Current status of precision medicine in pediatric oncology in Spain: a consensus report by the Spanish Society of Paediatric Haematology and Oncology (SEHOP)

The study analyzes the current status of personalized medicine in pediatric oncology in Spain. It gathers national data on the tumor molecular studies and genomic sequencing carried out at diagnosis and at relapse, the centers that perform these studies, the technology used and the interpretation and clinical applicability of the results. Current challenges and future directions to achieve a coordinated national personalized medicine strategy in pediatric oncology are also discussed. Next generation sequencing-based (NGS) gene panels are the technology used in the majority of centers and financial limitations are the main reason for not incorporating these studies into routine care. Nowadays, the application of precision medicine in pediatric oncology is a reality in a great number of Spanish centers. However, its implementation is uneven and lacks standardization of protocols; therefore, national coordination to overcome the inequalities is required. Collaborative work within the Personalized Medicine Group of SEHOP is an adequate framework for encouraging a step forward in the effort to move precision medicine into the national healthcare system.

Current status of precision medicine in pediatric oncology in Spain: a consensus report by the Spanish Society of Paediatric Haematology and Oncology (SEHOP).

The study analyzes the current status of personalized medicine in pediatric oncology in Spain. It gathers national data on the tumor molecular studies and genomic sequencing carried out at diagnosis and at relapse, the centers that perform these studies, the technology used and the interpretation and clinical applicability of the results. Current challenges and future directions to achieve a coordinated national personalized medicine strategy in pediatric oncology are also discussed. Next generation sequencing-based (NGS) gene panels are the technology used in the majority of centers and financial limitations are the main reason for not incorporating these studies into routine care. Nowadays, the application of precision medicine in pediatric oncology is a reality in a great number of Spanish centers. However, its implementation is uneven and lacks standardization of protocols; therefore, national coordination to overcome the inequalities is required. Collaborative work within the Personalized Medicine Group of SEHOP is an adequate framework for encouraging a step forward in the effort to move precision medicine into the national healthcare system.

Infratentorial congenital glioblastoma multiforme. A rare tumour with a still unknown biology. / Glioblastoma multiforme congenito infratentorial. Un tumor excepcional con una biologia aun desconocida.

INTRODUCTION: Congenital glioblastoma multiforme represents only 3% of congenital central nervous system tumours and an infratentorial location is unusual. CASE REPORT: A newborn with congenital glioblastoma multiforme with no mutation in the TP53 gene or p53 nuclear immunoreactivity that infiltrated practically the whole brainstem and also invaded supratentorial structures. CONCLUSIONS: As far as we know, only four cases with an infratentorial location have been reported previously, three in the cerebellum and one in the brainstem. The biology of congenital glioblastoma multiforme is not well known and, unlike glioblastoma multiforme in adults and children, mutations in the TP53 gene are uncommon. However, this is not associated with a more favourable prognosis. These observations suggest that specific biological processes underlie fetal glioblastoma multiforme development.

TITLE: Glioblastoma multiforme congenito infratentorial. Un tumor excepcional con una biologia aun desconocida.Introduccion. El glioblastoma multiforme congenito representa solo el 3% de los tumores congenitos del sistema nervioso central, y su ubicacion infrantentorial es excepcional. Caso clinico. Recien nacido con un glioblastoma multiforme congenito sin mutacion en el gen TP53 ni inmunorreactividad nuclear p53, que infiltraba practicamente todo el tronco cerebral e invadia tambien estructuras supratentoriales. Conclusiones. Hasta donde sabemos, solo se han referido previamente cuatro casos de localizacion infratentorial, tres en el cerebelo y uno en el tronco del encefalo. La biologia del glioblastoma multiforme congenito no se conoce bien y, a diferencia del glioblastoma multiforme en la edad adulta, las mutaciones en el gen TP53 son poco frecuentes, sin que eso parezca implicar un mejor pronostico. Estas observaciones sugieren que el glioblastoma multiforme con origen en la vida fetal tiene una biologia diferente del que se presenta en otras etapas de la vida.

Results of induction chemotherapy in children older than 18 months with stage-4 neuroblastoma treated with an adaptive-to-response modified N7 protocol (mN7)

Purpose. We report the response rate in children older than 18 months with stage 4 Neuroblastoma, using a modified dose-intensive, response-adaptive, induction mN7 protocol. Methods. From 2005 to 2012, 24 patients were treated with the mN7 protocol. Phase 1 included five MSKCC N7 cycles and surgery and two high-dose cyclophosphamide-topotecan (HD-CT) cycles for those who did not achieve complete remission (CR) and negative bone marrow (BM) minimal residual disease (MRD) status (CR+MRD-). Phase 2 consisted of myeloablative doses of topotecan, thiotepa and carboplatin plus hyperfractionated RT. Phase 3 included isotretinoin and 3F8 immunotherapy plus GM-CSF. BM MRD was monitored using GD2 synthase, PHOX2B and cyclin D1 mRNAs. Results. After 3 cycles, all patients showed BM complete histological clearance and 6 (25 %) were MRD-. Twenty of 21 s-look surgeries achieved macroscopic complete resection. After 5 cycles and surgery, 123I-MIBG scan was negative in 15 (62.5 %) cases, BM disease by histology was negative in 23 (96 %) and 10 (42 %) patients were MRD-. Twelve (50 %) pts were in CR, 2 in very good partial response (VGPR), 9 partial response (PR) and one had progressive disease. With 2 HD-CT extra cycles, 17 (71 %) pts achieved CR+MRD- status moving to phase 2. Overall and event-free survival at 3 years for the 17 patients who achieved CR+MRD- is 65 and 53 %, respectively, median follow-up 47 months. Seven (29 %) patients never achieved CR+MRD-. Univariate Cox regression analysis shows CR+MRD- status after mN7 induction as the only statistically significant prognostic factor to predict overall survival. Conclusions. mN7 induction regimen produced a CR+MRD- rate of 71 %. CR+MRD- status following induction was the only predictive marker of long-term survival (AU)

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Results of induction chemotherapy in children older than 18 months with stage-4 neuroblastoma treated with an adaptive-to-response modified N7 protocol (mN7).

PURPOSE: We report the response rate in children older than 18 months with stage 4 Neuroblastoma, using a modified dose-intensive, response-adaptive, induction mN7 protocol. METHODS: From 2005 to 2012, 24 patients were treated with the mN7 protocol. Phase 1 included five MSKCC N7 cycles and surgery and two high-dose cyclophosphamide-topotecan (HD-CT) cycles for those who did not achieve complete remission (CR) and negative bone marrow (BM) minimal residual disease (MRD) status (CR+MRD-). Phase 2 consisted of myeloablative doses of topotecan, thiotepa and carboplatin plus hyperfractionated RT. Phase 3 included isotretinoin and 3F8 immunotherapy plus GM-CSF. BM MRD was monitored using GD2 synthase, PHOX2B and cyclin D1 mRNAs. RESULTS: After 3 cycles, all patients showed BM complete histological clearance and 6 (25 %) were MRD-. Twenty of 21 s-look surgeries achieved macroscopic complete resection. After 5 cycles and surgery, (123)I-MIBG scan was negative in 15 (62.5 %) cases, BM disease by histology was negative in 23 (96 %) and 10 (42 %) patients were MRD-. Twelve (50 %) pts were in CR, 2 in very good partial response (VGPR), 9 partial response (PR) and one had progressive disease. With 2 HD-CT extra cycles, 17 (71 %) pts achieved CR+MRD- status moving to phase 2. Overall and event-free survival at 3 years for the 17 patients who achieved CR+MRD- is 65 and 53 %, respectively, median follow-up 47 months. Seven (29 %) patients never achieved CR+MRD-. Univariate Cox regression analysis shows CR+MRD- status after mN7 induction as the only statistically significant prognostic factor to predict overall survival. CONCLUSIONS: mN7 induction regimen produced a CR+MRD- rate of 71 %. CR+MRD- status following induction was the only predictive marker of long-term survival.

Rb and TP53 pathway alterations in sporadic and NF1-related malignant peripheral nerve sheath tumors.

SUMMARY: Karyotypic complexities associated with frequent loss or rearrangement of a number of chromosome arms, deletions, and mutations affecting the TP53 region, and molecular alterations of the INK4A gene have been reported in sporadic and/or neurofibromatosis type I (NF1)-related malignant peripheral nerve sheath tumors (MPNSTs). However, no investigations addressing possible different pathogenetic pathways in sporadic and NF1-associated MPNSTs have been reported. This lack is unexpected because, despite similar morphologic and immunophenotypic features, NF1-related cases are, by definition, associated with NF1 gene defects. Thus, we investigated the occurrence of TP53 and p16(INK4A) gene deregulation and the presence of microsatellite alterations at markers located at 17p, 17q, 9p21, 22q, 11q, 1p, or 2q loci in MPNSTs and neurofibromas either related (14 cases) or unrelated (14 cases) to NF1. Our results indicate that, in MPNSTs, p16(INK4A) inactivation almost equally affects both groups. However, TP53 mutations and loss of heterozygosity involving the TP53 locus (43% versus 9%), and p53 wild type overexpression, related or not to mdm2 overexpression (71% versus 25%), seem to mainly be restricted to sporadic MPNSTs. In NF1-associated MPNSTs, our microsatellite results are consistent with the occurrence of somatic inactivation by loss of heterozygosity of the second NF1 allele.

Analysis of the molecular species generated by MDM2 gene amplification in liposarcomas.

MDM2 gene is overexpressed in several tumors and its product may be processed into different isoforms, some of which have been demonstrated to possess transforming activity. In a panel of liposarcomas characterized by displaying 4 different combinations of mdm2/p53 immunoreactivity, molecular analysis of amplified MDM2 gene revealed a coexistence of mutated full-length MDM2 messenger RNAs, an out-of-frame splicing mRNA and finally aberrant spliced forms. Two of the latter are reported here for the first time. The molecular differences in this heterogeneous mRNA population seem to mirror distinct functional aspects of the altered encoded mdm2 proteins. In fact, besides the deleted transcripts defective in their ability to bind p53 and known to possess a transforming activity, here we describe both mutated full-length forms and deleted transcripts that still maintain the ability to bind p53 but, based on their mdm2+/p53+ immunophenotype, probably fail to signal its degradation. These aberrant forms, which are responsible for the accumulation and inactivation of p53, can contribute, together with the p53 independent transforming forms, to liposarcoma transforming pathway.

p53 gene status and response to platinum/paclitaxel-based chemotherapy in advanced ovarian carcinoma.

PURPOSE: The p53 gene plays a critical role in cellular response to DNA damage and has been implicated in the response to platinum compounds in ovarian carcinoma patients. Because taxanes could induce p53-independent apoptosis, we assessed the relevance of p53 gene status to response in ovarian carcinoma patients receiving paclitaxel and platinum-containing chemotherapy. PATIENTS AND METHODS: Forty-eight previously untreated patients with advanced disease received standard paclitaxel/platinum-based chemotherapy. In tumor specimens collected at the time of initial surgery, before therapy, p53 gene status and expression were examined by single-strand conformation polymorphism, sequence analysis, and immunohistochemical analysis. Microsatellite instability analysis was performed on available samples from 30 patients. RESULTS: Thirty-four (71%) of the 48 patients had a clinical response. Pathologic complete remission was documented in 13 (27%) of 48 patients. p53 mutations were detected in 29 (60%) of 48 tumors. Among the patients with mutant p53 tumors, 25 patients (86%) responded to chemotherapy. Only nine (47%) of 19 patients with wild-type p53 tumors responded to the same treatment. The overall response rate and the complete remission rate were significantly higher among patients with mutant p53 tumors than among patients with wild-type p53 tumors (P: =.008). Most of the tested tumors not associated with complete remission (10 of 12 tumors) were also characterized by microsatellite instability. The complete remission rate was higher among patients with tumors without microsatellite instability (five of seven patients). CONCLUSION: In contrast to the limited efficacy of treatment with paclitaxel in combination with standard platinum doses against wild-type p53 ovarian tumors, patients with mutant p53 ovarian tumors were more responsive to paclitaxel-based chemotherapy. The pattern of response to chemotherapy containing paclitaxel is different from that associated with high-dose cisplatin therapy. Determining p53 mutational status can be useful in predicting therapeutic response to drugs effective in ovarian carcinoma.

Biochemical uncovering of mdm2/p53 complexes in liposarcomas parallels their immunohistochemical detection.

Recent observations indicate the existence of pathogenetically distinct groups of well-differentiated (WD) dedifferentiated (DD) liposarcomas. In the retroperitoneal WD-DD liposarcomas, the predominant phenotype is represented by the aberrant (overexpressed) mdm2+/p53+ wild-type profile. At the nonretroperitoneal site, the WD liposarcomas present a wider association of MDM2/P53 gene expression; i.e., mdm2+/p53+, mdm2+/p53-, mdm2-/p53+ and mdm2-/p53-, and TP53 mutations seem to correlate with the dedifferentiation process. A biochemical study of mdm2-p53 association in 11 tumor samples characterized by the presence of different mdm2 and p53 immunophenotypes was performed. Immunoprecipitation assays using a p53-specific antibody were performed on tumor tissue and surrounding normal tissue; the immunoprecipitated material was then investigated for the presence of p53 (control) and of coimmunoprecipitated mdm2. This biochemical analysis showed that, in mdm2+/p53+/wild-type retroperitoneal liposarcomas, a band corresponded to mdm2 protein in the cellular lysates immunoprecipitated with a p53-directed antibody. In contrast, the mdm2+/p53- liposarcoma did not evidence the presence of mdm2 protein nor was p53 protein available to direct immunoprecipitation, as in the p53 mutant tumor samples with mdm2-/p53+ and mdm2-/p53- phenotypes. From the normal counterpart of retroperitoneal liposarcoma lysates, no p53 protein was immunoprecipitated. The findings in this study agree with the molecular data and they show the physical association of mdm2 and p53 in fresh liposarcoma surgical specimens.

Unilateral aneuploid dedifferentiated acinic cell carcinoma associated with bilateral-low grade diploid acinic cell carcinoma of the parotid gland.

A dedifferentiated acinic cell carcinoma (AciCC) of the right parotid gland with lymph node metastases occurred in a 36-year-old woman. The tumour was associated with a bilateral well-differentiated AciCC. The two components of this tumour had different (high and low) proliferative activity measured by Mib-1 and different (aneuploid and diploid) DNA content. Despite the presence of a high-grade component, TP53 mutations, microsatellite instability (MSI) and/or loss of heterozygosity (LOH) at the p53 locus were not detected. Although the follow-up of the patient is very short, the aggressiveness of the tumour is shown by a recurrence in the right parotid within 4 months and by the rapid development of regional metastases.

Limited role of TP53 and TP53-related genes in myxoid liposarcoma.

AIMS: Circumstantial evidence suggests that genetic changes may lead to tumor progression within the myxoid liposarcoma tumors (MLTs) carrying non-random chromosomal translocation t(12;16). METHODS: To address this subject an immunophenotypic analysis, applying antibodies against proteins encoded by TP53, MDM2 and CDK4 genes, complemented by molecular analysis of eight suitable cases, was performed on 104 consecutive cases. Chromosomal translocations were assessed either by cytogenetic analysis or by RT-PCR in 9 suitable cases and chimeric transcripts were found in all cases but two pleomorphic liposarcomas. RESULTS: Based on immunophenotyping and tumor site, the case material consisted of three groups. The first one was made up of 92 non-retroperitoneal cases carrying a null p53, mdm2, cdk4 immunophenotype, which remained unchanged over the time of recurrences and along the gamut of histologic subtypes. The second group was represented by five p53+, mdm2-, cdk4- non-retroperitoneal cases, 4 of which were further analysed by PCR-SSCP for p53 mutation. The immunophenotypic profile of these cases, complemented by the molecular findings, supported a role of TP53 in tumor progression in three high-grade MLTs. The third group, consisting of 7 retroperitoneal cases, showed a heterogeneous immunophenotype, sharing immunophenotypic and molecular features with the well-differentiated/evoluted (dedifferentiated) liposarcoma group. CONCLUSIONS: TP53 mutations seem to play a role in tumor progression in a few cases of MLTs (2.8%) showing more aggressive histologic characteristics. The unexpected finding that a number of retroperitoneal LMTs display the immunophenotypic profile of the well differentiated/evoluted (dedifferentiated) liposarcomas, deserves further investigation.

Molecular abnormalities in liposarcoma: role of MDM2 and CDK4-containing amplicons at 12q13-22.

It has recently been shown that mdm2 overexpression with stabilization of p53 represents a characteristic of retroperitoneal well-differentiated-dedifferentiated, here renamed evolved (WD-E), liposarcomas at the immunocytochemical, molecular, and cytogenetic level. This make-up appears to be confined to half the cases in non-retroperitoneal well-differentiated liposarcomas. Since in different tumours MDM2 amplification involves amplicons encompassing flanking genes, such as CDK4, the possibility was investigated that in these tumours, CDK4 could act as an alternative or additional gene involved in the transformation mechanism. Forty-one retroperitoneal (R)/non-retroperitoneal (NR) well-differentiated-dedifferentiated (WD-DD) and 33 myxoid/round cell liposarcomas were reanalysed by immunocytochemical, molecular (nine cases) and fluorescence in situ hybridization (FISH) (one case) techniques. The results showed that all but one R WD-E cases carried the mdm2+, p53+, cdk4+ immunophenotype. In NR-WD liposarcomas, this immunophenotype was shared in five cases and the remainder showed mdm2+, p53-, cdk4+ in four and mdm2-, p53-, cdk4+ in one case, showing ring chromosomes by FISH analysis. TP53 mutations are confirmed to be closely correlated with NR-DD liposarcomas and no CDK4 involvement was found in the myxoid/round cell liposarcoma group. As well as confirming the synergistic effect of MDM2 and CDK4, these results are consistent with the concept that amplicon(s) excluding MDM2 may contribute to transformation and support a role of CDK4 in opposing p53 function, particularly in NR WD liposarcoma.

Detection of TP53 mutation, loss of heterozygosity and DNA content in fine-needle aspirates of breast carcinoma.

Recent preclinical and clinical data suggest that TP53 status and TP53 mutations may be important in determining tumour aggressiveness and therapy response. In this study we investigate the feasibility of a structural and quantitative analysis of TP53 on fine-needle aspiration (FNA) material obtained from 31 consecutive female patients with breast carcinoma, enrolled in a primary chemotherapy protocol. Tumours were screened for p53 protein overexpression and TP53 mutations (exons 5-8) using immunocytochemistry, polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) and DNA sequencing analyses, and finally using fluorescence in situ hybridization (FISH) analysis. Positive nuclear staining was identified in six cases whereas mutations were detected in nine. Although the immunoreactive pattern fitted fully with the characterized TP53 mutation type, the considerable number of null p53 mutations (i.e. four) coupled with the lack of information regarding the localization of TP53 mutations make immunocytochemistry an inadequate indicator of TP53 function deregulation. Combining molecular and FISH analyses, we detected three cases with TP53 deletion and one case with deletion and mutation. Finally, DNA static-image analysis performed on 29 cases showed aneuploidy in 26 cases, which included all TP53-mutated cases. The present results show that FNA may assist clinical decisions by allowing the evaluation of a variety of biological parameters relevant for prognosis and treatment planning.

Distal intramural spread in adenocarcinoma of the lower third of the rectum treated with total rectal resection and coloanal anastomosis.

PURPOSE: This study was designed to evaluate the frequency of microscopic distal intramural spread in rectal adenocarcinoma and its correlation to other histopathologic prognostic factors. METHODS: We examined 55 patients with adenocarcinomas of the lower one-third of the rectum and measured the extent of distal intramural spread in the submucosa and/or muscular layer in comparison with Dukes Stage, diameter of tumor, distance of distal margin of resection from tumor, depth of infiltration into perirectal adipose tissue, nodal status, neoplastic infiltration of lymphatic vessels, blood vessels, and nervous branches. RESULTS: Distal intramural spread was found in 40 percent of patients, 77 percent of whom had advanced tumors with nodal metastases. Distal intramural spread appeared to be strictly related to tumor size (superior to 40 mm), infiltration of the perirectal adipose tissue, multiple positive lymph nodes, presence of neoplastic emboli in the intramural lymphatic vessels, and neoplastic invasion of the nervous branches. Local recurrence occurred in one Dukes Stage B patient with a positive distal margin of resection and in four patients with a negative distal margin of resection: three Dukes Stage C and one Dukes Stage B patients with neoplastic involvement of the circumferential margin of resection of the mesorectum. CONCLUSION: These preliminary data suggest that distal intramural spread may carry little importance in determining local recurrence of rectal adenocarcinoma.

Distinct mdm2/p53 expression patterns in liposarcoma subgroups: implications for different pathogenetic mechanisms.

Recent findings have indicated that TP53 inactivation in sarcomas may result from mutation and/or deletion of the TP53 gene or, alternatively, from binding to the MDM2 gene products. To investigate further a possible role of the two genes in sarcomas, 24 large and deep-seated lipomas and 74 liposarcomas of various subtypes were analysed for mdm2 and p53 overexpression by immunocytochemistry. Nineteen cases of the same series were also molecularly analysed for both MDM2 gene amplification and TP53 mutations, and a further ten cases for non-random chromosomal abnormalities. In the retroperitoneal well-differentiated-dedifferentiated (WD-DD) group, 15/16 WD and 8/8 DD liposarcomas displayed the mdm2+/p53+ phenotype, consistent with MDM2 gene amplification in the absence of TP53 mutations. In the non-retroperitoneal WD-DD group, 5/11 WD liposarcomas also retained the mdm2+/p53+ phenotype whereas all DD liposarcomas showed an immunophenotype and, when assessed, a genotype consistent with mutant TP53. Null mdm2 immunophenotype, coupled with evidence of a specific chromosome translocation t(12;16), was constantly observed in both the usual and the cellular subtypes of myxoid liposarcoma, three cases of which also showed TP53 alterations at the genetic or protein level. Neither mdm2 nor p53 overexpression was observed in the lipomas. The results show the existence of three main pathogenetically distinct groups of liposarcoma. The first retroperitoneal WD-DD group, which represents a novel class of tumours within a single histological category of sarcoma, where MDM2-mediated inactivation of p53 could be related to the pathogenetic mechanism. The second is the non-retroperitoneal WD-DD group, where the TP53 mutations appear to correlate with the dedifferentiation process. The third is the myxoid group, which is characterized by its own unique cytogenetic profile and never shows any involvement of TP53 or MDM2 genes. As for diagnostic significance, the absence of mdm2 and p53 reactivity in lipomas seems to represent a useful marker for differential diagnosis from lipoma-like WD liposarcomas.

Total rectal resection, mesorectum excision, and coloendoanal anastomosis: a therapeutic option for the treatment of low rectal cancer.

BACKGROUND: There is recent and sporadic evidence indicating that patients with very low rectal cancer may be treated via a sphincter-saving procedure, obviating the need for abdominoperineal resection and definitive colostomy. This study confirms these findings. METHODS: From March 1990 to October 1994, 79 patients affected with primary low rectal cancers were submitted for total rectal resection, mesorectum excision, and coloendoanal anastomosis. All lesions were located within 8 cm of the anal verge (within 6 cm in 64 cases). RESULTS: Eight patients relapsed at the pelvic level, and one patient only at the paraanastomotic site. Postoperative morbidity attributable to the procedure was low. A perfect continence was documented in 66% of cases after colostomy closure, and many patients (63%) had one or two bowel movements a day. Sixty-two patients of this series are alive, 49 without actual evidence of disease. Follow-up ranged from 2 to 56 months (median 23). CONCLUSIONS: The clinical and pathological data derived from this study suggest that radical mesorectum excision more than a large clearance margin of resection remains the most important factor in reducing the incidence of local relapse after low rectal cancer surgery and that total rectal resection and coloendoanal anastomosis is a suitable and safe option to traditional, demolitive surgical techniques.

Manual dissection of adenocarcinoma of the lower third of the rectum specimens for detection of lymph node metastases smaller than 5 mm.

BACKGROUND: The question of whether manual dissection when searching for metastatic lymph nodes from rectal cancer (less than 5 mm) is a reliable method remains controversial. METHODS: We examined 50 consecutive cases of primary adenocarcinoma of the rectum treated with a sphincter-sparing total rectum resection, total mesorectum excision, and coloanal anastomosis. We used a manual method for the detection of lymph nodes. RESULTS: One thousand seven hundred ninety-three lymph nodes were found (mean, 36 per patient). One hundred seventy-four contained metastases. Seventy-nine (45.4%) of the affected lymph nodes were less than 5 mm in greatest dimension. The percentage of metastases to small lymph nodes was similar to the percentage reported by Kotanagi (50%), but lower than the report of Herrera (78%), who used a clearing technique to search for regional lymph nodes. CONCLUSIONS: A median 17 months follow-up in these patients demonstrated that metastases in small lymph nodes are important in the accurate staging of rectal tumors and that a manual method of searching for small lymph nodes is reliable.