. Diagnosis: Wilms tumor with anaplasia, metastatic to one peri-aortic lymph node, Stage III, margins free of tumor, intralobar nephrogenic rests Discussion There are six critical components in the pathologic evaluation of a Wilms tumor (WT): gross examination, preparation of materials for protocol treatment, microscopic diagnosis, histologic subtyping, staging and recognition of precursor lesions (nephrogenic rests). Gross Examination and Procurement of Material for Protocol Therapy Appropriate management of a pediatric renal tumor begins well before the slides cross the microscope stage. WT is one of the more complicated pediatric tumors to handle in the gross room. WT are typically large, bulky, soft, friable masses that can extrude and leak over the surface of the kidney. The staging of WT can be affected by the selection of sections submitted for microscopic examination. Consultation with the surgeon regarding his/her intra-operative assessment is critical in cases with capsular rupture and renal vein invasion. Table 1.1 provides a list of key steps to follow (and document in the gross description) when handling pediatric nephrectomy specimens. Because the vast majority of Wilms tumor patients will be treated on a Children's Oncology Group (National Wilms Tumor Study) Protocol, pathologists and residents need to have an understanding of protocol requirements. Table 1.2 summarizes the requirements for submission of samples for molecular analysis and banking as well as preparation of materials for rapid central review. In our practice we routinely have 2 sets of slides cut up front on pediatric renal tumor cases to speed up the process. Diagnosis The majority of WT are easily recognizable from other pediatric renal tumors by virtue of their well-demarcated, non-infiltrative growth pattern, fibrous pseudocapsule and cellular components resembling the developing fetal kidney. WT may occur anywhere in the kidney and typically is composed of a variety of cell types which are classified as: 1) blastemal, primitive mitotically-active cells with hyperchromatic round, ovoid or angulated nuclei and high nuclear to cytoplasmic ratio arranged in cohesive nests, a serpentine pattern or a diffusely infiltrative pattern; 2) epithelial, cells showing differentiation along epithelial lines, typically tubular profiles or primitive glomerular structures, often arising at the periphery of blastemal nests; and 3) stromal, myxoid or collagenous areas containing cells with spindled profiles that may show skeletal muscle or other mesenchymal differentiation. The classic WT has a combination of these three cell types in varying proportions but a predominance of one or two patterns in not unusual. Heterologous elements including skeletal muscle, bone, cartilage, fat, squamous or mucinous epithelium are seen less commonly and are associated with tumors arising in the central portion of the kidney. Differential Diagnosis The differential diagnosis of WT becomes an issue in a small subset of cases usually in one of two circumstances, limited sampling or monophasic tumors. One of the most common diagnostic dilemmas occurs in differentiating a WT from a hyperplastic nephrogenic rest in a small needle biopsy (see below). In distinguishing WT from unrelated neoplasms it is important to know the other primary renal tumors occurring in childhood and their clinicopathologic characteristics. Congenital mesoblastic nephroma (CMN), clear cell sarcoma of kidney (CCSK) and malignant rhabdoid tumor (MRT) represent the most common pediatric renal neoplasms after WT. Primary renal lymphoma, renal primitive neuroectodermal tumor (PNET), papillary renal cell carcinoma, medullary renal cell carcinoma, angiomyolipoma and synovial sarcoma occur less frequently. Tumors originating outside the kidney such as neuroblastoma, desmoplastic small round cell tumor (DSRCT) and hepatoblastoma have some morphologic overlap with WT but can usually be distinguished by clinical and morphologic criteria and ancillary techniques. Blastemal predominant tumors, in particular the diffuse blastemal WT can have an infiltrative growth pattern and can present a diagnostic problem on small sample biopsies. These poorly differentiated, primitive tumors can resemble neuroblastomas, PNET and DSRCT. A positive WT-1 stain (with the N-terminal antibody) is helpful in confirming an infiltrative small round cell tumor is a blastemal WT. Stromal predominant tumors often raise questions about CMN, CCSK and synovial sarcoma. WT with exclusive tubular and papillary patterns can resemble metanephric adenoma or papillary renal cell carcinoma.Table 1.3 highlights the morphologic features and immunohistochemical stains useful in the differentiating WT from these other tumors of childhood. Histologic Subtyping WT are divided into two histologic groups, favorable histology and anaplasia. The assessment of anaplasia is critical as tumors with anaplasia will have a different treatment regimen from similar stage favorable histology tumors. Tumors are classified as anaplastic if they meet both of the following criteria: 1) increase in nuclear diameter at least 3 times that of an adjacent nucleus of the same cell type with nuclear hyperchromatism; and 2) enlarged, abnormal, usually multipolar mitotic figures. Each component (bar) of an anaplastic mitotic figure must be as large as or larger than a normal metaphase. Most anaplastic tumors can be categorized as diffuse anaplasia showing the characteristic microscopic features in multiple sections of the tumor. Even tumors with circumscribed areas of anaplasia are categorized as diffuse anaplasia if cells meeting criteria for anaplasia are present in any extrarenal site (extracapsular extension, vessels of the renal sinus or metastatic sites) or are present in random biopsy or post-treatment nephrectomy specimens. Tumors with localized abnormal mitotic figures but with moderate to severe nuclear enlargement and hyperchromatism seen throughout the tumor are also classified as diffuse anaplasia. In contrast, to classify a tumor as focal anaplasia, the area of anaplasia must be surrounded on all sides by non-anaplastic tumor or normal kidney, must not extend to the edge of the slide (unless you have an adjacent section on tumor map), cannot extend outside the kidney or in renal sinus vessels and are unassociated with changes of moderate or severe nuclear unrest in the remainder of the tumor. 10.8% of WT in National Wilms Tumor Study 5 (NWTS-5) showed anaplasia. Anaplasia is rare in children less than two years of age with the risk for anaplasia increasing with age. Anaplastic tumors are thought not only to be more resistant to chemotherapy than favorable histology tumors, but also may be inherently more aggressive. Even with aggressive therapy the relapse-free and overall survival estimates for patients with anaplastic tumors are significantly lower than the survival estimates for patients with favorable histology tumors. 4-year overall survival estimates for Stage 1 tumors are 82.6% for anaplastic tumors vs. 98.3% for favorable histology tumors. Survival estimates for anaplastic tumors stages II-IV are 81%, 72% and 35% respectively. A number of studies have shown that p53 immunostaining, with or without demonstrated mutations, is strongly associated with anaplasia in WT, while p53 staining is uncommon in favorable histology tumors. Since a functional p53 protein is important in directing cells with DNA damage into an apoptotic pathway, p53 mutations may explain the insensitivity of anaplastic WT to therapy. Adjuvant therapy does not compromise the histologic assessment of anaplasia as the anaplastic areas within a tumor persist after chemotherapy. Nuclear unrest is a term applied to a subset of favorable histology WT that show the nuclear changes of anaplasia without the abnormal, multipolar mitotic figures. One comparative study showed a risk of relapse for tumors with unrest between that of favorable histology and anaplastic tumors, but tumors with unrest retained their sensitivity to chemotherapy showing an overall survival indistinguishable from favorable histology tumors. They further showed that tumors with unrest were more like favorable histology tumors in their lack of p53 staining. The authors implied that tumors with nuclear unrest are appropriately grouped in the favorable histology WT category. The contribution of the cellular components within a WT to the prognosis is still being investigated. Within the favorable histology subgroup, tumors that are predominantly epithelial generally show a low degree of aggressiveness and more commonly present with low stage disease. But because of their low proliferative rate, these tumors can be relatively insensitive to chemotherapy. Because of these factors, small stage I epithelial WT may be amenable to surgery alone with close follow-up. In contrast to monophasic epithelial tumors, WT with a diffuse blastemal pattern show aggressive, infiltrative and rapid growth. These tumors have a higher incidence of relapse but remain responsive to chemotherapy. Occasionally situations arise in which one has to evaluate a specimen that underwent pre-operative chemotherapy (bilateral WT, unresectable WT). Tumors will be evaluated for favorable histology vs. anaplasia and then placed in one of four categories: 1) anaplastic tumors; 2) completely necrotic tumors (<1% viable tumor), 3) blastemal predominant tumors where >33% of tumor is viable and 67% of viable tumor is blastemal, and 4) intermediate tumors (all others). Staging follows the same scheme as for pre-therapy tumors. Staging The accurate staging of a WT is one of the more difficult tasks of the pediatric pathologist and is critical to risk-adapted therapeutic planning. A careful, thoughtful gross examination is the first and most important step. Appropriate use of ink, timely fixation, using only perpendicular cuts to assess margins (do not shave margins!) and judicious selection of sections will make this task more manageable. Remember to submit sections that include the triangular interface between the intrarenal tumor pseudocapsule, the extrarenal tumor pseudocapsule and the renal capsule to demonstrate extra-renal extension by tumor. Remember to submit several sections of the renal hilum to assess for vascular invasion. It is critical for WT staging to include an evaluation of lymph nodes. If lymph nodes have not been sampled separately, and none are found grossly in the hilar fat, all of the hilar fat should be submitted for microscopic examination. Table 1.4 summarizes the new staging system for WT. One new feature is that all tumor spills without regard to size or location are now considered Stage III. This includes cases that had a pre-operative or intra-operative needle biopsy. There are now two arms for treating Stage I favorable histology tumors. The "surgery only" arm requires that lymph nodes (hilar, peri-aortic or inguinal) must be sampled and examined histologically for patients to qualify, assuming they meet all of the other criteria (less than 24 months of age with kidney/tumor < 550 grams, and confined to kidney). Bilateral WT present a unique problem for histologic classification due to parenchymal sparing approaches to therapy. A recent study by Hamilton et al., reviewed 189 cases of bilateral (stage V) WT. The success rate at identifying anaplasia prior to starting therapy was 0/7 for core needle biopsies, 3/9 for open biopsy and 7/9 undergoing partial or complete nephrectomy. Despite the lack of concordance with pre and post-chemotherapy pathologic classification, no difference in event free survival at 5 years was demonstrable. Biologic Markers Loss of heterozygosity (LOH) for both chromosomes 1p and 16q have been shown to predict an increased risk of relapse and death for patients with favorable histology WT. These markers will now be used prospectively in conjunction with stage and histology to stratify patients into low and higher risk categories and will directly affect the therapeutic regimen. The analysis is done on snap-frozen tissue submitted to the Cooperative Human Tissue Network. Loss of 16q is more common in anaplastic Wilms tumor than favorable histology tumors (32.4% vs 17.4%). There was no association between frequency of LOH for either marker and stages II-IV, although stage I tiny tumors had a much lower frequency. Nephrogenic Rests Nephrogenic rests are regions of persistent embryonal tissue in the renal parenchyma and can be found in 30 to 44% of kidneys removed for WT, 4% of kidneys removed for dysplasia or urinary tract malformations and .21 to .87% of kidneys in pediatric autopsy series (higher incidence in infants < 3 months of age). The term nephroblastomatosis refers to multiple or diffusely distributed nephrogenic rests. The two fundamental categories of nephrogenic rests are based on the topography of the lesion. Perilobar rests (PLNR) are located at the periphery of the lobule and are usually subcapsular. They are often multiple and can be diffuse (diffuse perilobar nephrogenic rests or DPLN). Microscopically, perilobar rests are well demarcated but not encapsulated. They are typically composed of blastema and tubules with little intervening stroma. Similarly, tumors arising in association with PLNR are more likely to be blastemal or epithelial predominant. PLNR are associated with higher birthweights and overgrowth syndromes including Beckwith Wiedeman syndrome. PLNR serve as a marker of loss of imprinting or loss of heterozygosity for IGF-2. Intralobar rests (ILNR) are located deep within the lobule and are usually solitary. They have indistinct margins with respect to the normal kidney. ILNR contain blastemal, tubular and prominent stromal elements interspersed among normal glomerular and tubular elements. ILNR are also more often associated with early onset, stromal predominant WT or WT showing divergent (teratomatous) differentiation. ILNR are a morphologic indicator of WT1 mutation and are strongly associated with WAGR and Denys-Drasch syndromes. It is thought that ILNR result from an error earlier in nephrogenesis compared with PLNR, explaining the typical ILNR location deep within the lobule. Table 1.4 summarizes the clinical and pathologic features of both types of rests. From a diagnostic standpoint, it is important to recognize that hyperplastic (growing) nephrogenic rests may have many similar attributes as WT. Both can produce large masses, demonstrate rapid growth, and be multifocal. Microscopically, there are few reliable differences; be cautious. Both are composed of primitive nephrogenic elements. One characteristic that seems useful is that WT typically have a fibrous pseudocapsule, whereas nephrogenic rests do not. Needle biopsy specimens that do not include a portion of the periphery of the lesion may not be able to distinguish one from the other. Radiographic studies, preferably serial examinations, are generally more helpful in distinguishing rests from WT. Consideration of the radiographic features of the lesion should be a required component of pathologic examination of a needle biopsy specimen. Nephrogenic rests often conform to the shape of the kidney, or at least are non-spherical, while WT have a spherical growth pattern. Rests and WT also have differing patterns of enhancement on magnetic resonance (MR) following Gadolinium administration; rests are homogeneously enhancing while WT are typically heterogeneous. Not all nephrogenic rests are destined to become WT. Beckwith has outlined a number of potential alternative fates of a nephrogenic rest including hyperplasia, adenomatous change and regression. Chemotherapy can induce regression of hyperplastic nephrogenic rests and is a primary mode of therapy for DPLN. Dormant or sclerotic rests will not show an effect from chemotherapy. The presence of nephrogenic rests also has clinical implications by their association with genetic syndromes as well as the risk for development of contralateral WT. At particular risk is a patient whose WT was diagnosed prior to 12 months of age who also has perilobar or both perilobar and intralobar nephrogenic rests in the resected kidney. For example, if a patient < 1 year of age has a kidney removed for WT and that kidney has perilobar nephrogenic rests, that patient will have a 10% risk of developing WT in the opposite kidney (most occurring within 3 years from first diagnosis (range 0 to 13.1 years)). Table 1.1: Key steps in handling pediatric nephrectomy specimens Get the specimen intact. Avoid frozen sections. Locate and inspect key vascular and ureteral margins. Evaluate the external surface for evidence of capsular rupture. Inspect renal vein for tumor thrombus and evaluate for transection. Communicate with the surgeon in cases with renal vein tumor/thrombus or capsular rupture. Take artery, vein and ureteral margin cross sections. Photograph. Weigh. Ink before cutting!! Do not strip capsule!! Bivalve the specimen in a plane to best show tumor with respect to renal anatomy, using the renal pelvis as a guide. Try to avoid bivalving through any suspect capsular areas. Photograph cut surface (instant photo, printed digital photo, photocopy using a leak-proof bag, or diagram all work well). Measure tumor in three dimensions. Take fresh tumor and normal tissue for biological studies snap frozen in liquid nitrogen or cold isopentane. viable tumor, several pieces normal kidney nephrogenic rests (if grossly identifiable) Fix overnight in large container of formalin. Take sections and document location on photograph or diagram: Tumor with respect to renal capsule - take perpendicular sections that include the triangular interface between the intrarenal tumor pseudocapsule, the extrarenal tumor pseudocapsule and the renal capsule to demonstrate extra-renal extension by tumor Tumor with respect to renal hilum Tumor border with uninvolved kidney Any grossly distinctive areas Renal sinus/hilar soft tissue Any nephrogenic rests notable grossly Normal kidney (be generous) Hilar fat lymph nodes (if none identified grossly submit all hilar soft tissue) Adrenal gland or other organs (if present) Other helpful hints: One section per centimeter of tumor minimum. Take most sections from periphery of tumor to indicate relationship with capsule, hilum and margins. If tumor is multifocal, take sections of each nodule (one per centimeter diameter tumor). Try to avoid floaters by rinsing blade (or using new blade) after each cut, rinsing sections before placing in cassette and keeping area clean. Do not take "shave" margins!! Submit accompanying lymph nodes prior to bivalving the kidney or after the bench has Table 1.2: Protocol Requirements for new COG Renal Tumor Study Patient needs to be registered in Renal Tumor Classification and Banking Protocol (AREN0362) Save and submit snap frozen tissue (tumor, normal), formalin-fixed sections (tumor, normal) and oncologist-obtained samples (patient blood, urine; parents' blood) to Cooperative Human Tissue Network (CHTN) Submit complete set of recut slides, pathology report, gross photograph(s) to CHTN for rapid central review. If the tumor is a rhabdoid tumor, renal cell carcinoma or uncertain diagnosis submit unstained slides or block at the same time. Table 1.3: Wilms tumor differential diagnosis Tumor Peak age Age range Helpful features Wilms 2-4 yrs 0-6 yrs Most common pediatric renal tumor age 1-6 Well-demarcated from normal kidney with pseudocapsule Blastemal, stromal and epithelial elements Calcification uncommon Immunohistochemistry positive for CK, vimentin, WT1 protein, EMA can be positive Can be bilateral Can be associated with nephrogenic rests CMN 0-3 mos 0-1 yr Bland spindle cell lesion with infiltrative border Entrapped normal renal elements within tumor Immunohistochemistry positive for vimentin and smooth muscle actin CCSK 1-2 yrs .5-6 yrs Bland nuclear features with pale cytoplasm Arborizing capillary network Broad fibrous bands containing tubules, multiple subtypes Immunohistochemistry negative for epithelial markers, all are positive for vimentin, CD10 positive in 60% of cases MRT 0-1 yr 0-2 yrs Sheets of discohesive primitive cells Characteristic vesicular nuclei & prominent nucleoli Moderate amounts pink cytoplasm & rhabdoid inclusions Characteristic polyphenotypic immunohistochemistry: positive for vimentin, EMA, CK, CD99, NSE NB 0-3 yrs 0-6 yrs Malignant small round cells with range of differentiation; may show neuropil, rosettes, ganglion cell differentiation Calcification common Immunohistochemistry positive for NSE, synaptophysin, chromogranin; negative for CK, EMA ES/PNET Adolescents Wide range Diffuse malignant small cell tumor May have rosettes Immunohistochemistry positive for vimentin and diffuse membrane staining for CD99 RT-PCR positive for EWS-FLI1 or related fusion genes DSRCT Adolescents Wide range Nested malignant small cell tumor with desmoplastic stroma Immunohistochemistry positive for vimentin, CK, EMA, desmin, NSE and WT1 RT-PCR positive for EWS-WT1 or related fusion genes Metanephric Adenoma 41 yrs Wide range Pure epithelial tumor, small, differentiated glandular structures Bland, small nuclei, absent nucleoli, no mitoses Grossly circumscribed, unencapsulated tumor Immunohistochemistry positive for CK7 (patchy), WT1; negative EMA Table 1.4 Staging of Wilms tumor Stage I Confined to kidney and completely resected (all of the following must apply) Tumor cannot penetrate renal capsule (renal capsule has intact outer surface) Tumor not seen in vessels of the renal sinus No biopsy before nephrectomy (excluding fine needle aspirate) Lymph nodes must be examined to qualify for the surgery-only treatment arm* Stage II Tumor extends beyond kidney but completely resected (any of the following) Tumor extends through renal capsule Invasion of vessels outside the parenchyma (including renal sinus vessels or renal vein) AND Specimen margins uninvolved **Tumors extending beyond the kidney and invading adjacent organs may still be considered Stage II if removed en bloc with negative margins Stage III Gross residual tumor (any of the following) Involved surgical margin (grossly or microscopically) Transected tumor in renal vein or tumor thrombus firmly attached to or invading vein wall at margin where vein was severed Tumor in regional lymph nodes (abdomen or pelvis) Tumor penetrated through peritoneal surface or implants on peritoneal surface Prior core or open biopsy Tumor spill of any degree or localization either before or during surgery Tumor removed in more than one piece Local infiltration into vital structures not resectable Stage IV Disseminated disease Hematogenous metastasis or nodal metastases outside the abdomen Stage V Bilateral tumors Whenever possible, the substage of each tumor should be determined with the final designation indicating the highest substage lesion (e.g. Stage V, substage II) *Stage I tiny tumor treatment arm requirements (must meet all criteria) Patient less than 24 months of age Tumor/kidney < 550 gram Negative histologic lymph node examination Table 1.5 Perilobar vs. interlobar nephrogenic rests Feature Perilobar rests Intralobar rests Site in lobe Periphery, subcapsular Usually numerous, can be diffuse Parenchymal, usually deep Usually sparse Margins Circumscribed Indistinct, interdigitating Relation to normal renal elements Demarcated, no nephrons within rest Dispersed between nephrons Composition Blastema or tubules Scant or sclerotic stroma Blastema, tubules, stroma stroma usually predominates Median Age 36 months 23 months Associations BWS, Hemihypertrophy, Perlman syndrome, Trisomy 18, 13 Higher birthweights WAGR, Denys-Drash hypospadias, cryptorchidism Earlier onset tumors Risk for developing contralateral WT* 10% 5% WT features Blastemal or embryonal epithelial predominance in associated WT Stromal component predominates in associated WT *In patients < 1 year at diagnosis of unilateral WT where rests are found in the resected kidney BWS, beckwith-Wiedeman syndrome; WAGR, Wilms, aniridia, growth retardation Modified from: Beckwith JB. 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