The gene-reduction effect of chromosomal losses detected in gastric cancers

One hundred and forty-five patients with gastric cancer who underwent a curative surgical resection between February 1996 and June 2003 were enrolled in this study. The clinicopathologic and radiologic information was obtained by reviewing the detailed records of the patients. One hundred and sixteen cases were previously analyzed by multifocal genetic examination of the heterogeneous tumor sites [3]. The remaining 29 patients were examined using a single tissue block of each tumor.

The microscopic slides were reviewed and then a tumor site that was representative of the histologic feature was chosen. The histologic type of gastric cancer was defined as intestinal (glandular, cohesive or solid), diffuse, and mixed according to the Lauren classification [15, 16] and the degree of differentiation was graded according to the WHO classification. The tumor locations considered were the cardia, body, and antrum. The clinicopathologic tumor stage was determined according to the Tumor-Node-Metastasis (TNM) criteria [19]. Most gastric cancer patients (140 of 145, 97%) had undergone R0 gastrectomy and D2 or more extended lymphadenectomy. An overall mean of 28.6 (median: 33) lymph nodes were removed along with the specimen. None of the patients received pre-operative chemotherapy and radiation therapy.

A combination therapy of intravenous mitomycin, fluorouracil, and cytarabine (MFC) followed by oral fluorouracil was administered as a standard postoperative adjuvant treatment according to the physician's judgment on the overall prognosis of each case. During the follow-up period, a physical examination, laboratory tests, chest radiography, abdominal ultrasonography or computed tomography, and gastrofiberscopy were carried out every 3 or 6 months and disease relapse was histologically confirmed, if possible. When more than one recurrence site was detected at the first time of failure, the individual recurrences were counted separately. The recurrence site was categorized into hematogenous metastasis involving the lung, liver, or other distant organs, and non-hematogenous metastasis that included nodal involvement and peritoneal dissemination. Gastric remnant cancers were excluded from the recurrence pattern analysis.

The overall survival time was calculated from the date of surgical resection until either the day of the last follow-up contact or cancer-related death. The data on patients who died from other causes was censored at the time of death. Statistical analysis was performed in April 2007. The mean follow-up period of all the surviving patients was 40 months (range: 5 to 96 months) and was completed by 98% of the enrolled patients. During the survival analysis, 50 patients had died as a result of their cancers and 12 patients had died of other causes.

Five serial 7 μm-thick sections from each formalin-fixed paraffin-embedded tissue sample were deparaffinized and briefly stained with hematoxylin and eosin. A single tumor-cell-rich focus was chosen by microscopic examination and a tumor area ranging from 5 mm to 7 mm in diameter was manually dissected under a stereomicroscope (magnification, × 40) using a surgical scalpel. The microdissected tissue pieces were microscopically examined if the tumor cell content was > 70%, which had been confirmed to reflect a difference in the genetic content between the normal and tumor tissues [3, 7].

Approximately 1,000 microdissected tumor cells were incubated in 20 μL of DNA extraction buffer (0.5% Tween-20, 1 mM EDTA pH 8.0, 50 mM Tris pH 8.0, 0.5 mg/mL proteinase K) at 37°C for 24 hr. Formalin-fixed paraffin-embedded tissue DNA tends to be poorly amplified by PCR in older specimens. Most DNAs were extracted within five years after the preparation of the pathological specimens, which ensures the DNA's quality for PCR. The amount of DNA, with varying qualities, of the tissue lysate was determined based on the PCR band intensity with 5-10 ng/μL of the DNA, which was amplified using a microsatellite primer set, D19S226 (forward: 5' - CCA GCA GAT TTT GGT GTT GTC TA - 3'; reverse: 5' - ACA GAG CCA GAG CCA GTA GGA GT - 3'; amplicon size: 164 bp). The PCR amplification was performed under a hot-start condition with using a radioisotope (α-³²P dCTP, PerkinElmer, Boston, MA, USA) as described previously [3, 7]. Briefly, a total of 10 μL PCR mixture underwent 32 cycles of a serial amplification step that consisted of 94°C for 50 sec, a primer-specific annealing temperature for 50 sec, and 72°C for 1 min. The radioisotope-labeled microsatellite sequences were separated on a 6% polyacrylamide gel that contained 7 M urea and they were visualized by repeated exposures of each autoradiograph and using a radioluminograph scanner (BAS 2500, Fuji Photo Film Co. Ltd., Kanakawa, Japan).

The guidelines for scoring the status of LOH and MSI have been detailed in previous studies that used the same panel of microsatellite markers [7]. As a reference type of the microsatellite alleles, we retrieved the dinucleotide repeat markers that ranged from 88 bp to 247 bp in amplicon size and that had a heterozygous frequency >50%. The highly polymorphic microsatellite markers on 8 cancer-associated chromosomes (3p, 4p, 5q, 8p, 9p, 13q, 17p and 18q), which frequently suffered from LOH in gastric cancer [3, 7, 20], were used to increase the number of heterozygous alleles on each arm (Figure 1A). The five microsatellite markers on each chromosomal arm showed the clear PCR bands of the heterozygous alleles and spanned the entire length of the eight chromosomes [3, 5]. To ensure the chromosomal reduction, the chromosomal loss was assigned when the LOH event involved more than two microsatellite markers on one chromosomal arm [13]. Forty pairs of microsatellite primers were mixed in a total of 22 reaction tubes and each of which contained one (4 mixtures) or two (18 mixtures) primer sets that spanned different-sized amplicons at the same annealing temperatures. The resultant unequivocal microsatellite bands indicated the high specificity of the multiplex PCR condition, which was useful for small amounts of microdissected pathologic tissues.

Based on the same classification of the microsatellite genotypes that was applied in the previous study (Figure 1B) [7], the allelic profiles of the 40 microsatellite markers were analyzed for MSI at the homozygous markers that showed a few shadow or stutter bands in a pair of normal and tumor DNAs. Because MSI obscures the heterozygous allelic status, the allelic profiles of the 40 microsatellite sequences were initially analyzed for MSI at the homozygous markers. The LOH status was determined based on the allelic loss in the heterozygous marker (Figure 1B). The extent of chromosomal losses in each case was scored according to the number of constitutional chromosomal losses involving more than one microsatellite allele.

A total of 17,723 reference genes identified in a public database (http://​genome.​ucsc.​edu/​, March 2006 assembly) [21] were analyzed to calculate the number of genes per 1-Mb nucleotides segment. Serial analysis of gene expression (SAGE) data of normal gastric mucosa was obtained from a public database (http://​www.​ncbi.​nlm.​nih.​gov/​geo/​, "SAGE_Stomach_normal_B_antrum"). The transcriptional activity of individual genes was calculated by combining the reference gene map and the expressed gene tags. Based on a comparison of the microarray and SAGE data evaluating the gene expression profiles, the number of transcripts counted in the SAGE data was found to accurately estimate a great difference in the gene activity between the stomach-specific genes and housekeeping genes [21].

Fisher's exact test and χ ² tests were used to compare the clinicopathologic features with the microsatellite genotype of the gastric cancers. Probability curves were calculated according to the Kaplan-Meier method and compared using the log-rank test. Multivariate analysis was performed by the Cox's proportional hazards method with using stepwise procedures. Probability values were two-tailed, with a P value less than 0.05 being regarded as statistically significant. The statistical software package SPSS 11.0 (SPSS Inc., Chicago, IL, USA) was used for data analysis.

Most of the gastric cancers (116 out of 145 cases) were examined in a previous multifocal analysis on the heterogeneous tumor sites from a given gastric cancer [3]. Ninety five percent of the gastric cancers examined were found to have either a similar level of LOH or MSI commonly shared by heterogeneous tumor sites and then each gastric cancer was categorized into a single microsatellite genotype, and not a mixed genotype. The microsatellite genotypes of the 27 additional cases were scored according to the level of LOH and the presence of MSI, which were detected in a single tissue piece containing a representative histological component.

Each chromosomal loss was correlated with the clinicopathologic parameters of the gastric cancers (Table 1). Most chromosomal losses (4p, 5q, 9p, 13q, 17p and 18q) were associated with late-onset disease (P < 0.05). Two or more chromosomal losses were concurrently related with the antral location (9p and 18q losses), the intestinal-or mixed-type histology (5q, 17p and 18q losses), and a poor survival (3p, 9p and 13q losses) (P < 0.05). The 17p loss was most frequent in the intestinal-type cancers (67%). The 13q loss was most frequent in the diffuse-type cancers (47%).

A large fraction (52%) of diffuse-type gastric cancers had one or no chromosomal losses, which were classified into a baseline-level LOH (LOH-B), because most of the intestinal-and mixed-type cancers (86%) had two or more chromosomal losses (Figure 2) [7]. The high-level LOH cases with four or more chromosomal losses were most frequent in the mixed differentiation cases with both intestinal- and diffuse-type gastric cancers (44%). Overall, 15 LOH-B (10%), 65 LOH-L (45%), 50 LOH-H (35%), and 15 MSI (10%) cases were identified in the 145 surgical specimens.

The density of genes per 1-Mb segment, the top 100 active genes that were highly expressed in the stomach, and the frequency of the LOH genotypes detected in gastric cancers were analyzed on each chromosome (Table 2). In comparison of the eight cancer-associated chromosomes we examined and the remaining 14 chromosomes, the density of genes (4.8 genes versus 6.9 genes per 1-Mb segment) and the average number of the highly expressed genes (3.3 genes versus 5.3 genes) were lower in the cancer-associated chromosomes than in the other chromosomes. The mean number of transcripts estimated in each highly expressed gene was also low in the cancer-associated chromosomes (54 versus 69 transcripts per each gene). The individual autosomes were grouped in the order of gene densities. The chromosomes 2, 3, 4, 5, 8, 13 and 18 were categorized into the low-gene-density group of less than five genes per 1 Mb segment. The chromosomes 11, 16, 17, 19, 20 and 22 had a high density of genes of more than eight genes per 1 Mb segment. Consequently, of the eight cancer-associated genes that were examined, six (chromosomes 3, 4, 5, 8, 13 and 18) belonged to the gene-poor chromosomes, and the remaining two genes belonged to the intermediate-(the chromosome 9) and high-(the chromosome 17) gene-density groups. The chromosomes 17 and 13 contained high and low densities of genes (13.4 and 2.7 genes per 1-Mb segment), respectively. Chromosome 17 showed a high frequency of LOH in the LOH-L (49%) but not in the LOH-B cases (13%). The frequency of chromosome 13 loss was similar in the LOH-L (25%) and LOH-B cases (27%).

The 145 gastric cancers were analyzed for the relationships between the level of LOH and the clinicopathologic features (Table 3). Both the LOH-H and LOH-B gastric cancers were commonly associated with high-risk phenotypes such as lymphatic invasion (P = 0.006), venous invasion (P = 0.005), advanced stages (P < 0.0001), recurrence (P = 0.023) and a poor survival rate (P < 0.0001), whereas the LOH-L and MSI gastric cancers were correlated with well and moderate differentiation (P = 0.033) and an early tumor stage (P < 0.0001). The two genotypes related with high-risk-phenotype were dissimilar according to the anatomical site of occurrence and the pattern of recurrence. The LOH-H cases frequently occurred in the antral portion (70%) and they recurred through hematogenous spreading to the liver, lung and other distant organs (38%), as well as non-hematogenous metastasis such as peritoneal seeding and nodal involvement (36%). Most of the LOH-B cases were present in the cardiac and body portion (94%) and they relapsed in the peritoneal cavity (67%), rather than in distant organs (13%). The onset age of the LOH-B (45 years) and LOH-H (65 years) cases was significantly different (P < 0.0001).

Gastric cancers with the MSI genotype were positively correlated with late-onset disease (67 vs. 60 years, P = 0.038), female patients (73% vs. 34%, P = 0.008), intestinal- and mixed-type cancers (100% vs. 75%, P = 0.016) and an antral location (87% vs. 51%, P = 0.037), as compared with the LOH-L cases (Table 3). Favorable tumor behaviors such as an expanding and mixed growth pattern (100% vs. 57%), no venous invasion (100% vs. 83%) and good survival (91% vs. 87%) were more closely associated with the MSI genotype than with the LOH-L genotype.

The gastric cancers were analyzed for the progression (Figure 3A) and recurrence patterns (Figure 3B) of the microsatellite genotypes in relation to the tumor size. Small gastric cancers ≤ 2 cm in diameter with LOH-H (5 cases) and LOH-B (3 cases) often developed extraserosal invasion (two LOH-H), nodal metastasis (one LOH-B and two LOH-H), hematogenous recurrence (two LOH-H) and peritoneal seeding (one LOH-B). All the small gastric cancers ≤ 2 cm in diameter with LOH-L (9 cases) and MSI (1 case) were free of extraserosal invasion, lymph node metastasis and disease relapse. The LOH-L (21 cases) and MSI (3 cases) gastric cancers ≤ 3 cm in diameter showed no recurrence. Extraserosal invasion (LOH-L, 20 cases, 31%; MSI, 6 cases, 40%) and lymph node metastasis (LOH-L, 22 cases, 34%; MSI, 4 cases, 27%) were frequently detected in the large-sized LOH-L and MSI gastric cancers ≥ 5 cm in diameter.

The LOH-H and diffuse-type LOH-B genotypes were significantly associated with poor clinicopathological features when compared with LOH-L and MSI cases (Table 3). The gastric cancers were categorized into low-risk (MSI and LOH-L) and high-risk (LOH-H and diffuse-type LOH-B) tumor according to the relationships between the microsatellite genotypes and the clinicopathologic features. The Kaplan-Meier survival curves and the log-rank analysis demonstrated that the low- and high-risk genotypes were significantly associated with good and poor survivals in both stage II (P = 0.0005) and stage III (P < 0.0001) gastric cancers (Figure 4). The Cox's proportional hazards models that disclosed the microsatellite genotypes were the most significant prognostic factor (Table 4): the hazards ratios for cancer-related deaths in the patients with high-risk genotype cancer vs. the low-risk genotype cancer were 22.077 for stages II and III (model 1; 95% confidence interval, 6.57-74.18; P < 0.0001). The high-risk and low-risk genotypes were significant independent factors of survival in stage II (model 3; 15.42, 95% confidence interval, 1.71-139.5; P = 0.015) and stage III (model 5; 19.69, 95% confidence interval, 4.38-88.53; P < 0.0001). When removing the microsatellite genotypes from the Cox's models, the cancer stage, the tumor size, the growth pattern and venous invasion were the independent prognostic factors (models 2, 4 and 6).