Intratumor genomic heterogeneity in breast cancer with clonal divergence between primary carcinomas and lymph node metastases

Conflicting theories of epithelial carcinogenesis disagree on the clonal composition of primary tumors and on the time at which metastases occur. In order to study the spatial distribution of disparate clonal populations within breast carcinomas and the extent of the genetic relationship between primary tumors and regional metastases, we have analyzed by comparative genomic hybridization 122 tissue samples from altogether 60 breast cancer patients, including 34 tumor samples obtained from different quadrants of 9 breast carcinomas, as well as paired primary-metastatic samples from 12 patients. The median intratumor genetic heterogeneity score (HS) was 17.4% and unsupervised hierarchical clustering analysis comparing the genetic features to those of an independent series of 41 breast carcinomas confirmed intratumor clonal divergence in a high proportion of cases. The median HS between paired primary breast tumors and lymph node metastases was 33.3%, but the number of genomic imbalances did not differ significantly. Clustering analysis confirmed extensive clonal divergence between primary carcinomas and lymph node metastases in several cases. In the independent series of 41 breast carcinomas, the number of genomic imbalances in primary tumors was significantly higher in patients presenting lymph node metastases (median = 15.5) than in the group with no evidence of disease spreading at diagnosis (median = 5.0). We conclude that primary breast carcinomas may be composed of several genetically heterogeneous and spatially separated cell populations and that paired primary breast tumors and lymph node metastases often present divergent clonal evolution, indicating that metastases may occur relatively early during breast carcinogenesis.     

Clonal Heterogeneity in Human Esophageal Squamous Cell Carcinomas on DNA Analysis

Cancers are thought to arise through multistep accumulation of somatic mutations in the progeny of a single cell. Multiple mutations may induce molecular intratumor heterogeneity. Therefore, we examined molecular clonal heterogeneity in esophageal squamous cell carcinomas. Twenty-four esophageal squamous cell carcinomas and associated lymph node metastases were examined for microsatellite alterations, and abnormalities of the p53 and transforming growth factor-β type II receptor ( TGF-β RII ) genes. There were eight cases (33%) showing different patterns of loss of heterozygosity in primary tumors and metastatic lymph nodes with microsatellite markers. On the other hand, the abnormalities of p53 were identical in all these cases. No mutation was detected in the simple repeated sequences of the TGF-β RII gene. These results indicate that molecular clonal heterogeneity exists in esophageal squamous cell carcinomas. Therefore, care is necessary in preoperative genetic diagnosis using biopsy samples.     

Spatial and Temporal Heterogeneity of Panel-Based Tumor Mutational Burden in Pulmonary Adenocarcinoma: Separating Biology From Technical Artifacts

BackgroundTumor mutational burden (TMB) is an emerging biomarker used to identify patients who are more likely to benefit from immuno-oncology therapy. Aside from various unsettled technical aspects, biological variables such as tumor cell content and intratumor heterogeneity may play an important role in determining TMB.MethodsTMB estimates were determined applying the TruSight Oncology 500 targeted sequencing panel. Spatial and temporal heterogeneity was analyzed by multiregion sequencing (two to six samples) of 24 pulmonary adenocarcinomas and by sequencing a set of matched primary tumors, locoregional lymph node metastases, and distant metastases in five patients.ResultsOn average, a coding region of 1.28 Mbp was covered with a mean read depth of 609x. Manual validation of the mutation-calls confirmed a good performance, but revealed noticeable misclassification during germline filtering. Different regions within a tumor showed considerable spatial TMB variance in 30% (7 of 24) of the cases (maximum difference, 14.13 mut/Mbp). Lymph node–derived TMB was significantly lower (p = 0.016). In 13 cases, distinct mutational profiles were exclusive to different regions of a tumor, leading to higher values for simulated aggregated TMB. Combined, intratumor heterogeneity and the aggregated TMB could result in divergent TMB designation in 17% of the analyzed patients. TMB variation between primary tumor and distant metastases existed but was not profound.ConclusionsOur data show that, in addition to technical aspects such as germline filtering, the tumor content and spatially divergent mutational profiles within a tumor are relevant factors influencing TMB estimation, revealing limitations of single-sample–based TMB estimations in a clinical context.     

Spatial distribution of EGFR and KRAS mutation frequencies correlates with histological growth patterns of lung adenocarcinomas

Multiregional analysis provided first indications for morphological and molecular heterogeneity in lung adenocarcinomas (ADCs), but comprehensive morpho‐molecular comparisons are still lacking. The purpose of our study was to investigate the spatial distribution of EGFR and KRAS alterations systematically throughout whole tumor cross‐sections in correlation with the tumor cell content and the histopathological patterns. Central sections of 19 ADCs were subdivided into 467 segments of 5 mm × 5 mm. We determined the predominant histological growth pattern and the allele frequencies of driver gene mutations by digital PCR in every segment. We further quantified the absolute cell counts and proportions of tumor and non‐neoplastic cells in all segments to normalize the mutant allele frequencies. Driver gene mutations could be detected in >99% of the tumor containing segments, with high levels of inter‐ and intratumor heterogeneity regarding the mutant allele frequency (range: 0.04–19.36). Different patterns for the distribution of the variant allele frequency within a tumor were recognizable. While some cases showed ubiquitously low or high levels, others revealed regions with focally elevated frequencies. Differences between KRAS and EGFR alterations were not significant. The great majority of the analyzed tumor sections (16/19) exhibited two or more morphological growth patterns. Mutant allele frequencies were significantly higher in segments with a predominant solid pattern compared to all other histologies (p < 0.01). Our data indicate that driver gene mutations are present with high levels of inter‐ and intratumor heterogeneity throughout the whole tumor, with a correlation between the allele frequencies and histological growth patterns.     

Clonal origin of lymph node metastases in bladder carcinoma

BACKGROUND: Evidence of genetic heterogeneity within urothelial carcinomas of the bladder has raised questions about the clonal origin of urothelial carcinoma and its metastases. High-grade urothelial carcinoma of the bladder frequently metastasizes to multiple regional lymph nodes in the pelvis. Whether or not these multiple lymph node metastases originate from the same tumor clone is uncertain. Molecular analysis of microsatellite alterations and X-chromosome inactivation status of distinct tumor cell populations from the same patient may further our understanding of the genetic basis of carcinoma progression and metastasis. METHODS: The authors examined 24 patients who underwent radical cystectomy for urothelial carcinoma. All patients had multiple (from two to four) lymph node metastases. Genomic DNA samples were prepared from formalin fixed, paraffin embedded tissue sections using laser-assisted microdissection. Loss of heterozygosity (LOH) assays for 3 microsatellite polymorphic markers on chromosome 9p21 (D9S171, region of putative tumor suppressor gene p16), 9q32 (D9S177, putative tumor suppressor gene involved in urothelial carcinoma tumorigenesis), and 17p13 (TP53, the p53 locus) were performed. In addition, X-chromosome inactivation analysis was performed in primary tumors and metastases from 10 female patients. RESULTS: In total, 79 tumors were analyzed. The overall frequency of allelic loss was 67% (16 of 24 tumors) in the primary urothelial carcinomas and 79% (19 of 24 tumors) in the metastatic carcinomas. The primary urothelial carcinoma showed LOH at the D9S171, D9S177, and TP53 loci in 39% (9 of 23 tumors), 30% (6 of 20 tumors), and 30% (7 of 23 tumors) of informative samples, respectively. LOH in > or = 1 lymph node metastases was seen at the D9S171, D9S177, and TP53 loci in 35% (8 of 23 tumors), 45% (9 of 20 tumors), and 48% (11 of 23 tumors) of informative samples, respectively. Eleven tumors demonstrated identical allelic loss patterns at all DNA loci both in the primary carcinoma and in all corresponding lymph node metastases. Three tumors showed allelic loss in the metastatic carcinoma but not in its matched primary carcinoma. Six tumors demonstrated a different LOH pattern in each of its lymph node metastases. Clonality analysis showed the same pattern of nonrandom X-chromosome inactivation both in the primary urothelial carcinoma and in all of the lymph node metastases in five of nine informative tumors studied. Four tumors showed a random pattern of X-chromosome inactivation in both the primary carcinoma and in the metastases. CONCLUSIONS: LOH and X-chromosome inactivation assays showed that multiple lymph node metastases and matched primary urothelial carcinomas of the bladder had the same clonal origin, suggesting that the capability for metastasis often arises in only a single clonal population in the primary tumor. The variable LOH patterns observed in some of the tumors likely reflect genetic divergence during the clonal evolution of urothelial carcinoma.     

Tumor Evolution and Intratumor Heterogeneity of an Oropharyngeal Squamous Cell Carcinoma Revealed by Whole-Genome Sequencing

Head and neck squamous cell carcinoma (HNSCC) is characterized by significant genomic instability that could lead to clonal diversity. Intratumor clonal heterogeneity has been proposed as a major attribute underlying tumor evolution, progression, and resistance to chemotherapy and radiation. Understanding genetic heterogeneity could lead to treatments specific to resistant and metastatic tumor cells. To characterize the degree of intratumor genetic heterogeneity within a single tumor, we performed whole-genome sequencing on three separate regions of an human papillomavirus (HPV)-positive oropharyngeal squamous cell carcinoma and two separate regions from one corresponding cervical lymph node metastasis. This approach achieved coverage of approximately 97.9% of the genome across all samples. In total, 5701 somatic point mutations (SPMs) and 4347 small somatic insertions and deletions (indels)were detected in at least one sample. Ninety-two percent of SPMs and 77% of indels were validated in a second set of samples adjacent to the discovery set. All five tumor samples shared 41% of SPMs, 57% of the 1805 genes with SPMs, and 34 of 55 cancer genes. The distribution of SPMs allowed phylogenetic reconstruction of this tumor''s evolutionary pathway and showed that the metastatic samples arose as a late event. The degree of intratumor heterogeneity showed that a single biopsy may not represent the entire mutational landscape of HNSCC tumors. This approach may be used to further characterize intratumor heterogeneity in more patients, and their sample-to-sample variations could reveal the evolutionary process of cancer cells, facilitate our understanding of tumorigenesis, and enable the development of novel targeted therapies.     

Clonal heterogeneity influences drug responsiveness in renal cancer assessed by ex vivo drug testing of multiple patient-derived cancer cells

Renal cell cancer (RCC) has become a prototype example of the extensive intratumor heterogeneity and clonal evolution of human cancers. However, there is little direct evidence on how the genetic heterogeneity impacts on drug response profiles of the cancer cells. Our goal was to determine how genomic clonal evolution impacts drug responses. Finding from our study could help to define the challenge that clonal evolution poses on cancer therapy. We established multiple patient-derived cells (PDCs) from different tumor regions of four RCC patients, verified their clonal relationship to each other and to the uncultured tumor tissue by genome sequencing. Furthermore, comprehensive drug-sensitivity testing with 460 oncological drugs was performed on all PDC clones. The PDCs retained many cancer-specific copy number alterations and mutations in driver genes such as VHL, PBRM1, PIK3C2A, KMD5C and TSC2 genes. The drug testing highlighted vulnerability in the PDCs toward approved RCC drugs, such as the mTOR-inhibitor temsirolimus, but also novel sensitivities were uncovered. The individual PDC clones from different tumor regions in a patient showed distinct drug–response profiles, suggesting that genomic heterogeneity contributes to the variability in drug responses. Studies of multiple PDCs from a patient with cancer are informative for elucidating cancer heterogeneity and for the determination on how the genomic evolution is manifested in cancer drug responsiveness. This approach could facilitate tailoring of drugs and drug combinations to individual patients.     

Multiscale quantification of morphological heterogeneity with creation of a predictor of longer survival in glioblastoma

Intratumor heterogeneity is a main cause of the dismal prognosis of glioblastoma (GBM). Yet, there remains a lack of a uniform assessment of the degree of heterogeneity. With a multiscale approach, we addressed the hypothesis that intratumor heterogeneity exists on different levels comprising traditional regional analyses, but also innovative methods including computer-assisted analysis of tumor morphology combined with epigenomic data. With this aim, 157 biopsies of 37 patients with therapy-naive IDH-wildtype GBM were analyzed regarding the intratumor variance of protein expression of glial marker GFAP, microglia marker Iba1 and proliferation marker Mib1. Hematoxylin and eosin stained slides were evaluated for tumor vascularization. For the estimation of pixel intensity and nuclear profiling, automated analysis was used. Additionally, DNA methylation profiling was conducted separately for the single biopsies. Scoring systems were established to integrate several parameters into one score for the four examined modalities of heterogeneity (regional, cellular, pixel-level and epigenomic). As a result, we could show that heterogeneity was detected in all four modalities. Furthermore, for the regional, cellular and epigenomic level, we confirmed the results of earlier studies stating that a higher degree of heterogeneity is associated with poorer overall survival. To integrate all modalities into one score, we designed a predictor of longer survival, which showed a highly significant separation regarding the OS. In conclusion, multiscale intratumor heterogeneity exists in glioblastoma and its degree has an impact on overall survival. In future studies, the implementation of a broadly feasible heterogeneity index should be considered.     

Multiregion whole-exome sequencing of matched primary and metastatic tumors revealed genomic heterogeneity and suggested polyclonal seeding in colorectal cancer metastasis

BackgroundDistant metastasis accounts for 90% of deaths from colorectal cancer (CRC). Genomic heterogeneity has been reported in various solid malignancies, but remains largely under-explored in metastatic CRC tumors, especially in primary to metastatic tumor evolution.Patients and methodsWe conducted high-depth whole-exome sequencing in multiple regions of matched primary and metastatic CRC tumors. Using a total of 28 tumor, normal, and lymph node tissues, we analyzed inter- and intra-individual heterogeneity, inferred the tumor subclonal architectures, and depicted the subclonal evolutionary routes from primary to metastatic tumors.ResultsCRC has significant inter-individual but relatively limited intra-individual heterogeneity. Genomic landscapes were more similar within primary, metastatic, or lymph node tumors than across these types. Metastatic tumors exhibited less intratumor heterogeneity than primary tumors, indicating that single-region sequencing may be adequate to identify important metastasis mutations to guide treatment. Remarkably, all metastatic tumors inherited multiple genetically distinct subclones from primary tumors, supporting a possible polyclonal seeding mechanism for metastasis. Analysis of one patient with the trio samples of primary, metastatic, and lymph node tumors supported a mechanism of synchronous parallel dissemination from the primary to metastatic tumors that was not mediated through lymph nodes.ConclusionsIn CRC, metastatic tumors have different but less heterogeneous genomic landscapes than primary tumors. It is possible that CRC metastasis is, at least partly, mediated through a polyclonal seeding mechanism. These findings demonstrated the rationale and feasibility for identifying and targeting primary tumor-derived metastasis-potent subclones for the prediction, prevention, and treatment of CRC metastasis.     

E-cadherin and DAP kinase in pancreatic adenocarcinoma and corresponding lymph node metastases

E-cadherin and DAP kinase have been implicated as 'invasion suppressor' genes in human cancer. The aim of this study was to analyze the methylation status of E-cadherin and DAP kinase and the expression of the protein in the metastatic lesions and to compare it with the expression in the primary tumor. Methylation-specific PCR of the DAP kinase and E-cadherin promoter was performed in 28 primary adenocarcinomas of the pancreas and in 13 corresponding regional lymph node metastases. The presence of E-cadherin and DAP kinase protein was assessed by immunohistochemistry. Metastatic lymph nodes showed a significant different expression profile from the primary tumor. E-cadherin methylation was observed in 8/28 (29%) and loss of protein expression was observed in 16/28 (57%) of pancreatic carcinomas. E-cadherin methylation was observed in 7/13 (54%) and loss of protein expression was observed in 11/13 (85%) lymph node metastases (p=0.047). DAP kinase methylation occurred in 11/28 (39%) pancreatic carcinomas and loss of protein expression was observed in 13/28 (46%). DAP kinase was methylated in 6/13 (46%) lymph node meta-stases and loss of protein expression was observed in 10/13 (77%) (p=0.039). Comparing primary tumor and corresponding lymph node metastases in 13 cases, the status of E-cadherin methylation was discordant in 2 cases. The protein expression pattern of E-cadherin and DAP kinase was discordant in 4 and 3 cases respectively. Unmethylated tumor samples did not express E-cadherin in 12 and DAP kinase protein in 6 cases. Our results demonstrate that reduction of E-cadherin and DAP kinase expression is more frequent in lymph node metastases than in the primary tumor and methylation of the promoter region contributes to this reduction; however, an alternative mechanism of inactivation seems to exist.     

Allelic imbalance in the clonal evolution of prostate carcinoma

BACKGROUND: To understand better the genetic basis of the clonal evolution of prostate carcinoma, the authors analyzed the pattern of allelic loss in 25 matched primary and metastatic prostate tumors. METHODS: Twenty-five cases were selected from the surgical pathology files of the Mayo Clinic from patients who had undergone radical retropubic prostatectomy and bilateral lymphadenectomy between 1987-1991. All patients had regional lymph node metastases at the time of surgery. DNA samples for the analysis of allelic loss pattern were prepared from primary tumors and matched synchronous lymph node metastases by tissue microdissection. The oligonucleotide primer pairs for the microsatellite DNA markers were D8S133, D8S136, D8S137, ANK1 on chromosome 8p12-21, LPLTET on chromosome 8p22, and D17S855 (intragenic to the BRCA1 gene) on chromosome 17q21. One case was not informative at any of the loci tested and was excluded from further analysis. RESULTS: The overall frequency of allelic imbalance was 79% in primary tumors and 88% in paired metastases. Of 24 informative cases, 14 patients (58%) showed the same pattern of allelic loss or retention in matched primary and metastatic tumors at all marker locus; discordant allelic loss was observed in the remaining 10 patients (42%). Four patients showed loss of the same allele at one or more marker loci in both primary and metastatic tumors, but discordant allelic loss was observed at other marker loci. Five patients showed allelic loss in at least one genetic marker in the metastatic tumor but not in its matched primary tumor. Five patients displayed loss of one allele at one or more marker loci in a primary tumor but not in the matched metastases. There was no significant difference in the frequency of allelic imbalance between primary and metastatic tumors at any marker analyzed (P>0.05). CONCLUSIONS: These data suggest that different patterns of allelic deletion may be acquired during cancer progression to metastases. The differences in genetic composition between primary prostate carcinoma and its metastases may be related to intrinsic cancer heterogeneity, overall genetic instability, and clonal divergence.     

Maintenance of head and neck tumor gene expression profiles upon lymph node metastasis

Spread of cancer and development of solid metastases at distant sites is the main cause of cancer-related deaths. To understand and treat metastases, it is important to determine at which stages the most pivotal steps for development of metastases occur. In head and neck squamous cell carcinoma (HNSCC), metastasis nearly always occurs first in local lymph nodes before development of distant metastasis. Here, we have investigated gene expression patterns in HNSCC lymph node metastases using DNA microarrays. Several types of analyses show that the gene expression patterns in lymph node metastases are most similar to the corresponding primary tumors from which they arose, as long as samples contain sufficient proportions of tumor cells. Strikingly, gene expression patterns of metastatic primary HNSCC are largely maintained upon spread to the lymph node. Only a single gene, metastasis-associated gene 1 (MTA1), was found to show consistently changed expression between a large number of matched primary tumor-lymph node metastasis pairs. The maintained expression pattern includes the predictive signature for HNSCC lymph node metastasis. These results underscore the importance of the primary tumor gene expression profile for development and treatment of metastasis. The findings also agree with the concept that disseminated cancer cells alter the surrounding tissue into a metastatic environment that resembles the primary tumor microenvironment.     

Regional DNA content heterogeneity in colonic adenocarcinoma: prognostic significance in patients with liver metastases.

We retrospectively examined by flow cytometry the DNA ploidy pattern in tissue blocks from 25 primary colon adenocarcinomas and their lymph node and liver metastases. Intratumoral heterogeneity was present in 22% of primary tumors and 21% of metastatic liver deposits. Intertumoral heterogeneity, measured between the primary tumor and its lymph node and liver metastases, was 0% and 20%, respectively. Of 24 patients who underwent successful resection of their liver metastases, 8 neoplasms had uniformly diploid DNA content, while 16 tumors had aneuploid DNA pattern in either the primary tumor, the metastases, or both. Five-year survival was better in the diploid group (38% vs. 7%, P = 0.10 by log rank analysis). Three of eight patients in the diploid group remain free of disease, while all 16 patients with aneuploid cell populations have died of recurrent disease.     

Genetic differences between primary larynx and pharynx carcinomas and their matched lymph node metastases by multiplex ligation-dependent probe amplification

Lymph nodes metastasis is a major risk factor related to poor survival in larynx and pharynx carcinomas. The aim of this study is to search for markers of lymph node involvement analyzing the genetic differences between primary larynx and pharynx squamous cell carcinomas and their corresponding lymph node metastases. Twenty-five primary tumors and their corresponding lymph node metastases were examined. DNA copy number changes of 37 genes were analyzed by multiplex ligation-dependent probe amplification (MLPA). Loss of CDKN2A (9p21) occurred in 14 out of 25 pairs (56%) of primary tumor and lymph node metastases. Loss of LMNA (1q21) was exclusively detected in 8 lymph node samples (32%). Loss of CTNNB1 (3p22) and gain of CDKN2D (19p13) were also significantly more frequent in lymph node metastases. Other aberrations related to lymph node metastases were loss of MFHAS1 (8p23), RECQL4 (8q24) and gain of N33 (8p22) and TP53 (17p13). Primary tumor and corresponding lymph node metastases showed common genetic changes. However, the lymph node metastases presented with a number of additional alterations. Acquisition of these alterations may play a role in lymphatic metastasis development.     

11q23 allelic loss is associated with regional lymph node metastasis in melanoma.

Genetic alterations of the long arm of chromosome 11 have been implicated in melanoma pathogenesis, and we recently identified two distinct regions of common allelic loss in chromosomal band 11q23. To establish the point in time of melanoma tumorigenesis at which these two putative tumor suppressor loci become relevant, we investigated allelic loss [loss of heterozygosity (LOH)] in both chromosomal regions in tumors of progressing patients. We analyzed 102 tumor samples from 23 patients for whom at least two (10 patients) or three (13 patients) tumor samples from different clinical progression steps (such as primary tumor and/or in-transit metastasis and/or regional lymph node metastasis and/or distant metastasis) were available. We detected no 11q23 LOH at any stage in 3 of 23 patients and detected LOH at all stages tested in 8 of 23 patients. In 8 of the remaining 12 (67%) patients with 11q23 LOH at some stage during tumor progression, we found this to occur first at regional lymph node metastasis. Two of these patients retained constitutional heterozygosity in several in-transit metastases that developed up to 7 months after lymph node metastases that already had loss. We therefore conclude that 11q23 LOH is associated with regional lymph node metastasis in melanoma. Finally, we detected an allele shift restricted to a histomorphologically distinct part of a primary melanoma and found that the same parental chromosome was affected by allelic loss in a subsequently occurring lymph node metastasis. These findings support our conclusion and give additional evidence for the hypothesis of molecular heterogeneity of early tumor cell populations in melanoma.     

Dedifferentiation in the metastatic progression of prostate carcinoma.

BACKGROUND: Dedifferentiation is a distinctive feature of cancer progression. Detailed histologic analysis of primary prostate carcinoma and synchronous lymph node metastases may improve our understanding of the complex process of cancer progression and metastasis. METHODS: The authors studied 242 regional lymph node positive prostate carcinoma patients who underwent radical prostatectomy and bilateral lymphadenectomy between 1987 and 1992 at the Mayo Clinic. Patients ranged in age from 47-79 years (median, 66 years). The median follow-up was 6.1 years. Gleason scores of lymph node metastases and primary tumors were compared and correlated with systemic disease progression. Histologic dedifferentiation was defined as a higher Gleason grade in the lymph node metastases than in the primary tumor. Systemic disease progression was defined as the presence of distant metastases documented by biopsies, abdominal computed tomography, plain radiograph, or bone scan. RESULTS: The 5-year systemic progression free survival (PFS) rate was 90%. The Gleason score in the lymph node metastases was higher than in the primary tumor in 45% of patients, lower in 12% of patients, and matched exactly in 43% of patients. The 5-year PFS was significantly different between patients with histologic dedifferentiation (88% +/- 3) and those without dedifferentiation (94% +/- 2) (P = 0.04). Adjusting for the Gleason grade of the primary tumor and total lymph node tumor volume, the relative risk for disease progression associated with dedifferentiation was 1.8 (95% confidence interval, 0.7-4.7; P = 0.25). CONCLUSIONS: The findings of the current study demonstrate the morphologic heterogeneity of metastases from prostate carcinoma. There is a trend toward histologic dedifferentiation when prostate carcinoma metastasizes to regional lymph nodes. This dedifferentiation, although univariately significant, was not associated with disease progression when adjusted for lymph node tumor volume.     

Failure of cryosurgical treatment of experimental intradermal tumors to eradicate microscopic lymph node metastases in guinea pigs.

Inbred Sewall Wright strain 2 male guinea pigs with established intradermal tumors and microscopic lymph node metastases were treated either by local excision, cryosurgery, or intralesional injection of BCG. Cryosurgery and local excision were effective in eliminating growth of the primary tumor but failed to prevent growth of lymph node metastases. In contrast, intralesional injection of BCG caused regression of primary tumors and prevented growth of lymph node metastases.     

鼻咽癌弥散加权成像临床研究

目的 探讨弥散加权成像(DWI)对鼻咽癌原发灶及其转移性淋巴结的诊断价值,建立ADC值对鼻咽癌原发灶和颈部转移性淋巴结的诊断阈值.方法 连续对56例鼻咽癌初诊患者和55例健康志愿者行MRI常规扫描和DWI扫描.鼻咽癌患者均行鼻咽部活检和图像引导下颈部淋巴结穿刺.检测56例鼻咽癌原发灶及其颈部转移性淋巴结和55例志愿者的鼻咽壁及其颈部淋巴结的ADC值、eADC值并行比较.结果 56例患者原发灶均经病理诊断为非角化型癌,其中51个颈部淋巴结阳性.健康志愿者检出75个颈部淋巴结.鼻咽癌患者原发灶及其转移性颈部淋巴结的ADC值均低于健康志愿者,eADC值均高于健康志愿者.ADC值≤0.809×10-3 mm2/s作为诊断鼻咽非角化型癌原发灶的阈值,其敏感性为80.4%,特异性为74.5%,阴性预测值为79.2%,阳性预测值为77.6%,准确性为78.4%.ADC值≤0.708×10-3 mm2/s作为诊断淋巴结转移的阈值,其敏感性为43.1%,特异性为93.3%,阴性预测值为70.7%,阳性预测值为81.5%,准确性为73.0%.结论 弥散加权成像对鼻咽癌原发灶及其转移性淋巴结有一定诊断价值.     

Intratumor genetic heterogeneity in advanced human colorectal adenocarcinoma.

Colorectal carcinogenesis is widely accepted as one of the best-characterized examples of stepwise progression. The existing colorectal carcinogenesis model assumes genetic homogeneity of individual tumors for the main known genetic alterations: K-ras and p53 genes point mutations and loss of heterozygosity (LOH) of chromosome 5q and 18q. The object of the present study was to demonstrate the existence of an intratumor genetic heterogeneity in advanced sporadic colorectal carcinoma for these genetic alterations. Using improved tissue microdissection and DNA extraction, for each tumor, amplifiable DNA was obtained from 15 to 20 areas, of which 1 to 2 concerned lymph node metastases (LNM). This study revealed that 10 of 15 (67%) analyzed tumors were heterogeneous for at least 1 genetic alteration, with between 2 and 6 genotypically different clones detected per tumor. No correlation was observed between the genotype of these subclones and histological differentiation or invasive propensity. Intratumor heterogeneity was more frequently observed for LOH than for point mutations, 67% and 58% for LOH at APC and DCC locus, and 20% for mutation of either the K-ras or p53 gene. In 5 of the 9 (56%) heterogeneous cases with available LNM, the genotype observed in the LNM was different from that of the main clone in the primary tumor, and moreover, 2 of the LNM displayed a genotype undetected in the primary tumor. In conclusion, intratumor genetic heterogeneity was demonstrated in advanced sporadic colorectal carcinoma and was represented as topographically distinct genotypic subclones. Taking into account such a significant genetic heterogeneity of colorectal tumors, the use of genetic markers for prognosis management should be reconsidered.