Abstract The relative contribution of tumour histologyor molecular changes, compared with invasion pattern orstage, to prognostic assessment of gastric cancer was in-vestigated in a series of 185 advanced (T2 to T4, stageIB to IV) cancers that had undergone intentionally cura-tive surgery at Varese General Hospital. Survival analy-sis of the histological types considered in commonlyused classifications, such as Lauren, Kubo, the WorldHealth Organization (WHO) and related classifications,allowed separation of a small high-grade (Hg, 12 cases)group of adenosquamous, anaplastic and small cell endo-crine carcinomas from a large cohesive group (C, 86glandular or solid cancers) and from another large (87cases) group of tumours with dissociated cells [29 dif-fuse (D) and 58 mixed (M) tumours]. Univariate andmultivariate analysis showed the independent prognosticvalue of this C/M+D/Hg classification approach, whichproved superior to other classifications and to cell disso-ciation at the growing front or angio, lympho and neuro-invasion. Expression of sialyl Lewisc, the DUPAN-2 an-tigen, proved to be an independent predictor of worse
survival among tumours beyond stage I, showing an ex-clusively or predominantly cohesive structure. Microsat-ellite instability (MSI) predicted favourable survival inpurely cohesive tumours of intermediate (II) stage, espe-cially of solid/medullary and lymphoid stroma/lympho-epithelioma-like structure, among which two distinct tumour subsets were characterised, one MSI-positive andthe other Epstein-Barr virus positive. T2N0M0 (stage IB)tumours showed mostly favourable survival indepen-dently from histological type, invasive pattern, DUPAN-2 or MSI status. It is concluded that an appropriate histo-logical evaluation, coupled with sialylated glycoproteinshistochemistry and, for stage-II tumours, MSI tests maycontribute significantly to prognostic assessment of tumours beyond stage I. However, the stage itself, withspecial reference to lymph-node metastases and invasionlevel beyond subserosa, remains the most importantprognostic clue for gastric cancer.
Histological findings relevant to the prognostic evalua-tion of gastric cancer have been investigated extensively[12, 20, 31, 35, 38, 42, 48, 55]. In general, factors such as local lymph-node or distant metastases, level of gastricwall invasion or angioinvasion have been found to bemore important than histological classification of histo-chemically assessed tumour cell phenotype [8, 20, 49].However, in some studies, gland-forming “intestinal”cancers, somewhat mimicking colorectal tumours,showed better survival than “diffuse” cancers made up ofdispersed cells, minute cellular aggregates or microglands[48, 55]. Among minor tumour subsets, solid (medullary)
C. Capella (✉ )Department of Pathology, Ospedale di Circolo, viale Borri 57,21100 Varese, Italye-mail: [email protected].: +39-332-278231, Fax: +39-332-278599
A.M. Chiaravalli · M. Cornaggia · D. Furlan · C. CapellaDepartment of Pathology, University of Insubria, Varese, Italy
R. FioccaDepartment of Pathology, University of Genova, Italy
G. TagliabueU.O. Registro Tumori, Istituto Nazionale Tumori, Milano, Italy
C. KlersyMedical Statistics Unit, IRCCS Policlinico San Matteo, Pavia, Italy
E. SolciaDepartment of Pathology, University of Pavia and IRCCS Policlinico San Matteo, Pavia, Italy
Anna Maria Chiaravalli · Matteo CornaggiaDaniela Furlan · Carlo Capella · Roberto FioccaGiovanna Tagliabue · Catherine Klersy · Enrico Solcia
The role of histological investigation in prognostic evaluation of advanced gastric cancerAnalysis of histological structure and molecular changes compared with invasive pattern and stage
cancer with cohesive cells, forming well-defined cellularaggregates devoid of gland lumina [34, 54], indolent mu-coid cancer with well-formed glandular structures sittingin lakes of extracellular mucous [9] or cancers with lym-phoid stroma with or without lymphoepithelial-type his-tology [11, 37, 60] have been associated with better sur-vival, whereas adenosquamous cancer [39, 42], hepatoidcarcinoma [40], choriocarcinoma [28] or poorly differen-tiated small cell (neuroendocrine) carcinoma [36, 50]showed a more ominous outcome. In addition, tumourswith an expanding type of growth, partly overlappingwith Lauren’s intestinal cancers, showed more favourablebehaviour than those with infiltrative type, which includemost of the diffuse cancers [38, 48]. More recently, thestudy of tumour cell dissociation at the invasion front ofthe neoplasia proved predictive of patient outcome [21].
To our knowledge, no study investigated systematicallythe predictive potential of all the different histologicaltypes, subtypes or growth patterns incorporated in the vari-ous classification systems so far developed. However, ba-sic information on histology seems now necessary not onlyfor prognostic assessment of the tumour disease but also asa prerequisite for correct interpretation of the role of genet-ic lesions and other molecular changes in its developmentand progression, an issue to which many studies are pres-ently devoted. Indeed, different genetic changes, with spe-cial reference to microsatellite instability (MSI) and p53 orcadherin E gene mutations, have been detected between in-testinal and diffuse cancers [5, 10, 33, 44, 47, 58], whichmay have epidemiological, pathogenetic and prognosticrelevance [53]. However, the precise interplay of geneticfactors with the wide spectrum of histological patterns dis-played by gastric cancer deserves further investigation.
Sialyl-glucoconjugates, such as sialyl-Lewisa (de-tected by the CA-19–9 antibody) and sialyl- Lewisx havebeen associated with poor prognosis [3, 26, 41]. TheDUPAN-2 antibody is known to react with the sialyl-Lewisc epitope (a non-fucosylated biosynthetic precursorof sialyl-Lewisa epitope), which is mostly expressed onMUC1, a large transmembrane mucin also bearing sial-yl-Lewis a and b [24, 27, 30]. The antibody reacts withnormal pancreatobiliary ducts, while showing scarce orno reactivity for normal uninflamed gastric mucosa [7,51]. DUPAN-2 has been found to be a selective markerfor pancreatic tumours of ductal origin, but it has neverbeen investigated systematically in gastric cancer.
In this paper, the main histological criteria commonlyused in gastric cancer classification and a few histology-related molecular factors have been extensively investi-gated using univariate and multivariate analysis as pre-dictors of patient outcome against a background of gas-tric wall invasion and local or distant metastases.
Materials and methods
A series of invasive (T2–T4) gastric cancers operated on at VareseGeneral Hospital from January 1980 to April 1987 were takenfrom the files of the Anatomic Pathology Service. All cases hadbeen operated in the two surgical units of the hospital, using com-
parable guidelines and procedures. No chemotherapy was given.Stained sections and paraffin blocks of primary tumour tissue(three specimens as a mean), peritumour gastric wall, resectionmargins, lymph nodes and the original macroscopic and micro-scopic reports were available for all cases. All documents pertain-ing to the clinical history of the patient, including endoscopic andradiological examinations, description of surgical intervention, periodic ambulatory or distant follow-up, family doctor and homecare and Varese Province Tumor Registry data were consulted andcarefully evaluated. Special attention was paid to the stage of tu-mour disease at the time of surgery. The assessment of metastaseswas based on extensive clinical, X-ray and ultrasonography inves-tigation, in addition to intraoperative exploration and histologicalsampling. In particular, negative liver ultrasonography and X-raylung examinations were necessary requirements to include the pa-tients among those lacking evidence of metastases. Patients whohad undergone palliative surgery were excluded from the study. Atotal of 185 patients who had undergone potentially curative sur-gery entered the study. For most surviving persons, the follow-upwas prolonged up to 4 April 2000. The cause of death was recon-structed from clinical and anatomical pathology records for thosewho died during hospitalisation. Tumour registry records and afamily doctor interview were added for those who died at home.
Paraffin sections were stained with haematoxylin and eosinand with the alcian blue–periodic acid-Schiff method. For eachcase, archival and newly stained histological sections were reas-sessed independently by at least two of us (M.C. and A.M.C.) andthan revised under the supervision of a third pathologist (C.C.),whose final judgement was taken in the few cases where the origi-nal two pathologists failed to agree. The histological classifica-tions of Lauren [31], Kubo [29], Ming [38], Watanabe et al. [61]and Gabbert et al. [21] were applied. In addition, a classificationof tumours into purely or predominantly glandular, purely or pre-dominantly diffuse, heavily mixed (glandular and diffuse with atleast 40% of each component) and mucinous (with at least 50% ofextracellular mucinous component) was applied [19], with solidand adenosquamous tumours forming minor separate subtypes. Asimplified classification scheme already adopted for molecularstudies [32] was also tested; it considered glandular (Fig. 1), dif-fuse (Fig. 2) and mixed (Fig. 3) cancers, with the last group show-ing both glandular and diffuse components in excess of 10%, solidtumours being grouped with glandular and adenosquamous tu-mours being grouped with mixed cancers. Cancers with lymphoidstroma (CLS) [60], with or without a lymphoepithelioma-like pat-tern [11], and poorly differentiated small cell (neuroendocrine)carcinomas [50] were characterised as previously reported. Tu-mour stage was assessed using the tumour node metastasis (TNM)system according to the International Union Against Cancer(UICC) [52]. Tumours with invasion limited to muscularis propria(T2m) or subserosa (T2) were separated from those involving theserosa (T3) or adjacent organs (T4).
Tumour sections were stained with the avidin–biotin immuno-peroxidase technique using monoclonal (Biogenex, San Ramon,Calif.) directed against the DUPAN-2 antigen [51]. Endocrine dif-ferentiation was searched in tumours showing solid medullary ormixed solid-diffuse histology using chromogranin A, synaptophy-sin, PGP 9.5 or neuron-specific enolase antibodies in immunohis-tochemical tests or with Grimelius silver, as previously reported[50]. Human leukocyte antigen (HLA)-DR expression was testedin lymphoepithelioma-like and medullary tumours using LN3 anti-bodies (Biotest, Dreich, Germany) while CD31 antigen immuno-staining (M823 antibody, Dako, Glostrup, Denmark) of endotheli-al cells was applied whenever conventional histology was insuffi-cient to confidently assess vessel invasion. In addition, nuclear ac-cumulation of the P53 oncoprotein was investigated with D07monoclonal antibodies (Dako), recognising both wild and mutatedmolecular forms, and the expression of hMLH-1 (G168–15 anti-body, Pharmingen, San Diego, Calif.) and hMSH-2 (FE11 anti-body, Oncogene, Cambridge, Mass.) mismatch repair genes wastested on tumour specimens using adjacent non-tumour mucosa as control tissue. All tumours showing frank (unreactivity of alltumour nuclei coupled with reactivity of non-tumour cells) or
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questionable (reduced reactivity of tumour cells) loss of nuclearimmunostaining for the latter gene products were tested for MSI atthe molecular level [13].
For the identification of MSI, tumour and non-tumour tissuewas carefully dissected out from haematoxylin–eosin-stained andconsecutive unstained sections under direct microscopic control.Polymerase chain reaction (PCR) products of DNA extracted from tumour and non-tumour tissue were obtained using specificprimers (BAT 26, BAT 25, BAT 40) which were end labelled with6-FAM phosphoramidites [64]. The PCR product was analysed inan Applied Biosystems 310 automated DNA sequencer, using theGene Scan 672 (version 1.2) software for fragment sizing analysis.According to Zhou et al. [64], MSI was present when Bat26 or, alternatively, Bat 25 and Bat40 were unstable.
The Epstein-Barr viral (EBV) genome was sought in tumoursections with a nonradioisotopic in situ hybridisation assay [46]using the digoxigenin-labelled 19-base synthetic oligonucleotideGAC AAC CAC AGA CAC CGTC [complementary to a portionof messenger (m)RNA expressed by the EBER-1 gene, known tobe actively transcribed in latently infected cells] on deparaffinisedsections treated with 0.25 mg/ml proteinase K and predenatured at95°C for 5 min. After overnight hybridisation at 37°C and carefulwashing, the sections were incubated with 1:500 diluted sheep anti-digoxigenin antibody conjugated with calf intestine phospha-tase (Boehringer, Mannheim, Germany) and developed with nitro-blue tetrazolium salt. EBV-transfected B95.8 cells (Enzo Diagnos-tics) were used as a positive control and, when hybridised with anon-correlated probe, as a negative control.
Statistics
Data were described as mean and standard deviation (SD) or me-dian and quantiles if skewed and absolute and relative frequenciesfor continuous and categorial variables, respectively. The consid-ered outcome was death from disease. All other cases were cen-sored at time of last observation or at time of death from othercauses (including perioperative death, up to 2 months after sur-gery). Death rates were calculated as events per 1000 personmonths and reported together with their 95% confidence interval(95% CI). Cox survival models were fitted in order to compute the
Fig. 1 Gastric carcinoma of glandular (cohesive) type (haematox-ylin–eosin; original magnification ×100)
Fig. 2 Gastric carcinoma of diffuse type (haematoxylin–eosin;original magnification ×200)
Fig. 3 Gastric carcinoma of mixed type (haematoxylin–eosin;original magnification ×200)
Fig. 4 Cohesive gastric carcinoma (glandular) with intense cyto-plasmic immunostaining for DUPAN-2 (immunoperoxidase; origi-nal magnification ×200)
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relative hazard (and its 95% CI) for a series of candidate prognos-tic factors. Cox model assumptions were assessed graphically bycomparing the observed survival with survival predicted by themodel. A series of tumour variables of physiopathological rele-vance (histological classification, lympho, neuro and angioinva-sion, DUPAN-2 antigen expression, MSI, diameter, level of gastricwall invasion, lymph-node involvement and distant metastases)were included in a multivariate model after checking for colineari-ty. To evaluate the role of different histological classifications,separate models were fitted. For model validation, we computedthe likelihood-based explained variation and the LeCessie–vanHouwelingen shrinkage (noise) coefficient. Discriminative capaci-ty was assessed graphically by comparing quartiles of the predic-tor indexes. Variables were included in a logical physiopathologi-cal sequence. Hierarchical models were compared by means of a likelihood ratio test. Some subgroup analysis was performed. In these cases, both the 95% CIs and the P values are to be con-sidered in the framework of an exploratory analysis. Stata 6.0(College Station, Tex.) was used for computation. A P value lessthan 0.05 was retained for statistical significance.
Results
Histological classification
With the Lauren system, the 185 tumours investigatedwere classified as reported in Table 1. At univariate Coxregression analysis, the classification as a whole gave nosignificant prognostic information, although intestinaltumours showed a slight trend toward more favourablesurvival than diffuse cancers. The World Health Organi-zation (WHO) classification showed no significant dif-ference among its numerous types and subtypes (univari-ate Cox for the model: P=0.061). However, trends werefound for the “undifferentiated” (mostly solid) carcino-mas to show a better outcome and for the adenosqua-mous and mixed signet ring/tubular cancers to show aworse prognosis. There was no substantial difference insurvival between well and poorly differentiated tumoursamong tubular cancers, while papillary and mixed signetring/tubular tumours showed a trend for more severe be-
haviour than tubular cases as a whole. Kubo’s classifica-tion showed a significant prognostic value, with more fa-vourable survival of medullary tumours and well-differ-entiated adenocarcinomas, 34 cases in all, comparedwith the remaining 151 tumours (P=0.013). There wasno substantial difference between moderately and poorlydifferentiated adenocarcinomas or between these andmucoid tumours showing large, overwhelming extracel-lular deposits of mucin. Among diffuse cancers, the signet ring subtype was slightly more favourable thanthe desmoplastic one, whereas the anaplastic subtypewas more ominous (Table 2).
To simplify Kubo’s diagnostic approach, while keep-ing as much as possible of its prognostic value, we addedmedullary to well-differentiated adenocarcinomas, poorlyto moderately differentiated adenocarcinomas, signet ringto desmoplastic diffuse cancers and anaplastic to adeno-squamous and poorly differentiated endocrine car-cinomas. As extracellular mucin was found to be largelyirrelevant in terms of survival, mucoid tumours wereclassified according to the pattern of their epithelial com-ponent. The resulting “simplified” Kubo classification is reported in Table 3. Univariate Cox analysis showedimproved survival predictive power compared with theoriginal classification, especially due to the significantlyworse prognosis of high-grade (Hg) tumours. However,the difference between the two largest groups, namelymoderately to poorly differentiated adenocarcinomas anddiffuse cancers and between the former and well-differ-entiated tumours, remained non-significant.
Previous histopathological, histochemical and molec-ular findings [12, 19, 32] suggested the opportunity totest whether mixed glandular/diffuse tumours might havea worse prognosis than purely glandular cancers. Indeed,the 60 mixed (M, Fig. 3) tumours with 10–90% dissoci-ated, diffusely growing cells (death rate 14.95, 10.97–20.38) showed significantly worse prognosis (P=0.005)than the 86 glandular (Fig. 1) to solid (cohesive, C) can-
10. Small cell (p.d. endocrine) 2 2 (100) 140.93 35.25–563.50
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cers, while resembling that of the 34 diffuse (D, Fig. 2)cancers. The resulting CMD classification is shown inTable 4, where five adenosquamous tumours were in-cluded in the mixed group.
To assess whether the extent of tumour cell dissocia-tion among the 60 mixed (D/C) cancers was prognostic-ally relevant, 22 tumours with more extensive tumourcell dissociation (>40%; MD tumours) were separatedfrom 38 tumours showing frank predominance of the co-hesive component (MC tumours). Only a slight non-sig-nificant trend for more severe prognosis was observed inthe former group (death rate 18.57, 11.20–30.81 vs13.38, 9.04–19.81), whose behaviour proved essentiallyidentical to that of purely diffuse cancers (death rate19.27, 12.81–29.00). Addition of all mixed tumoursshowing at least 10% of the dissociated component tothe diffuse cancers of CMD classification gave a largegroup of 99 cases (death rate 17.24, 13.61–21.82) show-ing obviously worse prognosis (P<0.001) than that of the86 cohesive tumours. When 12 Hg tumours (correspond-ing to group 4 of Table 3, simplified Kubo classification:five adenosquamous, five anaplastic and two poorly dif-ferentiated, small cell endocrine carcinomas) were sepa-rated from the remaining 87 M+D cases, the highly pre-dictive C/M+D/Hg classification of Table 5 was ob-tained. Univariate Cox analysis of the remaining 173 tu-mours showed that both CMD (model P=0.0073, ex-plained variation 0.0552) and C/M+D (model P=0.0019,explained variation 0.0542) classifications maintainedpredictive ability, although reduced in respect to that ofthe whole series.
Attempts to identify a low-grade group gave onlynon-significant trends for better survival of seven cyto-logically grade-1 well-differentiated adenocarcinomas,eight solid cancers and ten cohesive CLS than the re-maining cohesive cancers.
The pattern of invasive growth investigated accordingto Ming [38] showed only a non-significant trend forbetter survival of the 51 expanding than 134 infiltrativecancers (model, P=0.1812). An investigation of tumourcell dissociation at the invasion front according to Gab-bert and co-workers [21] gave more informative prog-nostic indications (model, P=0.040), with significant dif-ferences between 78 grade-3 (predominantly dissociated)tumours and 46 grade-0 (compact, 12 cases) or grade-1(with single dissociated cells, 34 cases) tumours(P=0.030) or 61 grade-2 (with partial dissociation of
tumours glands or cell complexes) tumours (P=0.044).However, no significant differences were found amonggrade-0, -1 or -2 tumours in our series, where the prog-nostic value of the Gabbert classification was essentiallydue to the significant difference (P=0.012) of grade-3cases compared with the remaining cases. The Gabbertsystem correlated with CMD-based classifications (Pear-son χ2>100, P<0.001). In particular, 31 of 34 (91%) dif-fuse and 44 of 65 (68%) mixed cancers of CMD classifi-cation fit in the Gabbert grade-3 group, compared withonly 3 of 86 (3%) of cohesive tumours. This suggeststhat cell dissociation of cancer as a whole does not differsubstantially from that occurring at its invasion front.
DUPAN-2 antigen expression
DUPAN-2 antigen was expressed by at least 1% of cellsin 122 of 185 (65.9%) tumours and by more than 5% in72 (38.9%) tumours (Fig. 4). Using univariate Cox anal-ysis, the antigen was found to predict a worse survival,with more significant results when expressed by morethan 5% of tumour cells, either in the whole series or
Table 3 Simplified version of the Kubo classification. UnivariateCox: model P=0.0001. Group 1: P=0.084 vs group 2, P=0.007 vsgroup 3 and P<0.001 vs group 4; group 2: P=0.129 vs group 3,
P<0.001 vs group 4; group 3, P=0.002 vs group 4. C.I. confidenceinterval, w.d. well-differentiated, m.d. moderately differentiated,p.d. poorly differentiated
Histological type No. Cancer deaths Death rate 95%C.I.
a Corresponding groups of the original classification
Table 4 Cohesive, mixed, diffuse (CMD) classification. Univari-ate Cox: model P=0.001; explained variation=0.072. 1 vs 2,P=0.002; 1 vs 3, P=0.001; 2 vs 3, P=0.5341; 1 vs 2+3, P<0.001.C, 77 glandular and nine solid; M, five adenosquamous and twoanaplastic; D, three anaplastic and two small cell endocrine carci-noma. CI confidence interval
Histological No. Cancer Death 95% C.I.type deaths
1. C 86 41 (48) 6.98 5.14–9.482. M 65 44 (68) 15.74 11.71–21.153. D 34 25 (74) 20.70 13.99–30.64
Table 5 Cohesive (C)/mixed (M)+diffuse (D)/high-grade (Hg)classification. Univariate Cox: model P<0.0001; explained varia-tion =0.1143. 1 vs 2, P=0.003; 1 vs 3, P<0.001; 2 vs 3, P=0.001.C.I. Confidence interval
Histological No. Cancer Death 95% C.I.type deaths rate
among cohesive cancers and mixed cancers with pre-dominance of the cohesive component (MC tumours) butnot among diffuse or mixed cancers with more than 40%dissociated tumour cells (MD tumours) (Table 6). In aparallel analysis, expression of P53 protein by more than10% of cells (64 of 185 tumours, 34.6%) showed no sig-nificant influence on survival, even when analysis wasrestricted to histological types (glandular and mixed) ofhigher expression.
The lack of DUPAN-2 effect among both MD and Dtumours suggests a need to group together all tumoursshowing more than 40% diffuse cells, irrespective of thepresence or absence of a cohesive component, whilekeeping MC tumours as a separate group with prognosissignificantly different from that of purely cohesive can-cers (P=0.016). This would offer the possibility of iden-tifying the particularly ominous subgroup of DUPAN-2-positive MC cancers significantly worse than their DU-PAN-2-negative counterpart (P<0.001) and diffuse, MDor DUPAN-2-positive cohesive cancers (P<0.05), a be-haviour mostly confirmed also after separation of the Hggroup (Table 7).
Microsatellite instability
Immunostaining for MLH-1 and MSH-2 of the 185 casesidentified 143 cases whose tumour cells retained normalnuclear reactivity for both factors, 30 cases with obvious
loss of MLH-1 (Fig. 5) and two cases with loss of MSH-2. In ten tumours, immunohistochemical tests forone (MLH-1 in four cases and MSH-2 in one case) orboth (five cases) factors failed for technical reasons orgave inconclusive results. A search for MSI at the mo-lecular level in 86 cases, including all immunohisto-chemical tests failures, gave instability in all 32 casesshowing frank loss of MLH-1 or MSH-2 immunostain-ing, in 2 of 43 cases (4.7%) retaining immunostaining ofboth factors and in 2 cases w ith inconclusive MLH-1coupled with positive MSH-2. Three of the five caseslacking reactivity for both factors and one lacking onlyMLH-1 also failed to react with the molecular test andwere dropped out of MSI analysis. The other four caseswith immunohistochemical failure of one or both factorsproved stable by means of molecular analysis. The re-maining 99 cases showing normal nuclear immunostain-ing for both MLH-1 and MSH-2 products, which shouldimply less than 5% probability of MSI (as shown usingmolecular analysis of the 86 cases), were assumed to bestable. Thus, proven MSI-positive cases turned out to be36 out of 181 (20%) successfully tested.
MSI-positive cases were preferentially concentratedamong solid tumours (six of eight cases, 76%) and glan-dular to solid CLS (four of ten, 40%) tumours (oftenshowing a prominent, partly Crohn-like lymphoid cellinfiltration), compared with the remaining glandular (19of 67, 28%), mixed (5/55, 9%) or diffuse (0) tumours ofCMD classification (Fisher exact test for MSI+ distribu-
Table 6 Stratification of DU-PAN-2 expression by histologi-cal type. C cohesive, M mixed,D diffuse, C.I. confidence in-terval. Bivariate Cox: modelP<0.0001. C, positive vs nega-tive, P=0.001; MC, positive vsnegative, P<0.001; C vs MC,P=0.012
tion among subtypes of CMD classification: P<0.001).Among subgroups of Kubo classification, MSI-positivecases were more represented among medullary tumours(12 of 17, 71%) than adenosquamous cancers (2 of 5,40%), well- (2 of 15, 13%), moderately (12 of 52, 23%)or poorly (8 of 37, 22%) differentiated adenocarcinomasor mucoid (0/5) cancers.
Univariate Cox analysis showed a significant im-provement of survival in 36 patients with MSI-positivetumours versus 145 patients with MSI-negative tumours
Fig. 5 Microsatellite-instable gastric carcinoma negative forMLH-1. Normal glandular and stromal cells are positive (immu-noperoxidase; original magnification ×400)
Fig. 6 In situ hybridisation for Epstein-Barr virus RNA (EBER-1)in a gastric carcinoma with lymphoid stroma. Intense nuclear stainis present in neoplastic epithelial cells (nitroblue tetrazoliumsalt–methyl green, original magnification ×400)
Fig. 7 Lymphoinvasion in a gastric carcinoma of diffuse type(haematoxylin–eosin; original magnification ×200)
Fig. 8 Neuroinvasion in a gastric carcinoma of mixed-type (hae-matoxylin–eosin; original magnification ×200)
(P=0.003). The improvement, however, was restricted tocohesive tumours (P=0.007 compared with correspond-ing MSI negative tumours). A trend for negative associa-tion (Fisher exact test, P<0.001) found between MSI andDUPAN-2 positives and their opposite effects on surviv-al suggested their combined use in a tentative diagnostic algorithm. As shown in Table 7, MSI-positive cohesivecases differ sharply from MSI-negative/DUPAN-2-posi-tive cases (P<0.001); however, they do not differ signif-icantly from MSI-negative/DUPAN-2-negative cases(P=0.112). Among MSI-negative tumours, the differencebetween DUPAN-2-positive and -negative cases remainssignificant (P=0.012).
EBV detection
EBV in situ hybridisation was performed on 66 tumours,inclusive of all CLS, all MSI-positive and 24 non-CLSMSI-negative cases. Eight cases were shown to be EBV-positive (Fig. 6), of which seven were among the 13 CLStumours (54%) and one (an MSI positive case) was among
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53 (2%) non-CLS tumours (Fisher exact test, P<0.001), i.e.1 of 31 MSI-positive and 0 of 24 MSI negative. Interest-ingly, among the CLS tumours, five of the six EBV-nega-tive (83%) and none of the seven EBV-positive (Fisher ex-act test, P=0.005) proved to be MSI positive. HLA-DR an-tigen was found to be widely expressed in all CLS tu-mours, with or without lymphoepithelioma-like pattern andirrespective of their EBV or MSI status. None of the EBV-positive cases and only four of 36 (11%) MSI-positivecases showed P53 protein immunostaining in more than10% tumour cells, against 60 of the remaining 139 tumours(43%; Fisher, P<0.001 vs MSI-positive cases).
Tumour stage
Tumour diameter less than 4 cm was found to signifi-cantly improve survival (death rate 5.14, 3.35–7.89)compared with those of 4–10 cm (death rate 14.82,11.98–18.33; P=0.002 vs <4 cm), while tumours of morethan 10 cm significantly worsened survival (death rate66.48, 24.95–177.14; P=0.035 vs 4–10 cm).
The influence of the level of gastric wall invasion or lymph-node involvement on survival is reported in Table 8. Stratification of lymph-node involvement as 1–3,4–8 and above 8 proved superior to the classical TNMsystem (1–7, 8–15, >15) both in terms of comparable nu-merosity of groups and of improved separation of prog-nostic classes. MSI was found to be negatively associated(Fisher exact test, P=0.006) and DUPAN-2 expressionpositively associated with lymph-node (Fisher, P=0.030)metastases, while both showed only marginal, non-signif-icant association with the level of wall invasion. On thecontrary, high histological grade was significantly associ-ated with both lymph-node involvement (Fisher, P=0.002)and level of wall invasion (Fisher, P=0.004).
(P=0.042) all proved predictive of worse survival atunivariated Cox analysis. In addition, a correlation wasfound of lymphoinvasion and neuroinvasion with bothlevel of wall invasion and lymph-node metastases (Fisher, P<0.001 in all cases), while angioinvasion corre-lated only with lymph-node metastases (P=0.002). Neu-roinvasion was also related with extranodal metastasesfound at the time of surgery (P=0.006).
Intrabdominal extranodal metastases were detected atthe time of surgery in 37 of the 185 patients (20%). Theycarried highly significant (P<0.001) worsening of surviv-al (death rate 42.48, 29.33–61.53, vs 8.89, 7.16–11.04 ofpatients without metastases). DUPAN-2 expression corre-lated positively (Fisher, P=0.008) and MSI negatively, ifany (Fisher, P=0.062), with metastases. A significant as-sociation was also found between metastases and M+Dhistology of CMD classification (Fisher, P<0.05) or Hghistology of C/M+D/Hg classification (Fisher, P<0.001).
The relationship between TNM stage and survival isshown in Table 8. It should be outlined that the prognos-tic influence of DUPAN-2 expression, MSI, lymphoinva-sion, neuroinvasion (but not angioinvasion) and histolog-ical type (CMD or C/M+D/Hg) lost significance among38 advanced tumours of stage-IB (T2N0M0). The favourable prognostic effect of MSI reached statisticalsignificance only among stage-II tumours (death rate3.26, 1.55–6.84, vs 12.96, 9.35–17.96), while the worsening effect of DUPAN-2 expression, lymphoinva-sion, neuroinvasion and M/D or Hg histology retainedsignificance among stage-II, -III and -IV tumours.
Multivariate analysis
Diameter: less than 4, 4–10 or greater than 10; level ofwall invasion: T2a, T2b or T3/4; lymph-node status: N0,N1 to N7, N greater than 7; lymphoinvasion, neuroinva-sion and angioinvasion; Kubo, simplified Kubo, CMD,
Table 8 Relationship of inva-sion level, lymph node metas-tases and tumour node metasta-sis (TNM) stage with survival.T2a vs T2b, P=0.031; T2b vs T3/4, P<0.001. LR x2 (3)= likelihood ratio test.N0 vs N1 to 7, P<0.001; N1 to 7 vs N8 to 15, P=0.016;N8 to 15 vs N>15, P=0.054.N0 vs N1 to 3, P<0.001; N1 to 3 vs N4 to 8, P=0.020;N4 to 8 vs N>8, P=0.003. IB vs II, P<0.001; II vs III, P=0.103; III vs IV, P<0.002.C.I. confidence interval
a Univariate Cox models; + formodels with A or B in additionto N0
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T2 invasion level lost significance only in the presenceof lymph-node metastases. Omission of DUPAN-2 ormetastases, though reducing significantly the informa-tion given by the model as a whole, did not change sub-stantially the contribution of the remaining variables.
Discussion
Understanding the role of a plethora of new molecularfactors in the progression of gastric cancer requires aprecise knowledge of the natural history of this complexnepotistic disease, with special reference to the impact ofhistological patterns, invasion pathways and tumourstage on patient outcome. In this study, many effortshave been dedicated to a careful dissection of the varioushistological types and subtypes of gastric cancer consid-ered in commonly used classifications and to their uni-variate and multivariate analysis from the point of viewof patient outcome, in an attempt to clarify which histo-logical structure (e.g. glandular vs solid/medullary ordiffuse) or cellular factor (cell grade, cohesion vs disso-ciation, functional differentiation) might provide a clueto prognostic evaluation. For this purpose, Kubo’s classi-fication proved superior to the essentially descriptiveWHO scheme [61] or to the popular Lauren’s classifica-tion.
By dismissing the mucoid type from a previous four-group scheme we had modulated from Kubo’s classifica-tion [19] and by classifying all tumours as gland-formingto solid (cohesive), diffuse or mixed [12, 32] based onthe cohesive or dissociated pattern of tumour cells, wefound that the cohesive-type was significantly predictiveof a more favourable prognosis than both diffuse andmixed cancers. The resulting CMD (or C/M+D) classifi-cation proved superior to Lauren and Kubo classifica-tions at both uni- and multivariate analysis and providedinformation independent from classical stage parameters.Concerning the mechanism through which the above histological patterns influence prognosis, it seems likelythat tumour cell dissociation, as found in diffuse ormixed cohesive/diffuse cancers, favours local and lymph-node invasion [21] and peritoneal involvement [17],while the degree of differentiation of glandular structureis, by itself, devoid of significant prognostic value, pro-vided cohesivity of tumour cells is retained.
Attempts to separate histologically low-grade (solid,CLS or grade-1) tumours were not successful as theirrelatively low death rate did not differ significantly fromthat of the remaining cohesive cancers. Staging, withspecific reference to stage IB (T2N0M0), proved to bemuch more effective in identifying cases with favourableprognosis among advanced gastric cancers. A smallgroup of histologically Hg tumours (6.5% of our series),formed by adenosquamous, anaplastic and poorly differ-entiated endocrine (small cell) carcinomas, proved to beprognostically worse than all other histological groups.The worse prognosis of adenosquamous [39, 42] orpoorly differentiated endocrine (small cell) carcinomas
C/M+D, C/M+D/Hg and Gabbert histological classifica-tions; greater than 5% DUPAN-2 expression; MSI andextranodal metastases were included in the multivariatemodel. Colinearity was observed between each of thehistological classifications. Thus, several models of mul-tivariate analysis were developed, where one of the his-tological classifications was combined with the remain-ing factors. Lymph-node involvement, T3/4 invasion lev-el, metastases, Hg histology of C/M+D/Hg or simplifiedKubo classification and adenosquamous and anaplastictumours of original Kubo classification and DUPAN-2antigen expression proved to be independent predictorsof poor survival (Table 9) in all models. M or M+D his-tology of CMD or C/M+D classifications and poorly dif-ferentiated adenocarcinoma of the original Kubo systemalso retained independent predictive power; however,these parameters lost independent predictive power whenthe 12 Hg cases were excluded from the analysis. Themodel with C/M+D/Hg classification proved superior toall other systems for its high-explained variation withlowest noise and equal only to that with original Kuboclassification in providing the best quartile discrimina-tion by graphical check (data not shown). The latter,however, was found to be unpractical because of unduecomplexity (ten or more subtypes considered).
At bivariate and stepwise-multivaried analysis of fac-tors included in the multivaried model with theC/M+D/Hg classification, it was found that angioinva-sion and neuroinvasion retained significance only incombination with diameter, while M+D histology lostsignificance with further addition of MSI and a diametergreater than 10 cm with the addition of invasion level.Diameter from 4 cm to 10 cm, lymphoinvasion, MSI and
Table 9 Multivariate analysis with the cohesive (C)/mixed(M)+diffuse (D)/high-grade (Hg) classification (181 tumours).Model, P<0.0001 (with C/M+D classification: P<0.0001;M+D vsC: Hazard ratio 1.66, P=0.048, 95% C.I. 1.00–2.75). Model explained variation =0.458 (0.395 with C/M+D); noise in model=0.126 (0.143 with C/M+D). C.I. confidence interval;MSI microsatellite instability
Hazard P value 95% C.I.ratio
Histology – <0.001 –M+D vs C 1.33 0.277 0.80–2.20Hg vs C 10.87 0.000 4.72–25.04
4–10 cm vs <4 cm 1.20 0.521 0.69–2.08>10 cm vs <4 cm 1.40 0.587 0.42–4.72
Invasion – 0.018 –T2b vs T2a 2.19 0.095 0.87–5.49T3 vs T2a 3.43 0.011 1.33–8.84
Lymph-node involvement – 0.001 –N1–7 vs N0 2.73 0.003 1.41–5.28N>7 vs N0 4.22 0.000 1.93–9.20
Metastases 1.74 0.028 1.06–2.87
[36, 50] have been repeatedly documented. In addition,the poor outcome of “anaplastic medullary” cancers partly mimicking embryonal carcinoma [45] or chorio-carcinoma [28] and of solid hepatoid carcinoma [40],which may also fit in our Hg group, has been outlined.Careful identification and separation of these rare tu-mour species from ordinary cancers is mandatory, be-cause their Hg histology seems to represent a strong, in-dependent predictor of poor outcome and to account fora substantial part of (though not all) the prognostic valueof mixed, diffuse and related histological types of com-mon classifications.
Improved separation of high and low survival tumourscame from the study of DUPAN-2 expression and MSI ormismatch repair gene products, whose effect on patient survival proved, however, to be, at least in part,histology dependent. DUPAN-2 expression, which waswell represented in all histological groups (with the ex-ception of solid, CLS and anaplastic tumours) significant-ly worsened survival of cohesive tumours and of mixedcancers with predominance of the cohesive component,while showing no influence on mixed tumours withprominence of the diffuse component or on purely diffusecancers. MSI and/or lack of repair gene expression,which were more frequently found in solid/medullary orglandular tumours compared with mixed cancers, whilelacking in purely diffuse cancers, showed a favourableprognostic influence on cohesive but not on mixed tu-mours. Thus, selective addition of DUPAN-2 expressionand/or MSI pattern to histological groups allowed signifi-cantly improved prediction of their behaviour.
Analysis of the stage dependence of prognostic factorsshowed that the influence of histological type, DUPAN-2expression, MSI, lymphoinvasion and neuroinvasion wassubstantially negligible among T2N0M0 tumours, whichformed a low malignancy group, accounting for about20% of the whole series. The favourable outcome of N0cancers with invasion limited to muscularis propria [23] orsubserosa [1] has already been outlined. The prominentrole of lymph-node involvement in determining the prog-nosis of advanced gastric cancer has been stressed byseveral investigators [4, 8, 42, 49]. Of interest is also our
confirmation of previous findings suggesting separation of cases with a limited number (up to three according toIchickura et al. [25]; Adachi et al. [2] and present studyand up to four according to Wu et al. [62]) of metastaticlymph nodes from those with more extensive involvement.This is an important difference with respect to the one to seven involved lymph nodes fitting in the N1 group according to the TNM system [52], especially consideringthat a 0/1 to 3 (or 4)/4 (or 5) to 8/≥9 categorisation hasbeen found to be prognostically more informative than theTNM or Japanese Rules staging systems [2, 25, 62].
The prominent negative influence of DUPAN-2 expression on survival that we have observed in thisstudy may be accounted for in part by its correlationwith lymph-node and extranodal metastases. However,DUPAN-2 antigen expression emerged as an indepen-dent prognostic factor in multivariate analysis also when
both kinds of metastatic spread were part of the model.Thus, a more specific effect of the DUPAN-2 epitope islikely to operate. Indeed, sialylated surface carbohy-drates of cancer cells have been shown to favour cancerinvasiveness by (1) inhibiting homotypic cellular aggre-gation and adhesion to extracellular matrix, thus favour-ing local invasion, lymphatic channel permeation andlymph-node involvement [41, 56], (2) acting as ligandsof E- and P-selectins in the vascular endothelium, thusfavouring tumour extravasation [59] or (3) promotingangiogenesis and haematogenous metastases [3, 43, 57].It seems likely that the biochemically related DUPAN-2epitope may act through similar mechanisms and that itsdetection in a primary cancer of purely or prevalentlyglandular structure may signal a high potential for tumour invasion and metastatic spread.
Altered expression of cadherin E, another factor play-ing a role in intercellular adhesion, has also been shown ingastric cancer to correlate with shortened survival, inde-pendently from invasion level, lymph-node involvementand blood or lymphatic vessel invasion [22]. Preliminaryinvestigation in our patient series (data not reported)showed no correlation between DUPAN-2 expression andcadherin E loss, the latter being mostly concentrated indiffuse cancers or the diffuse component of mixed can-cers, two types of tumour where, as shown in this study,DUPAN-2 expression did not influence survival. Thus,these two prognostically unfavourable changes impairingcell adhesion seem to be unrelated to each other.
The better prognosis shown by cancers with MSI con-firms previous investigations [16, 63]. However, its favourable effect on prognosis seems mostly restricted tosolid and glandular tumours of intermediate (II) stage,being overcome by the strongly favourable effect of lowstage (IB) and by the strongly unfavourable effect ofhighly advanced stage (III and IV). This stage-restrainedand histology-related pattern, in addition to its lack of independent power in multivariate models inclusive oflymph-node involvement, limits the prognostic value ofMSI, despite its obvious pathogenetic relevance [32, 44,63]. MSI may have a role in preventing lymph-node (anddistant) metastases during tumour progression. Its higherfrequency in purely cohesive and, especially, in solid/medullary tumours, scarce occurrence in mixed and lackin purely diffuse tumours suggest some negative correla-tion with factors (mutation or suppressed expression ofcadherin E and other adhesins) known to favour cell dis-sociation and a positive correlation with factors promot-ing cell cohesion without necessarily forming glands.
Our study confirms the occurrence of a prominent,partly Crohn-like lymphoid cell infiltration in MSI-posi-tive tumours [15], which has been attributed to the ex-pression, due to genetic instability, of a number of mutat-ed proteins with antigenic potential, causing a specificcytotoxic immune response directed against neoplasticcells. The widespread expression by tumour cells of cathepsin E, which may have a crucial role in antigenprocessing [6, 18] and of class II HLA molecules, with aknown role in antigen presentation, may support this in-
167
terpretation. A distinct subset of lymphoid cell-rich gas-tric cancer showed EBV genetic material, in addition toextensive expression of cathepsin E and HLA-DR anti-gen. This subset resembled more closely classic oropha-ryngeal lymphoepithelioma in its histological pattern,arose preferentially in proximal stomach or gastric stump(against the preferential antral origin of MSI positive tumours), and showed a negative correlation with MSI.A negative association between EBV and MSI was alsoobserved by Chong et al. [14] in a Japanese series ofgastric cancers. It seems likely that the two kinds of lym-phoid cell-rich tumours, both of which are negativelycorrelated with P53 protein expression, represent twoaetiologically separate types of cancer causing cellularimmune response. The latter may possibly contribute totheir relatively favourable prognosis.
In conclusion, the classification of a series of advancedgastric cancers on the basis of their cohesive (glandular tosolid) or dispersed (diffuse) pattern of cellular aggregation,identification of mixed tumours (showing both cohesiveand diffuse components) prognostically more akin withpurely diffuse cancers and separation of a small group ofhistologically Hg tumours led to substantial improvementof the outcome predictive power of histology, which ac-quired an independent prognostic value. Careful compari-son with previous investigations from the literature gavesupport to our findings and to the proposed classificationcriteria. Addition of DUPAN-2 antigen expression and, forstage-II tumours, of MSI, further improved the outcomepredictive power of histology. Precise stage assessment re-mains the main factor in prognostic evaluation of mostgastric cancers. In particular, lymph node-negative tumoursstrictly confined to the gastric wall have a mostly favour-able outcome, which is largely independent of histology,invasive pattern, DUPAN-2 expression or MSI.
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