Gian Franco Zannoni

Maria Grazia Prisco

Gaetano Valerio

Ilaria Vellone

Giovanni De Stefano

Daniela Scambia

Gallo

Franco

Maria Grazia

Gian Franco Zannoni

Valerio Gaetano Vellone

Ilaria De Stefano

Giovanni Scambia

Dr Daniela Gallo
email: d.gallo@rm.unicatt.it

cancer in elderly patients with vulvar squamous cell carcinoma: a pilot study

Keywords: Vulva, Premalignancy, Cancer, Oestrogen, E-cadherin Vulva, Premalignancy, Cancer, Oestrogen, E-cadherin

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It accounted for 26,800 cases, with the incidence rate higher than 1.5 per 100,000 women in North America, South America and Europe, and lower than 1.0 per 100,000 in developing countries. [START_REF] Sankaranarayanan | Worldwide burden of gynaecological cancer: The size of the problem[END_REF] According to NCI's SEER Cancer Statistics Review, an estimated 3,460 cases of vulvar cancer were expected to be diagnosed in the USA in 2009, with about 900 women dying of it (http://www.cancer.gov/cancertopics/types/vulvar). Notably, vulvar carcinoma is encountered most frequently in post menopause, with more than 50% of cases diagnosed over the age of 70, and incidence rate peaking in women aged 75 and older. [START_REF] Sankaranarayanan | Worldwide burden of gynaecological cancer: The size of the problem[END_REF] There are two different types of vulvar SCC.

The less common one consists of mainly non-keratinizing carcinomas, and mainly affects younger women. It is caused by infection with high-risk human papillomavirus (HPV), predominantly HPV 16 and 18, and it is associated with usual vulvar intraepithelial neoplasia (VIN, also known as Bowenoid or classic VIN, basaloid or warty subtype). [START_REF] Van De Nieuwenhof | Review of squamous premalignant vulvar lesions[END_REF] On the other hand, the most common vulvar SCC occurs in elderly women, leads to mostly differentiated keratinizing SCC, and develops following a HPV-negative pathway. Its premalignancy, differentiated (or simplex) VIN (dVIN) can be difficult to distinguish from benign vulvar lesion or normal epithelium; 2 dVIN is highly proliferative, and it is assumed that it might rapidly progress into an invasive neoplasm, since it is seldom found without vulvar carcinoma, and it is often adjacent to HPV-negative vulvar SCC. [START_REF] Yang | Vulvar intraepithelial neoplasia of the simplex (differentiated) type: a clinicopathologic study including analysis of HPV and p53 expression[END_REF][START_REF] Fox | Recent advances in the pathology of the vulva[END_REF] Within recent years, it has also clearly emerged that HPV-negative invasive SCC in elderly women is often associated with squamous cell hyperplasia (SCH) or lichen sclerosus (LS) in the adjacent tissue. The nature of this interrelationship is obscure, and these two conditions are usually considered to be of a fully benign, non-neoplastic nature. Indeed, longitudinal studies report only a slight tendency for LS to evolve into SCC (2-6%); similarly, some Authors claim a role for SCH in the development of HPV-negative vulvar SCC, but the exact pathway remains to be elucidated. [START_REF] Van De Nieuwenhof | Review of squamous premalignant vulvar lesions[END_REF] Overall, relatively little is known about molecular changes in the genesis of vulvar cancer, and only a few studies have fully taken into account the heterogeneous nature of this form of neoplasia; in The lack of knowledge of vulvar SCC oncogenesis, and the malignant potential of preneoplastic lesions, result into the absence of an evidence-based protocol for the optimal treatment and followup of patients.

In this context, we have been intrigued by the observation that the highest incidence of conventional keratinizing SCC is observed in patients with low oestrogen physiological state (postmenopausal women), this suggesting that hormonal factors could actually play a role in the development of cancer. It has been previously reported that the epithelial malignancies arising from the vulva are usually not hormonally sensitive, since no ERα expression was recorded in vulvar cancer. [START_REF] Mosny | Immunohistochemical investigations of estrogen receptors in normal and neoplastic squamous epithelium of the vulva[END_REF] However, the recent discovery of a second oestrogen receptor, termed ERβ, has shed new light on the oestrogen signalling pathway, powerfully indicating that the mechanism of oestrogen action is far more complex than anticipated. Human ERβ has a structure highly homologous to the previously known human ER (now termed ERα), and oestrogen are known to bind ERβ with affinity similar to ERα. Both isoforms regulate gene transcription by direct binding to the oestrogen-regulated element (ERE), or indirectly, by protein-protein interaction with transcription factor activator protein-1 (AP-1), and Sp1: depending on cell type, promoter context, and ligand, ERβ may antagonize ERα-induced transactivation. [START_REF] Heldring | Estrogen receptors: how do they signal and what are their targets[END_REF] Both oestrogen receptors have been identified in the normal vulvar epithelium, by biochemical, immunohistochemical, and molecular techniques, [START_REF] Schwartz | The oestrogen receptor (ER) in vulva, vagina and ovary[END_REF][START_REF] Taylor | Differential expression of oestrogen receptor isoforms and androgen receptor in the normal vulva and vagina compared with vulval lichen sclerosus and chronic vaginitis[END_REF] but, so far, no studies have investigated their possible role in the transition from normal epithelium to invasive vulvar cancer.

The aim of the present study was to examine the modulation of ERα, ERβ and PR in the normal vulva and in the SCC developing in elderly patients, extending the evaluation to vulvar lesions adjacent to SCC. In addition, since recent studies have identified E-cadherin as a novel transcriptional target of oestrogen signalling, [START_REF] Helguero | Different roles of estrogen receptors alpha and beta in the regulation of E-cadherin protein levels in a mouse mammary epithelial cell line[END_REF][START_REF] Park | Estrogen regulates Snail and Slug in the downregulation of E-cadherin and induces metastatic potential of ovarian cancer cells through estrogen receptor alpha[END_REF] the expression of this EMT marker was also studied. The expression of proliferation marker Ki67, and apoptotic marker p53 was assessed, and used as molecular indication of vulvar premalignant and malignant lesions. The results obtained suggest that changes in the expression and localization of oestrogen receptors might be key events in human vulvar tumorigenesis, although further studies are needed to confirm these preliminary observation, and to clarify the involved pathways. In addition, it would be helpful to verify whether routine assessment of their expression might serve as a marker of risk of disease progression.

MATERIAL AND METHODS

This study was conducted on 34 patients who underwent surgical resection of vulvar cancer at the Gynaecologic Oncology Unit, Catholic University of Rome. In our Institution a written informed consent is routinely requested to patients for collection of their clinical data, and paraffin embedded sections for research use. Vulvar specimens included exclusively wide excisions, hemivulvectomy and radical vulvectomy undertaken for cure. No patient received preoperative chemotherapy or radiotherapy. Carcinomas were graded according to Frankman and colleagues [START_REF] Frankman | Prognostic factors in invasive squamous cell carcinoma of the vulva[END_REF] into grade 1 well differentiated neoplasms (G1), grade 2 moderately differentiated (G2) and grade 3 poorly differentiated (G3). Staging of vulvar SCCs was performed according to the FIGO Classification. [START_REF] Vulva | American Joint Committee on Cancer: AJCC Cancer Staging Manual[END_REF] A representative block containing both normal and tumour tissue, as well as the SCC-associated vulvar lesion, was chosen for each patient. None of the samples displayed morphological evidence of HPV infection. Median age was 73 years (range 62-87). The patients, all in menopause for at least 10 years, had never undergone hormone replacement therapy. The body mass index (BMI) was calculated using the National Institutes of Health on-line BMI calculator (http://www.nhlbisupport.com/bmi/). Median BMI was 28.3 (range 20-33.2). Information concerning depth of invasion and nodal metastases was obtained from a review of pathology reports. The overall characteristics of the patients are listed in Table 1A.

Immunohistochemistry

Three-micrometer-thick paraffin sections were mounted on Superfrost coated slides and dried overnight. The sections were deparaffinised in xylene and rehydrated in graded solutions of ethanol;

the endogenous peroxidase was blocked with 3% H 2 O 2 for 5 min. Conditions for antigen retrieval, incubation times and primary antibodies used are described in Table 1B. To reduce non specific binding, the sections were incubated with 20% normal goat serum for 30 min, at room temperature.

Sections were incubated with the secondary anti-mouse/rabbit EnVision System-HRP (DakoCytomation, Carpinteria, CA, USA) for 30 min, at room temperature. The slides were

Evaluation of immunohistochemical staining

The analysis of all tissue sections was performed by two of the authors (GFZ and VGV). In case of disagreement, sections were submitted to a rejoint evaluation by a multiheaded microscope.

Expression was evaluated by considering the percentage of cells exhibiting immunoreaction as well as the intracellular localization of signalling. In normal tissues and in SCC-associated vulvar lesions, analysis was restricted to the epithelial component, where the number of positive (brown stained) cells in a random field of 300 cells was counted; the reactivity pattern of cell positivity was also recorded (i.e. basal, lower-, middle-, upper-third of the epithelium, or diffuse). In tumours, the number of positive cells was counted in five separate fields of 100 cells each (total number 500 tumour cells). Ki67 and p53 staining index was expressed as the mean percentage of positively stained nuclei in relation to the total number of epithelial-or tumour-cell nuclei considered. Ecadherin staining index was expressed as the mean percentage of cells with positive membrane staining in relation to the total number of epithelial-or tumour-cell nuclei considered. Scoring of ERs and PR was evaluated as previously reported [START_REF] Hayat | Microscopy, immunohistochemistry, and antigen retrieval methods for light and electron microscopy[END_REF][START_REF] Gallo | Estradiol and phytoestrogens differently influence the rodent postmenopausal mammary gland[END_REF] . Briefly, the mean percentage of stained cells (both nuclear and cytoplasmic staining) was categorized as follows: 0=0%, 1=1-10%, 2=11-33%, 3=34-66%, 4=67-100%. The intensity of staining was also evaluated, and graded from 0-3, where 0=no staining, 1=weak staining, 2=moderate staining, and 3=strong staining. The two values obtained were multiplied to calculate a receptor score (maximum value 12).

Statistical analysis

Statistical analysis was performed using the GraphPad Prism5 Software. Differences between NE and SCC in the percentage of Ki67-, p53

Ki67

Ki67 is a nuclear antigen expressed in cells during proliferative activity (Fig. 1B). In NE, 62% of cases showed Ki67 positivity, with a mean value of 2.1±0.2% labelled cells (Fig. 1C); positivity was restricted to the lower third of the epithelium and consistently characterized by a negative basal cell layer. Very strong Ki67-staining was observed in all SCCs (100%), with a mean value of 32.6±3.0% labelled cells (Fig. 1C). These differences achieved statistical significance (p<0.001).

Ki67 expression was recorded in 94, 100 and 100% of SCHs, LSs and dVINs adjacent to SCC, with mean values of 8.2±3, 3.2±1.0, and 13.6±3.0% labelled cells, respectively (Fig. 1C). Notably, positivity in SCHs was almost confined to the lower third of the epithelium, with basal layer cells occasionally stained; in LSs, basal layer cells were consistently stained; in dVINs immunopositivity was seen in both the lower and the middle third of the epithelium, with noticeable staining in the basal layer in all cases. p53 immunoreactivity was recognized as a nuclear staining signal (Fig. 2A). In NE, 32% of cases were positive for p53, with a mean value of 2.5±0.3% labelled cells (Fig. 2B); positivity was restricted to the lower third of the epithelium, showing expression in the basal layer only in 5 cases.

All but one SCCs showed immunopositivity (97%), with a mean value of 66±5.2% labelled cells (Fig. 2B). These differences achieved statistical significance (p<0.001). The absence of p53 expression in one of the SCCs could be due to several factors, as a complete gene deletion, or a frameshift, or a nonsense gene mutation, which has altered the structure of the p53 protein, so that it is no longer detectable by the primary antibody used.

p53 expression was recorded in 31, 80 and 100% of SCHs, LSs and dVINs adjacent to SCC, with mean values of 6.0±1.0, 14.5±6.0, and 46.0±4.0% labelled cells, respectively (Fig. 2B). Positivity in lesions was mainly evident in the lower and the middle third of the epithelium, with basal cell layer labelling observed in LSs and dVINs; in dVINs, p53 staining occasionally extended to the upper epithelium.

ERα α α α

ERα immunoreactivity was recognized as a nuclear staining signal (Fig. 3A). In NE, 44% of cases showed expression, with a mean receptor score value of 4.6±0.8 (Fig. 3B). Positivity was evident in the lower and the middle third of the epithelium, with basal cells consistently stained (Fig. 3A).

Conversely, only one case was stained in SCCs (p<0.001 vs NEs), with a receptor score of 9 (Fig. 3B).

Immunopositivity for ERα was recorded in 31, and 20 % of SCHs, and LSs adjacent to SCC, with receptor score values of 7.2±1.8, and 3, respectively. Signal was evident in the lower and the middle third of the epithelium. No expression was seen in dVINs associated with SCC (Fig. 3B). A really intriguing finding was observed in one case, which showed an abrupt interruption of labelling at the point of morphological transition from NE to dVIN (Fig. 3C). showed only cytoplasmic staining, and 15% exhibited both nuclear and cytoplasmic staining, (Fig. 4A andB). The corresponding mean receptor score values were 10.2±0.6, 8.5±0.5, and 12.0±0 for nuclear, cytoplasmic and nuclear/cytoplasmic localization, respectively (Fig. 4A andB). In all cases examined positivity extended to the whole epithelium. On the other hand, 82% of SCC cases showed low cytoplasmic immunopositivity, while the remaining 18% exhibited nuclear/cytoplasmic localization (p<0.001 vs NEs), with a receptor score of 6.3±0.7, and 9.7± 1.4, respectively (Fig. 4A andB).

Immunopositivity for ERβ in the SCHs adjacent to SCC was seen to be nuclear in 25%, cytoplasmic in 50%, and nuclear/cytoplasmic in 25% of cases; the mean receptor score was 7.5±0.9, 5.7±1.1, and 8.0±4.0 in the different intracellular compartments, respectively (Fig. 4B). In LSs, staining was nuclear in 40%, cytoplasmic in 20%, and nuclear/cytoplasmic in 40% of cases; the receptor score was 7.3±3.3, 8.0, and 3.0±0, in the different intracellular compartments (Fig. 4B).

Finally, in dVINs, staining was cytoplasmic in 23%, and nuclear/cytoplasmic in 77% of cases, with a mean receptor score of 7.3±2.4 and 8.0±1.2, in the different intracellular compartments (Fig. 4B).

In all lesions examined, staining covered the entire epithelium.

PR (A+B)

An antibody which would recognize the N-terminal region of both PRA and PRB isoforms, was used in this study. In keeping with previous findings [START_REF] Hodgins | An immunohistochemical study of androgen, oestrogen and progesterone receptors in the vulva and vagina[END_REF] , we found only occasional PR-positive cells in the epithelium of either normal tissues, SCC-associated vulvar lesions or SCCs examined. Due to the scantiness, it was not possible to perform quantification (Fig. 4C).

E-cadherin

E-cadherin immunoreactivity was recognized as a membrane staining signal (Fig. 5A). In NE, all cases (100%) showed positivity, with a mean value of 77.4±2.3% labelled cells, mainly in the lower and the middle third of the epithelium, and with expression in the basal cell layer; staining intensity was low to moderate (Fig. 5A andB). Conversely, only 26% of cases were stained in SCCs, with a mean value of 26.7±7.9% labelled cells (Fig. 5A andB). These differences achieved statistical significance (p<0.001).

All SCC-associated vulvar lesions displayed immunopositivity for E-cadherin in the lower and the middle third of the epithelium, with mean values of 60.0±4.4, 48.0±10.0, and 57.7±4.9% labelled cells, in SCHs, LSs, and dVINs, respectively (Fig. 5B).

DISCUSSION

Results from the present study demonstrated that changes in both ERα and ERβ protein expression tightly characterize the transition from normal epithelium to invasive cancer in elderly patients with vulvar squamous cell carcinomas. These changes were associated with a significant reduction in the expression of E-cadherin in tumours when compared to normal epithelium.

ERβ was the predominant steroid receptor in normal vulvar epithelium, with almost all patients showing strong nuclear positivity; on the other hand, positivity for ERα was limited to only 44% of cases. Overall, our data is in keeping with previous findings showing that ERβ prevails over ERα in the vulvar epidermis as in the skin . [START_REF] Taylor | Differential expression of oestrogen receptor isoforms and androgen receptor in the normal vulva and vagina compared with vulval lichen sclerosus and chronic vaginitis[END_REF][START_REF] Johannesson | Steroid receptor expression in the vulvar vestibular mucosa--effects of oral contraceptives and menstrual cycle[END_REF][START_REF] Thornton | The biological actions of estrogens on skin[END_REF] One of the most striking observations to emerge in our study was the shift from a mainly nuclear to a mainly cytoplasmic ERβ staining in the transition from normal epithelium to vulvar malignancy, this change being associated with a reduced protein expression. A deregulated profile was also noticed in SCC-associated vulvar lesions. Cytoplasmic ERβ staining has been reported in several studies and it is likely to be genuine and not a staining artefact. Indeed, other Authors have already reported a cytoplasmic ERβ localization in different tumours, as breast [START_REF] Shaaban | Nuclear and cytoplasmic expression of ERbeta1, ERbeta2, and ERbeta5 identifies distinct prognostic outcome for breast cancer patients[END_REF] and lung [START_REF] Zhang | Estrogen receptor beta functions through nongenomic mechanisms in lung cancer cells[END_REF] , suggesting that, in cancer cells, endogenous ERβ could be sequestered in the cytoplasm by a splice variant of metastatic tumour antigen-1 (MTA1s). [START_REF] Zhang | Estrogen receptor beta functions through nongenomic mechanisms in lung cancer cells[END_REF] Besides, a decreased ERβ expression compared with normal tissue has been observed also for other cancers, as mammary, [START_REF] Roger | Decreased expression of estrogen receptor beta protein in proliferative preinvasive mammary tumors[END_REF] prostatic, [START_REF] Horvath | Frequent loss of estrogen receptor-beta expression in prostate cancer[END_REF] ovarian, [START_REF] Lazennec | Estrogen receptor beta, a possible tumor suppressor involved in ovarian carcinogenesis[END_REF] and colorectal carcinoma. [START_REF] Foley | Selective loss of estrogen receptor beta in malignant human colon[END_REF] Noteworthy, we have recently reported that a reduction in the ERβ levels of mouse skin high-risk papillomas (premalignant lesions progressing to skin SCC) could, at least partially, account for an increased susceptibility to skin SCC observed in ovariectomized female mice when compared to intact controls. [START_REF] Mancuso | Modulation of basal and squamous cell carcinoma by endogenous estrogen in mouse models of skin cancer[END_REF] A clear reduction in the percent of ERα immunoreactive specimens was another significant finding observed in the present study, when comparing NEs to SCCs, with the same trend noticed in SCCassociated vulvar lesions, particularly dVINs, which were always negative. Overall, our data is in keeping with previous results showing that expression of ERα is completely lost in vulvar cancer, which, for this reason, is considered not hormonally sensitive. [START_REF] Mosny | Immunohistochemical investigations of estrogen receptors in normal and neoplastic squamous epithelium of the vulva[END_REF] What mechanism accounts for the altered expression of ERs in tumours, and what mechanism allows ERs dysfunction, especially loss of expression, to promote tumour progression, remain open questions. In order to partially address this issue, we evaluated the profile of E-cadherin expression in NEs, SCC-associated vulvar lesions, and tumours. It has long been recognized that the cell-cell adhesion receptor, E-cadherin, is an important determinant of tumour progression, serving as a suppressor of invasion and metastasis in many contexts. Accordingly, loss of E-cadherin function has been found widespread in sporadically arising epithelial tumours and is thought to be an important step in the development of most, if not all, epithelial-derived tumour types. [START_REF] Jeanes | Cadherins and cancer: how does cadherin dysfunction promote tumor progression?[END_REF] Besides, and importantly, there is a growing body of evidence supporting the existence of a strong connection between oestrogen signalling and E-cadherin expression, although the molecular mechanisms and the final outcomes of this connection remain ambiguous. A most credited hypothesis suggests that unliganded-and liganded-ERα exert opposite effects on E-cadherin gene transcription (stimulation and inhibition, respectively), while ERβ always acts to maintain high E-cadherin protein level. [START_REF] Helguero | Different roles of estrogen receptors alpha and beta in the regulation of E-cadherin protein levels in a mouse mammary epithelial cell line[END_REF][START_REF] Park | Estrogen regulates Snail and Slug in the downregulation of E-cadherin and induces metastatic potential of ovarian cancer cells through estrogen receptor alpha[END_REF][START_REF] Cardamone | ERalpha as ligand-independent activator of CDH-1 regulates determination and maintenance of epithelial morphology in breast cancer cells[END_REF] In this context, we could speculate that the deregulation in oestrogen receptor expression observed in vulvar cancer can be a key event in suppressing E-cadherin levels in tumour cells, thus promoting cancer progression. However, further studies are warranted to support this hypothesis, and clarify the mechanisms responsible for the decreased expression of ERs in tumours. Besides, also the possible role of different endogenous oestrogen levels on the final vulvar tumour outcome should be addressed in greater detail in future studies. Indeed, the possibility of non-ovarian sources of oestrogen could not be excluded in our study since more than 80% of patients were overweight or obese (according to their BMIs), and data stratification according to BMI values was hardly achievable, considering the limited number of case series, and the homogenous distribution of patients. Other findings observed in the present study confirm previous literature data. As expected, a very low positivity of Ki67 and p53 was noticed in NEs, while a high positivity of both proteins was seen in SCCs, in keeping with findings by other Authors. [START_REF] Chiesa-Vottero | Histopathologic study of thin vulvar squamous cell carcinomas and associated cutaneous lesions: a correlative study of 48 tumors in 44 patients with analysis of adjacent vulvar intraepithelial neoplasia types and lichen sclerosus[END_REF][START_REF] Van Der Avoort | MIB1 expression in basal cell layer: a diagnostic tool to identify premalignancies of the vulva[END_REF] Interestingly, we noticed that NEs adjacent to dVINs tended to show higher levels of both proteins (data not shown), this suggesting that normal-looking epithelium of cancer patients can harbour occult genetic alterations, which can function as markers of disease extension. As expected, and consistently to previous data, a tendency towards a deregulated expression pattern was also observed in SCC-associated vulvar lesions (particularly LSs and dVINs) [START_REF] Chiesa-Vottero | Histopathologic study of thin vulvar squamous cell carcinomas and associated cutaneous lesions: a correlative study of 48 tumors in 44 patients with analysis of adjacent vulvar intraepithelial neoplasia types and lichen sclerosus[END_REF][START_REF] Van Der Avoort | MIB1 expression in basal cell layer: a diagnostic tool to identify premalignancies of the vulva[END_REF] , where, however, a possible effect of the close tumour microenvironment cannot be discounted. In this regard, in order to ensure that a non-invasive lesion is being assessed, future studies should examine cases of dVIN distant from invasive cancers or not concurrently associated with tumour.

In conclusion, data from the present study, although needing a confirm in a larger population, has led us to the striking results that ERs expression is altered when vulva turn cancerous, thus putting the ER signalling pathway into the spotlight as a potentially important player in vulvar carcinogenesis. Assessment of the oestrogen receptor status profile should now be considered in translational arms of vulvar cancer trials, where its potential clinical role can be addressed more rigorously. Besides, additional experimental studies are needed to understand the molecular basis of this disease.

INTRODUCTION

  It has been estimated that in 2002 vulvar cancer represented about 3% of all gynaecologic cancers.

  pathogenesis from LS to differentiated VIN, and subsequent SCC, is unknown.

  with diaminobenzidine (DAB substrate System, DakoCytomation), counterstained with Mayer's Haematoxylin, dehydrated in ethanol and xylene and finally mounted.

  -specific polyclonal ERβ antibody, which would recognize all ERβ isoforms, was used in this study. In NE, strong ERβ staining was found in all examined specimens, although with a different intracellular localization: 79% of cases showed an exclusively nuclear localization, 6%

Figure 1 -

 1 Figure 1 -A) HE staining of normal vulvar epithelium (NE), squamous cell hyperplasia (SCH),

Figure 2 -

 2 Figure 2 -A) Representative pictures for p53 immunostaining showing no expression in NE, and

Figure 3 -

 3 Figure 3 -A) Representative pictures for ERα immunostaining showing nuclear protein expression

Figure 4 -

 4 Figure 4 -A) Representative pictures for ERβ immunostaining showing a strong and mainly

Figure 5 -

 5 Figure 5 -A) Representative pictures for E-cadherin immunostaining showing high expression in

Published on behalf of the British Division of the International Academy of Pathology Histopathology

Acknowledgements

We thank Dr. Ferdinando Coronetta (Department of Obstetrics and Gynecology, Catholic University of the Sacred Heart, Rome, Italy) for his skilful technical assistance in the experiments.

Author Disclosure

GF Zannoni, MG Prisco, VG Vellone, I De Stefano, G. Scambia, and D Gallo, no conflicts of interest.


MD PhD LLM James E Boers
email: j.e.boers@isala.nl

Harriëtte Meeuwissen

Natalie Methorst

two rabbit monoclonal antibodies (SP3 and 4B5) and two in situ hybridisation methods (FISH and SISH

Keywords: esophageal adenocarcinoma, gastric adenocarcinoma, HER2, immunohistochemistry, in situ hybridisation

HER2 status in gastroesophageal adenocarcinomas assessed by two rabbit monoclonal antibodies (SP3 and 4B5) and two in situ hybridisation methods (FISH and SISH)

INTRODUCTION

Therapies directed against tumors overexpressing the transmembranous HER2 receptor as a result of HER2-amplification has become widely available in the last decade for breast carcinomas. HER2positivity is reported in other carcinomas, most notably gastric and esophageal adenocarcinomas [START_REF] Marx | HER-2 amplification is highly homogenous in gastric cancer[END_REF][START_REF] Bang | HER2-positivity rates in advanced gastric cancer: results from a large international Phase III trial[END_REF][START_REF] Hofmann | Assessment of a HER2 scoring system for gastric cancer: results from a validation study[END_REF][START_REF] Reichelt | Frequent homogeneous HER-2 amplification in primary and metastatic adenocarcinoma of the esophagus[END_REF][START_REF] Ross | The HER-2/neu oncogene in tumors of the gastrointestinal tract[END_REF][START_REF] Tapia | Close association between HER-2 amplification and overexpression in human tumors of non-breast origin[END_REF][START_REF] Kanta | Topoisomerase IIalpha gene amplification in gastric carcinomas: correlation with the HER2 gene. An immunohistochemical, immunoblotting, and multicolor fluorescence in situ hybridization study[END_REF][START_REF] Kim | Evaluation of HER-2 gene status in gastric carcinoma using immunohistochemistry, fluorescence in situ hybridization, and real-time quantitative polymerase chain reaction[END_REF][START_REF] Park | HER-2/neu amplification is an independent prognostic factor in gastric cancer[END_REF][START_REF] Miller | Gene amplification in esophageal adenocarcinomas and Barrett's with high-grade dysplasia[END_REF][START_REF] Tanner | Amplification of HER-2 in gastric carcinoma: association with Topoisomerase IIalpha gene amplification, intestinal type, poor prognosis and sensitivity to trastuzumab[END_REF][START_REF] Flejou | Expression of c-erbB-2 oncogene product in Barrett's adenocarcinoma: pathological and prognostic correlations[END_REF][START_REF] Tsugawa | Amplification of the c-erbB-2 gene in gastric carcinoma: correlation with survival[END_REF][START_REF] Takehana | Status of c-erbB-2 in gastric adenocarcinoma: a comparative study of immunohistochemistry, fluorescence in situ hybridization and enzyme-linked immuno-sorbent assay[END_REF][START_REF] Motojima | erbB-2 expression in welldifferentiated adenocarcinoma of the stomach predicts shorter survival after curative resection[END_REF][START_REF] Brien | HER-2/neu gene amplification by FISH predicts poor survival in Barrett's esophagus-associated adenocarcinoma[END_REF][START_REF] Jaehne | Expression of Her2/neu oncogene product p185 in correlation to clinicopathological and prognostic factors of gastric carcinoma[END_REF][START_REF] Risio | HER2 testing in gastric cancer: molecular morphology and storage time-related changes in archival samples[END_REF][START_REF] Barros-Silva | Association of ERBB2 gene status with histopathological parameters and disease-specific survival in gastric carcinoma patients[END_REF] .

A large phase III trial employing trastuzumab, directed against the HER2 protein, has been conducted

for advanced gastric carcinomas showing clinical benefit (ToGA trial [START_REF] Van Cutsem | Efficacy results from the ToGA trial: A phase III study of trastuzumab added to standard chemotherapy in first-line human epidermal growth factor 2 (HER2)positive advanced gastric cancer[END_REF] )

Adenocarcinomas of the distal esophagus, esophago-gastric junction (EGJ) and gastric cardia carcinomas share many risk factors and the incidence of these tumors has risen dramatically in the developed world 21, 22 . On the other hand, gastric carcinomas situated in the body or antrum are epidemiologically and biologically distinct from adenocarcinomas situated at or near the EGJ. While the incidence of distal gastric carcinomas is decreasing in industrialized countries, they still constitute a major global health problem. A significant proportion of patients with distal esophageal or gastric carcinomas presents in an advanced disease stage resulting in poor overall survival 23, 24 . A first trial with anti-HER2 therapy in advanced gastric adenocarcinoma showed clinical benefit and with other ongoing trials in advanced gastric and esophageal adenocarcinomas, reliable HER2 status assessment in both esophageal and gastric adenocarcinomas is likely to become as important as in breast cancer.

HER2 status is usually determined by immunohistochemistry (IHC) and/or in situ hybridisation (ISH).

With IHC the original 4-tiered scoring system originally described for the FDA-approved HercepTest (Dako, Glostrup, Denmark) is widely used irrespective of the IHC-method employed. Samples scored as 0 and 1+ are negative, 2+ as equivocal and 3+ as positive. In the original algorithm for breast cancer, only cases with 2+ score had to be retested with ISH. However CAP/ASCO guidelines 25 require in house validation of 1+ and 3+ samples with ISH before a certified laboratory can confine ISH retesting to 2+ samples.

Recently, a modification of the HercepTest scoring system for gastric carcinomas was proposed [START_REF] Hofmann | Assessment of a HER2 scoring system for gastric cancer: results from a validation study[END_REF] .

The original system required circular staining for a 2+ / 3+ score, and staining of > 10% tumor cells in breast cancer. As noncircular basolateral IHC staining was frequently observed in gastric carcinomas, as well as strong (3+) staining of < 10% tumor cells in biopsies, these elements were added to the original HercepTest system. Novel rabbit monoclonal HER2 antibodies have been recently introduced claiming higher avidity and lower background staining. The 4B5 antibody (Ventana Medical Systems, Tucson, AZ) is directed against the extracellular domain of the HER2-receptor and is FDA-approved. Another antibody is SP3 (Labvision, Thermo Fisher Scientific, Fremont, CA) directed against the intracellular domain providing clearer staining but possibly lower sensitivity [START_REF] Milanezi | EGFR/HER2 in breast cancer: a biological approach for molecular diagnosis and therapy[END_REF] . Both antibodies claim an excellent correlation with ISH No results have been previously published using SP3 and/or 4B5 IHC or SISH in gastric or esophageal adenocarcinomas. We conducted a single institution study in 146 patients using the 2 antibodies with SISH. In addition, all cases showing 1+ immunoscore or higher were retested with Dako FISH. The objective was to determine the predictive value of both antibodies for and the incidence of HER2-amplification. Esophago-gastric Region (EGR) was defined as the distal esophagus, the esophago-gastric junction or gastric cardia. Distal stomach was defined as gastric body or antrum. On the newly cut slides stained with haematoxylin-eosin (HE), the tumor was typed as 'intestinal', 'diffuse', 'mucinous' or 'mixed' using the Laurén classification.

HER2 IHC studies were carried out on the NeXes stainer (Ventana) using 3 µm slides after antigen retrieval (10 mM citrate, pH=7,3, boiling time 25 min.). On all slides, 3 breast cancer tumor samples with an immunoscore of 0, 1+ and 3+ were used as controls. The rabbit monoclonal antibody SP3 was used in a dilution of 1:20 with a 32 min incubation time at 37ºC. Subsequently, iView DAB (Ventana) was carried out for visualisation. In our practise, 300 cases of breast cancer specimens were compared with this SP3 protocol in comparison with DAKO FISH (protocol: see below) yielding excellent concordance. Similarly, 4B5 was applied in prediluted form as provided by the manufacturer.

For scoring of HER2 immunoreactivity the modified Herceptest™ 4-tiered scoring system developed for gastric adenocarcinoma by Hofmann James Boers -HER2 in gastroesophageal adenocarcinomas p.8

RESULTS

Tumor characteristics (table 1)

Histologic characterics and the anatomical location of the 146 gastro-esophageal tumours are presented in table 1. Biopsies of 72 adenocarcinomas were from the Esophago-Gastric Region (EGR); 44 (61%) from the distal esophagus and 28 (39%) from the gastric cardia. 63 were classified as intestinal type, 6 as mixed and 3 as mucinous. 74 adenocarcinomas were from the distal stomach; 24 (32%) in the gastric body and 50 (68%) in the antrum. 54 were of intestinal type, 9 mixed, 9 diffuse and 2 of mucinous type.

HER2 immunostains (table 2)

SP3 immunoreactivity (figure 1) was completely absent in 125 (86%) cases. An immunoscore of 1+ was seen in 4, 2+ in 6 and 3+ in 11 cases. Membranous 4B5 immunoreactivity (figure 2) was absent in 106 (72%) cases; immunoscores of 1+ were present in 17 cases, 2+ in 6 and 3+ in 17 . All cases with immunoscores of 1+ and higher showed immunoreactivity in at least 10% of tumor cells. U-shaped basolateral staining was quite often noted. Background staining was enterily absent with the SP3antibody. The 4B5 stains showed invariably extensive cytoplasmatic background staining of the gastric foveolar layer (figure 5A); intestinal metasplasia when present also showed this staining pattern but less substantial. In addition, cytoplasmatic background staining of tumor was noted in 40 (27%) cases of which 7 (5%) strong with nuclear immunoreactivity. In additional 2 cases, only strong background staining was noted without notable membranous staining (figure 5B).

HER2 SISH / FISH versus Immunoscore (tables 3A and 3B)

SISH was performed in all 146 cases (table 3A; figure 3). In some cases of SISH some nuclear haze was present, possibly as a result of fixation time of approximately 6h (figure 5C). All 17 cases with SP3 immunoscores 2+/3+ were amplified as were 5 out of 129 (4%) cases with negative SP3 immunohistochemistry (immunoscores 0/1+). Only 1 James Boers -HER2 in gastroesophageal adenocarcinomas p.9

SP3-immunoscore 1+ were 2+ in the 4B5 in 2 cases, and 1+ in one case. Two cases with strong 4B5 background staining of nuclei (but no membranous staining) were not amplified.

Sensitivity for amplification was 77.3% for SP3 and 95.5% 4B5 ' IHC positive' cases as defined by immunoscores 2+/3+. Specificity was 100% for SP3-positive IHC and 98.4% for 4B5-positive IHC, respectively .The positive predictive value (PPV) of SP3-positivity was 100% and the negative predictive value (NPV) 96.1%. For 4B5 the PPV was 91.3% and the NPV 99.2%.

Of 42 performed FISH procedures (figure 4) the results were identical to SISH. 22 cases were amplified (table 3B). Granular red background staining was seen in a minority of cases in both tumor and normal epithelial cells (figure 5D).

Heterogeneity of HER2 positivity (table 4)

Heterogeneity of HER2-immunoreactivity was the dominant pattern, and areas of HER2

DISCUSSION

In a study of 146 cases with biopsies of primary esophagus and gastric adenocarcinomas, frequent HER2-immunoreactivity using 2 novel rabbit monoclonal antibodies was identified. Both SP3 and 4B5

immunostaining had a high positive predictive value for HER2-amplification. 4B5 had a much higher negative predictive value than SP3. SISH -when compared with FISH in selected cases -yielded identical results compared with FISH. HER2-amplification was most often present in biopsies of esophagus and gastric cardia adenocarcinomas (27% and 18%, respectively); amplification was less frequent in adenocarcinomas of the body and antrum region (7%).

Many studies have been published on the concordance of HER2 immunohistochemistry and in situ hybridisation in breast cancer [START_REF] Wolff | American Society of Clinical Oncology/College of American Pathologists Guideline Recommendations for Human Epidermal Growth Factor Receptor 2 Testing in Breast Cancer[END_REF] , usually focusing on the rabbit polyclonal antibody A408 (used in HercepTest) or the mouse monoclonal CB11 (used in the previous generation of Ventana Pathway®), both FDA-approved kits. In daily practice the application of these antibodies results in a relatively high percentage of equivocal 2+-immunoscores. In order to achieve an unequivocal 3+immunoscore pattern in cases with high amplification a high background in normal tissue has to be accepted precluding easy and reproducible assessment of immunohistochemistry. The new generation of more avid rabbit monoclonal antibodies has been developed in order to produce better signal-to-background patterns. In the field of Estrogen Receptor-immunohistochemistry, the new SP1antibody has become an acclaimed test. SP3 and 4B5 are new rabbit monoclonal antibodies directed against the intracellular and extracellular of the HER2-receptor, respectively.

Ricardo et al [START_REF] Ricardo | HER2 evaluation using the novel rabbit monoclonal antibody SP3 and CISH in tissue microarrays of invasive breast carcinomas[END_REF] James Boers -HER2 in gastroesophageal adenocarcinomas p.13 junction had a HER2-positivity of 20.9% and 33.2%, respectively. This is substantially higher than most reports including the present study. In contrast to the present study, they found 4.9% FISH positive cases in which IHC was completely negative, and a 15% amplification rate when immunoscore was 1+. Subsequent analysis failed to show clinical benefit of trastuzumab in the FISHpositive but immunonegative subgroups. Another possible explanation might be that patients with advanced disease only were accrued for the ToGA trial while HER2-amplification could be to be a late event in oncogenesis. The average HER2-positivity rates of the patients included in the ToGA at the time of the report of Bang et al. did not vary according the geographical region (Europe or Asia) but was a function of histological type in concordance with our study.

The few larger studies with in situ hybridisation confirmed HER2 status in esophageal adenocarcinoma report a HER2-positivity rate of at least 15%. Our 27% HER2 amplification rate is in line with substantial higher HER2-positivity in esophageal carcinoma rather than (distal) gastric adenocarcinoma. Unfortunately, questions concerning definitions, segmentation of oncological entities, division of treatment groups and different research interests hinder a comprehensive view of HER2-positivity rates of the anatomical region comprising the distal esophagus and the entire stomach.

An important difference in HER2 immunoreactivity and amplification between breast cancer and gastro-esophageal adenocarcinomas is the striking heterogeneity of HER2-positivity in the latter. The 2007 ASCO/CAP HER2 Guidelines [START_REF] Wolff | American Society of Clinical Oncology/College of American Pathologists Guideline Recommendations for Human Epidermal Growth Factor Receptor 2 Testing in Breast Cancer[END_REF] in breast cancer demands HER2-immunoreactivity/amplification in at least 30% of breast cancer tumor areas in order to render a HER2-positivity as a conclusion.

Hofmann et al [START_REF] Hofmann | Assessment of a HER2 scoring system for gastric cancer: results from a validation study[END_REF] propose to allow even less than the original HercepTest 10% 3+ pattern of immunoreactivity in biopsies of gastric carcinomas; in resection specimens the 10% threshold is retained. The considerable heterogeneity of HER2-positivity -73% of all HER2-amplified cases in our study -suggests that HER2 amplification is a late event in gastro-esophageal adenocarcinomas.

Nevertheless, a recent report of a large phase III trial using trastuzumab (ToGA trial [START_REF] Van Cutsem | Efficacy results from the ToGA trial: A phase III study of trastuzumab added to standard chemotherapy in first-line human epidermal growth factor 2 (HER2)positive advanced gastric cancer[END_REF] ) showed clinical benefit, especially in the group with both FISH positivity and an immunoscore of at least 2+ using Herceptest. The magnitude of clinical benefit is similar to metastatic breast carcinomas. This is remarkable considering the heterogeneous HER2-positivity in gastric adenocarcinomas in contrast to breast cancer, in which more than 95% of HER2-positive cases are homogeneously HER2-amplified.

Recently, Marx et al. [START_REF] Marx | HER-2 amplification is highly homogenous in gastric cancer[END_REF]

reported highly homogeneous HER2 amplification in multiple sections obtained

Deleted: However, HER2positivity was defined in this study as IHC immunoscore 3+ and/or FISH positive.

Deleted: At least one other phase III trial (www.clinicaltrials.gov; NCT00680901) is in progress using the drug lapatinib (directed against both HER1 and HER2). James Boers -HER2 in gastroesophageal adenocarcinomas p.14 from 8 highly amplified cancers. Possibly, this is a selection, as they studied 166 surgically resected gastric cancers cases, 27 of which showed unequivocal amplification.

Silver in situ hybridisation is a recently introduced technique being somewhat similar to CISH in that a conventional microscope can be used to assess the probe signals. Reports are still limited 29, 36 with less than 150 cases examined; so far, good correlations between FISH and SISH are described. In our study, 42 cases yielded identical results using both techniques. Assessment of HER2/Chromosome 17 signal in the multiple biopsies was much faster and easier with SISH in comparison with FISH, and allows good correlation between IHC and SISH using conventional techniques as ink-marking.

Most contemporary studies outside trials use the very cost-effective tissue micro-array (TMA)

technique. However, given the high incidence of heterogenous HER2-immunoreactivity/amplification, we felt that using the TMA technique in this field would yield an underestimation of the incidence of HER2-amplification rates. While we did not study resection specimens, the claim of Marx et al. [START_REF] Marx | HER-2 amplification is highly homogenous in gastric cancer[END_REF] that HER2 amplification is highly homogenous is contradicted by our findings in biopsy material.

In conclusion, HER2 amplification is present in a significant proportion of esophago-gastric region adenocarcinomas (24%) but at a much lower rate in the distal stomach (7%). Both rabbit monoclonal antibodies SP3 and 4B5 can be used for initial screening for possible amplification though the 4B5 antibody has the highest negative predictive value. FISH and SISH yields identical results. The SISH assay offers relatively easy and fast assessment. Our findings provide important guidance for HER2testing in gastroesophageal adenocarcinomas for patients where anti-HER2 treatment is considered. NOTE: we discarded original panel 3&4, which is and was in .eps format as required. However, the new panel 3&4 can now not be uploaded for .eps file format was not accepted. We would like to know how we can upload the new panel 3&4. As for now, we have a low resolution net figure 3D as a example to show to the reviewer. 240x199mm (100 x 100 DPI)

  case out of 123 with 4B5 immunoscores 0/1+ was amplified, and 21 out of 23 (91%) cases with immunoscores 2+/3+. The 2 amplified cases with SP3-immunoscore 0 were scored 2+ in the 4B5 immunohistochemistry. The 3 amplified cases with Deleted:

Figure 3 / 4 .

 34 Figure 3 / 4. Figure 3 (SISH) and 4 (FISH) with identical cases. 3A and 4A: high amplification; 3B and 4B: low amplification; 3C and 4C: (lower magnification) area with (white arrow) and without (yellow arrow) magnification; 3D: Chromosome 17 SISH: polysomy with > 3 copies chromosome 17 per nucleus; 4D: polysomy. FISH: red dots = HER2; green dots = Chromosome 17.

Figure 5 .

 5 Figure 5. A: 4B5 IHC: background staining in foveolar layer of gastric epithelium; B: 4B5 IHC: strong nuclear and cytoplasmic background staining in a diffuse type adenocarcinoma; C: HER2 SISH: nuclear haze; D: FISH: red granular background staining of some nuclei (encircled) among nuclei showing high amplification.

Figures 1 / 2 .Figure 5 .

 125 Figures 1 / 2. Figures 1 (SP3 IHC) and 2 (4B5 IHC) show identical cases. 1A: basolateral staining in SP3 (Score 1+) but in 2A a 3+ staining pattern in 4B5; 1B and 2B: 1+ staining in both SP3 and 4B5; 1C and 2C (low magnification): heterogeneous staining with 3+ pattern in the right half of the tumor and a 1+ staining in the left side, identical in SP3 and 4B5; 1D and 2D: detail of the 1+ / 3+ transition zone. 144x219mm (600 x 600 DPI)

  The study includes biopsies of 178 consecutive patients with the diagnosis of adenocarcinoma of the stomach or distal esophagus (study period 1999 -2007). Sufficient material for immunohistochemistry (IHC) and in situ hybridisation (ISH) studies was available in 146 cases with formalin-fixed, paraffinembedded primary tumour biopsies. The average number of biopsies per case was 5.8 (range, 2 -14, S.D. 2.4). Location of the tumor was noted (distal esophagus, gastric cardia, body, or antrum).

	PATIENTS AND METHODS
	Deleted:
	F o r
	P
	e
	e r
	R
	e
	v i e
	w

HER2 amplification and anatomical location/histological type (table5) Of

  James Boers -HER2 in gastroesophageal adenocarcinomas p.10Of the tumors situated in the EGR, 17 (24%) were HER2 amplified: 12 out of 44 distal esophageal adenocarcinomas and 5 of 28 cardia carcinomas. In the distal stomach, 5 (7%) showed HER2 amplification: 2 of 24 body and 3 of antrum carcinomas.

	James Boers -HER2 in gastroesophageal adenocarcinomas p.11
		Deleted: (27%)
		Deleted: (18%)
		Deleted: (8%)
		Deleted: (6%)
		Deleted: 4
		Deleted: in FISH
		Deleted: 5
	F o r F o r	Deleted: As presented is table 5, the single case of nonamplified SP3-immunoscore 1+ was 80% positive; the amplified cases 30%, 40% and 90% 1+ immunoreactivity,
		respectively. The amplified SP3
	P P e e e r e r	2+ cases contained 50 -90% HER2-positive tumor area; SP3 3+ cases were in 7 cases 80-100% positive and in 4 cases 20-30% positive. The sole amplified 4B5 1+ case contained 30% immunoreactive tumor area; the 16 nonamplified cases had 10%-100% 1+ immunoreactive areas. In 2 cases of nonamplified 4B5
		2+ cases, 20% resp. 50% was
	R R	immunoreactive while the 4 amplified cases contained 10%-90% positive tumor areas.
	e e v i e v i e w w	Deleted: : Deleted: in Deleted: and 2 Deleted: (17%) in non-amplified Deleted: . In these cases, HER2 copy number > 5/nucleus in amplified cases but ≤ 5/nucleus when amplification was not present.
		Deleted: A
		Deleted: s
		Deleted: 6 and 7
		Deleted: (27%)
		Deleted: (4%)

amplification closely matched positive HER2-immunoreactivity. Percentage of tumor staining for an antibody without regard of immunoscore, the number of positive biopsies and/or calculated H-score did not predict amplification status. The only generalisation that could be made was that most amplified cases had a 2+/3+ immunoscore in SP3, and 3+ (any percentage) or 2+ (> 50% tumor area) in 4B5 IHC.

Polysomy

Polysomy defined as ≥ 3 chromosome 17 copies per cell was present in 28 cases (19.2%). Polysomy was noted in 7 out of 22 (32%) amplified cases showing invariably ≥ 5 HER2 signals per nucleus. In contrast, polysomy present in 21 out of 104 cases had < 5 HER2 signals per nucleus Therefore, assessment of polysomy -quite often a striking finding in tumours in our study-did not contribute to the prediction of amplification. the total of 117 adenocarcinomas of the intestinal type, 20 (17%) showed HER2-amplification; 17 cases were located in the EGR and 2 in the distal stomach. Two out of 15 cases (13%) with mixed type type histology were also amplified located in the distal stomach. None of the 14 cases with diffuse or mucinous types showed amplification.

Deleted: (table

4

).

Table 1 .

 1 This study has been supported with an unrestricted scientific grant by Roche b.v., Woerden, The Histologic 37 classification and anatomical position of the adenocarcinomas.

	Deleted: A significant
	difference is that SISH is carried
	out on the Ventana automated
	stainers instead of a lengthy
	manual procedure. Also, a
	chromosome 17 probe is a
	feature, performed on a
	separate slide besides the HER2
	probe (though recently, an
	update of this technique came
	available allowing both probes to
	be visualised in one slide);
	although CISH offers a
	chromosome 17 probe as well
	usually only a HER2 in situ
	hybridisation is carried out.
	Deleted: In situ hybridisation
	remains a relatively costly
	technique (approx. € 300) and is
	laborious when performed
	manually. Even with SISH,
	assessment times are
	considerably longer than with
	IHC.

Table 2 .

 2 SP3 versus 4B5 HER2 immunoscores.

							Formatted Table
				4B5
	SP3	0	1+	2+	3+	total
	0	105	15	5	0	125 (86%)
							Deleted: 0
	1+	1	2	1	0	4 (3%)	Deleted: 2
	2+	0	0	0	6	6 (4%)
							Deleted: 1
	3+	0	0	0	11	11 (7%)
							Deleted: 5
		106	17	6	17		Deleted: 7
	total	(72%)	(12%)	(4%)	(12%) 146 (100%)

Table 3A .

 3A HER2 SP3 and 4B5 immunoscores versus Silver in situ hybridisation (SISH)

				James Boers -HER2 in gastroesophageal adenocarcinomas p.20
			ISH				ISH
	SP3	neg	pos	total	4B5	neg	pos		total
	0	123 (98%)	2 (2%)	125	0	106 (100%)	0 (0%)		106
	1+	1 (25%)	3 (75%)	4	1+	16 (94%)	1 (6%)		17
	2+	0 (0%)	6 (100%)	6	2+	2 (33%)	4 (66%)		6
	3+	0 (0%)	11 (100%)	11	3+	0 (0%)	17 (100%)	17
	total	124	22	146	total	124	22		146
	Table 3B. HER2 SP3 and 4B5 immunoscores versus FISH ISH SP3 neg pos total 4B5 neg 0 19 (90%) 2 (10%) 21 0 2 (100%) F o r	ISH pos 0 (0%)	total 2
	1+ 2+ 3+ total	1 (25%) 0 (0%) 0 (0%) 20	3 (75%) 6 (100%) 11 (100%) 22	4 6 11 42	1+ 2+ P 16 (94%) 2 (33%) 3+ 0 (0%) e total 20 e 17 (100%) 17 1 (6%) 17 4 (66%) 6 22 42 r
						R
							e
							v i e
									w

Table 4 .

 4 SP3 and 4B5 immunoscores versus percentage IHC positive cells in amplified/nonamplified

	tumors
				ISH negative					ISH positive
			% pos cells in IHC				% pos cells in IHC
			1-	11-	51-	76-	sub-		1-	11-	51-	76-	Sub-
	SP3	0	10	50	75	100	total	SP3 0	10	50	75	100	total
	0

Table 5 .

 5 Number (and percentage of biopsies) of HER2 amplified cases versus Laurén classification and anatomical location.

							Deleted: 6
							Formatted Table
				Anatomical position
				Total			Total
	Laurén	esophagus cardia	EGR	body	antrum	distal stomach
	intestinal 12 (30%) 5 (22%) 17 (27%) 1(7%) 2 (5%)	3(6%)
	mixed	0 (0%)	0 (0%)	0 (0%) 1 (17%) 1 (33%)	2 (22%)
	diffuse	0 (0%)	0 (0%)	0 (0%)	0 (0%) 0 (0%)	0 (0%)
	mucinous	0 (0%)	0 (0%)	0 (0%)	0 (0%) 0 (0%)	0 (0%)
	total	12 (27%) 5 (18%) 17 (24%) 2 (8%) 3 (6%)	5 (7%)

Table 6 .

 6 HER2 status in studies with ISH on adenocarcinomas 1C and 2C (low magnification): heterogeneous staining with 3+ pattern in the right half of the tumor and a 1+ staining in the left side, identical in SP3 and 4B5; 1D and 2D: detail of the 1+ / 3+ transition zone.

							Deleted: 8
							Deleted: Table 7. Percentage
		Reference	method	n (total) n (pos)	% (pos)	Formatted Table HER2-positivity ¶	... [2]
	Stomach	Ishikawa 38	FISH	105	19	18%
		Takehana 14	FISH	352	29	8%
		Risio	18	FISH	72	11	15%
		Varis	39	CISH	52	9	17%
		Tanner	11	CISH	131	16	12%
		Park	9	FISH/CISH	182	7	4%
		Yano 40	FISH	200	54	27%
		Kim	8			FISH	248	19	8%
		Barros-Silva 19	FISH	463	38	8%	Formatted: Font: (Default) Arial
		Marx 1	FISH	166	27	16%	Formatted: Font: (Default) Arial
							total	1.981	229 mean 12%
	Esophagus Brien 16	FISH	63	12	19%
		Tanner	11	CISH	100	24	24%
		Reichelt 4	FISH	110	16	15%
							total	273	52 mean 19%
	FISH: fluorescence in situ hybridisation; CISH: chromogenic in situ hybridisation

Table 4 .

 4 SP3 and 4B5 in relation to amplification

		SP3	4B5
	concordance	96,6% 97,9%
	discordance	3,4%	2,1%
	positive predictive
	value	100,0% 91,3%
	negative predictive
	value	96,1% 99,2%
	sensitivity	77,3% 95,5%
	specificity	100,0% 98,4%
		Page Break
	Page 22: [2] Deleted		James Evan Boers	3/20/2010 10:43:00 AM

Table 7 .

 7 Percentage HER2-positivity

			Anatomical position
							Total
				Total			distal
	Laurén	esophagus cardia	EGR body antrum	stomach
	intestinal	30%	22% 27%	7%	5%	6%
	mixed	0%	0% 0%	17%	33%	22%
	diffuse	0%	0% 0%	0%	0%	0%
	mucinous	0%	0% 0%	0%	0%	0%
	total	27%	18% 24%	8%	6%	7%

Published on behalf of the British Division of the International Academy of Pathology Histopathology

ACKNOWLEDGEMENTS

The authors wish to thank prof. Marc van de Vijver for providing comments on an earlier version on the manuscript.

Deleted: Table 4. SP3 and 4B5 in relation to amplification ¶ Deleted: 5

... [1]


MD Ben Vainer
email: ben.vainer@rh.regionh.dk

Birgitte Grønkaer Toft

Karen Ege Olsen

Grete Krag Jacobsen

Niels Marcussen

Handling of radical prostatectomy specimens: Total or partial embedding?

Keywords: prostate cancer, radical prostatectomy Gleason, pathology, partial embedding, prostate cancer, radical prostatectomy

come

Introduction

Proper examination and accurate reporting of radical prostatectomy specimens (RPS) is crucial in decision of post-surgical treatment and prediction of patient outcome. Important histological features are tumour grade, tumour volume, tumour stage and surgical margins.

There is widely international agreement that tumour grade should be assessed according to the Gleason criteria, which in 2005 was modified for better correlation with prognosis by an international working group under International Society of Urological Pathology (ISUP) 1, 2 . Macroscopical recognition of cancer areas within the prostatic gland is inaccurate, and macroscopical naked eye guidance for selection of sections for embedding may therefore not provide the correct areas for histopathological evaluation. Consequently, the entire RPS is sectioned and prepared for microscopical evaluation. Mostly, the gland is cut in serial horizontal sections, and the apex and the bladder neck area are cut parasagitally.

Consensus of the proper way to handle RPS has, however, not yet been obtained, clearly illustrated by a survey of American pathologists from 1994 and a recent European survey, conducted by the European Network of Uropathologists (ENUP) [START_REF] True | Surgical pathology examination of the prostate gland. Practice survey by American society of Clinical Pathologists[END_REF][START_REF] Egevad | Handling and reporting of radical prostatectomy specimens in Europe: a web-based survey by the European Network of Uropathology (ENUP)[END_REF] .

With the increasing demands to surgical pathology, the continuous difficulties in recruiting staff, and the limitations of economical resources it is inevitable to set priorities in the pathology service. Whether total embedding of RPS is in fact needed in order to provide the urologists with the correct information, or whether this information is sufficiently obtained with examination of only selected parts of the prostate has not been approached systematically. Thus, the aim of the present study was to evaluate whether essential information is lost when less than the whole prostate gland is embedded for histopathological examination.

Handling of the surgical specimen

When arriving at the pathology department, the prostate was partly divided by a cut in the mid-sagital plane through the anterior surface, separating the two lobes for optimal fixation. The gland was fixed for an additional 20 hrs in formic acid and 24 hrs in 4% buffered formalin. The procedure at Odense University Hospital was slightly different, as the initial mid-sagital cut was not performed, and decalcification was done after sectioning.

The gross examination included measurement in three dimensions, weighing the prostate after removal of the seminal vesicles, and separating the left from the right lobe after inking the anterior and the posterior halves with two different colours. Apical and basic sections of 5-10 mm depending of the total size of the RPS were cut horizontally, subsequently sliced parasagitally and placed in cassettes with often more than one section per cassette.

The remaining part of the prostate was cut horizontally in appr. 3 mm sections and placed in standard cassettes, ensuring laterality. Large sections were divided to fit standard cassettes. Finally, sections from the seminal vesicles (as a minimum the apex and a cross section) were embedded.

Post-fixation in 4% formalin and embedding in paraffin was followed by 4 µm sectioning and staining with haematoxylin and eosin.

Microscopical evaluation

The pathologist divided the glass slides from each specimen in two sets by removing the haematoxylin and eosin stained sections representing every second horizontal slice of the middle part of the prostate (Fig. 1). These glass slides were kept separately. In some cases, an uneven number of slides were withheld due to organ assymmetry and/or variation in slice thickness of the two lobes. The remaining glass slides, including those from the apex, the basis and the seminal vesicles, were evaluated by conventional light microscopy, if necessary with additional immunohistochemical staining with a cocktail of racemase P504S, high molecular weight-cytokeratin, and p63. A form was filled out for each prostate containing information of tumour type (WHO 2004 criteria [START_REF] Epstein | Tumours of the prostate[END_REF] ), Gleason score with elaboration of primary and secondary growth pattern according to the 2005 WHO/ISUP consensus report [START_REF] Epstein | The 2005 International Society of Urological Pathology (ISUP) consensus conference on Gleason grading of prostatic carcinoma[END_REF] with the clarification of how to deal with tertiary growth patterns (recently reviewed by Epstein [START_REF] Epstein | An update on the Gleason grading system[END_REF] ), rough estimation of the volume involved (%), diameter of the largest malignant focus (mm), laterality, multifocality, extracapsular extension, involvement of the resection margin, and tumour stage (pTNM system [START_REF] Epstein | Tumours of the prostate[END_REF] ).

Tertiary Gleason patterns were dealt with according to the precision recently described in a review Epstein. The extent of extraprostatic growth (stage pT3) was recorded as ≤2 or >2 high power fields. After completion of this assessment, the initially withheld slides were examined, and a final report was prepared for the urologists.

Statistics

Medians or means are presented depending on data distribution. Student's t-test was applied for group comparisons, and the Spearman test or the Kruskal Wallis test was used for assessment of trends. A p value of 0.05 or less was considered statistical significant.

The study was conducted according to the Helsinki Declaration and the Danish legislation.

Results

Details of the 238 radical prostatectomies are shown in Table 1. The age of the patients was 63.4 years (mean; SD 5.2, range 47-84) with no difference between the two centres.

The prostate glands with a median weight of 41.1 g (range 16-126 g) were embedded in median 38 cassettes (range 18-76), and between 3 and 26 slides (median 12) were initially withheld. This corresponded to 29.7% of the total slides (range 14.3-55.8%). The number of cassettes as well as the number of initially removed slides correlated with the prostatic weight (total no. of cassettes, R S =0.872; no. of initially removed slides, R S =0.776; both p<0.001, Spearman's test) (Fig. 1). Adenocarcinoma was demonstrated in all 238 RPS, of which 235 (98.7%) were classified as acinar. Alternative growth features were demonstrated in 29 acinar adenocarcinomas [START_REF] Epstein | The 2005 International Society of Urological Pathology (ISUP) consensus conference on Gleason grading of prostatic carcinoma[END_REF] (12.3%) (Table 1). The remaining 3 cases (1.2%) were either pure ductal (n=1) or mixed acinar and ductal (n=2) adenocarcinomas. Twenty-one (8.8%) of the carcinomas involved only one lobe, all of which involved less than half of the lobe corresponding to stage pT2a.

No stage pT2b tumours were registered. One-hundred-fifty (63.0%) were bilateral but confined to the prostate gland (pT2c), whereas extraprostatic growth, i.e. stage pT3, was observed in 67 (28.2%) with 48 (20.2%) having spread to the extraprostatic connective tissue only (pT3a) and 19 (8.0%) involving the seminal vesicles (pT3b). No stage pT4 tumours were diagnosed. Statistical testing revealed an inverse correlation between the tumour stage and the prostatic weight, i.e. high-stage carcinomas tended to be present in smaller prostate glands (R S =-0.137, p=0.035). Distribution of the tumour volume was strongly skewed to the right with a median of 10% (range 1-80%) and a median diameter of the largest cancer focus of 15.0 mm. The dominant Gleason score was 7 (n=173, 72.7%), including an upgrading from diagnostic biopsies in 58 cases (p<0.001). When examining the withheld slides, the Gleason score had to be changed in five cases, namely in one case from 3+4=7 to 3+5=8, in three cases from 4+3=7 to 4+4=8 and in 1 case from 3+4=7 to 4+5=9. Two cases were downgraded from 3+4=7 to 3+3=6 with a tertiary pattern 4 component. In the group of Gleason score 7 carcinomas, distribution of primary and secondary growth patterns changed in three cases, as the amount of the secondary pattern was dominating in the initially withheld sections (in two cases from 3+4 to 4+3 and in one case from 4+3 to 3+4).

In two cases (0.8%), small foci of extraprostatic growth were only detected in the withheld sections but not in the originally examined slides, which changed the stage from pT2c to pT3a, and in one RPS (0.4%) a small focus of adenocarcinoma was detected in the surgical margin only in the initially withheld slides. In six other cases, the stage was changed from pT2a to pT2c because of revealing of involvement of both lobes. No significant changes were found in tumour volume or in diameter of the largest tumour focus.

Discussion

Correct histopathological evaluation of RPS, including tumour grading and staging, is essential for the choice of the successive treatment and of prediction of prognosis [START_REF] Bostwick | Evaluating radical prostatectomy specimens: therapeutic and prognostic importance[END_REF] . to obtain this, correct tissue sampling from the specimens is of utmost importance. Ideally, all tissue should be examined under the microscope, but this is not always feasible due to shortage of staff, time and economy. Therefore, selection of tissue for microscopical analysis must be performed. Usually, pathologists select tissue sections of interest for microscopical assessment based on different, more or less reliable, macroscopical criteria.

Mostly, the gross appearance is an acceptable guidance, but in some cases this is not the case. Some neoplasias, such as prostate cancer, are macroscopically not easily recognisable within the specimen, and selection might therefore be more or less at random and, hence, seriously flawed.

The sampling technique for RPS and subsequent microscopical evaluation differs between centres, and consensus for the handling of RPS has not yet been reached. A recent survey among European uropathologists published in 2008 demonstrated that 71.6% of the institutions always embedded RPS in total, additional 17.6% performed total embedding in selected cases, and more than 10% of the institutions handled RPS always by partial embedding [START_REF] Egevad | Handling and reporting of radical prostatectomy specimens in Europe: a web-based survey by the European Network of Uropathology (ENUP)[END_REF] . The survey did not describe how the selection was done when embedding partially, i.e. systematically or guided by gross appearance or other specified criteria.

Several studies have been conducted in which embedding of whole mount sections have been compared to embedding of quadrants, the conclusion being that whole mount sampling is not superior to evaluation on standard-sized glass slides as to detection of adverse pathological features [START_REF] Hollenbeck | Whole mounted radical prostatectomy specimens do not increase detection of adverse pathological features[END_REF][START_REF] Cohen | Sampling of radical prostatectomy specimens. How much is adequate?[END_REF] . Also, as Srigley pointed out, whole-organ sectioning is expensive and requires additional technical expertise in the laboratory and important prognostic information may be lost primarily due to the thickness of the sections [START_REF] Srigley | Key issues in handling and reporting radical prostatectomy specimens[END_REF] .

The present study demonstrated that partial embedding of RPS with systematical selection of every second whole coronal section of the major part of the prostate and total embedding of the apical and basal sections loses only minor information important for the planning of post-surgical treatment and follow-up. This was corroborated by Kim et al [START_REF] Kim | Limited sampling of radical prostatectomy specimens with excellent preservation of prognostic parameters of prostate cancer[END_REF] who analyzed 148 RPS in order to evaluate the impact of five partial sampling methods on tumour stage, Gleason score, extraprostatic extension, resection margin, and number of cassettes. Alternative slices plus peripheral 3-mm rim of the remaining prostate was most efficient. This method detected all cases of extraprostatic extension and resection margin involvement and reduced the number of paraffin blocks by 25%.

For cases where cancer is not easily identified in the primarily examined histological slides, the remaining tissue should be examined and if malignancy is still not found cutting deeper should be done into the blocks corresponding to the location of malignancy demonstrated in the diagnostic needle biopsies. In the one case in the present investigation in which cancer was not detected in the initial slides, the remaining prostatic tissue would under any circumstance have been embedded for histological assessment.

Information for guiding the post-surgical approach and for prognosis was therefore lost in only three cases (1.2%) when embedding only half of the horizontal sections from the RPS, including two cases that changed tumour stage from organ-confined cancer (pT2) to cancer with minimal foci of extraprostatic extension (pT3) and one case in which positive surgical margins were demonstrated. In routine urology, such changes do not alone prompt a shift in the post-surgical approach.

The procedure for safe-keeping the initially unembedded prostate slices should be considered, depending on how detailed information, the urologists need in cases of minimal cancers not detected at first. Consecutive embedding of all slices in order and only processing half of the horizontal slices for microscopic examination will ensure a precise localisation of minimal cancer foci, but is obviously not as effective in decreasing the laboratory workload. We have concluded that separation of the right and the left lobe in separate containers is sufficient in these cases.

In some clinical trials, the inclusion criteria comprise separation of Gleason score 7 (3+4) from 7 (4+3). In the present study, advancement from 3+4 to 4+3 was observed in only two cases (0.8% of the total number of RPS; 1.2% of Gleason score 7 cancers), whereas one case was downgraded from 4+3 to 3+4. However, inclusion into such trials is always combined with other criteria, e.g. pT stage and surgical margin status often with inclusion of pT2c tumours with positive resection margins. This was the case of one of the two cases that changed from 3+4 to 4+3 when assessing the entire gland (0.4%). The Recognising the increasing demands to surgical pathology concerning both the increasing number of specimens and their complexity combined with the continuous difficulties in recruiting staff, it is important to decrease the workload whenever possible without compromising with the information needed to treat the patients optimally. In this respect, a reduction of tissue sections is a central issue. The approach with partial embedding of tissue from RPS used in this investigation will decrease the number of cassettes by approximately 30%, which in a cancer centre such as Rigshospitalet with more than 250 prostatectomies annually (increase of 50% from 2008 to 2009) will result in a fall in workload at the pathology laboratory equivalent to 75 RPS annually.

In conclusion, systematic partial embedding of RPS loses only minor information, which for the great majority of patients does not influence their post-surgical treatment or follow-up, but results in a significant reduction of the workload and of the resource requirement. Because of the small size of the prostate, the hemiprostate sections were not further divided into quadrants in this particular case.

  238 RPS were included constituting all prostatectomies performed at two Danish university hospitals (i.e. Rigshospitalet/Copenhagen University Hospital, and Odense University Hospital) during a 12 months period from September 2007 to August 2008.

  7 (3+4) vs. 7 (4+3) is not adequately documented, and the exclusion of such patients from participation in those clinical trials by the systematic partial embedding protocol applied in this study may therefore not influence the chance of survival for this patient.

Figure 1 .

 1 Figure 1. Gross examination and sectioning of a small radical prostatectomy specimen. After weighing and colour marking, the prostate was divided into the right and the left lobe by a mid-sagital cut, and the seminal vesicles were removed. Each lobe was sectioned separately by coronal slicing and further parasagital sectioning of the apical and basal horizontal slices. From the seminal vesicle the apex and a middle cross section was obtained. The photograph shows sectioning of the right lobe with a rest of the right seminal vesicle in the lower left corner and the entire left lobe with the seminal vesicle in the lower right corner. Sections withheld in the initial assessment are marked by red crosses.

Figure 1 .

 1 Figure 1. Gross examination and sectioning of a small radical prostatectomy specimen. After weighing and colour marking, the prostate was divided into the right and the left lobe by a midsagital cut, and the seminal vesicles were removed. Each lobe was sectioned separately by coronal slicing and further parasagital sectioning of the apical and basal horizontal slices. From the seminal vesicle the apex and a middle cross section was obtained. The photograph shows sectioning of the right lobe with a rest of the right seminal vesicle in the lower left corner and the entire left lobe with the seminal vesicle in the lower corner. Sections withheld in the initial assessment are marked by red crosses. Because of the small size of the prostate, the hemiprostate sections were not further divided into quadrants in this particular case. 155x117mm (72 x 72 DPI)

Table 1 .

 1 Characteristics of the radical prostactomies and corresponding pre-operative histopathology information. Unless otherwise stated, data are from evaluation of the radical prostatectomy specimens.

	No. of radical prostactomies	n=	238 (RH: 172, OUH: 66)
	Age of patients, years	(mean)	63.4 (range 47-84)
	Gleason, pre-op. biopsies (1)
	5	(n)	1 (0.4 %)
	6	(n)	100 (42.0 %)
	7	(n)	115 (48.3 %)
	8	(n)	10 (4.2 %)
	9	(n)	3 (1.3 %)
	Missing	(n)	9 (3.8 %)
	Prostatic weight	(g; median)	47.1 (range 16-126)
	No. of cassettes/total slides	(median)	38 (range 18-76)
	No. of slides initially removed	(median)	12 (range 3-26)
		(fraction -%)	29.9 (range 14.3-55.8)
	Type of carcinoma
	Acinar adenocarcinoma	(n)	235 (98.7 %)
	Conventional, no variants	(n)	206 (86.6 % of all carcinomas)
	Glomeruloid variant	(n)	4 (1.7 % of all carcinomas)
	Mucinous extravasation	(n)	8 (3.4 % of all carcinomas)
	Foamy cell variant	(n)	9 (3.8 % of all carcinomas)
	Colloid variant	(n)	6 (2.5 % of all carcinomas)
	Pseudohyperplastic variant	(n)	2 (0.8 % of all carcinomas)
	Ductal adenocarcinoma	(n)	1 (0.4 %)
	Mixed acinar and ductal	(n)	2 (0.8 %)
	Gleason (1)
	5	(n)	7 (2.9 %)
	6	(n)	44 (18.5 %)
	7	(n)	173 (72.7 %)
	3+4	(n)	112 (64.7 % of Gleason 7s)
	4+3	(n)	61 (35.3 % of Gleason 7s)
	8	(n)	11 (4.6 %)
	9	(n)	6 (2.5 %)
	Perineural growth	(positive; n)	185 (77.7 %)
	Tumour diameter	(mm; median)	15 (range 1-45)
	Tumour fraction of total gland (volume)	(%; median)	10 (range 1-80)
	Stage (pT) (2)
	pT2	(n)	171 (71.8 %)
	pT2a	(n)	21 (8.8 %)
	pT2b	(n)	0 (0.0 %)
	PT2c	(n)	150 (63.0 %)
	pT3		67 (28.3 %)
	pT3a	(n)	48 (20.2 %)
	pT3b	(n)	19 (8.0 %)
	pT4	(n)	0 (0.0 %)
	Involvement of surgical margins	(positive; n)	68 (28.6%)

Published on behalf of the British Division of the International Academy of Pathology Histopathology

Gleason grading according to the ISUP

modified scoring system (Ref. 1).[START_REF] Epstein | Update on the Gleason grading system for prostate cancer[END_REF] Tumour staging according to the WHO 2004 staging system (Ref. 7). SD = standard deviation (normal distribution assumed)

Published behalf of the British Division of the International Academy of Pathology Histopathology


Alexander Törnroos

Dr Hans Olsson
email: hans.olsson@lio.se

increases the number of lymph nodes recovered

Keywords: Colorectal cancer, Lymph nodes, Methylene blue Colorectal cancer, lymph nodes, methylene blue injection. Running title: Methylene improves lymph node recovery

Postoperative intra-arterial methylene blue injection of colorectal cancer specimens increases the number of lymph nodes recovered

Introduction

Adjuvant chemotherapy significantly reduces mortality and the risk of recurrence in stage III (T1-4, N1-2, M0) colorectal cancer relative to surgery alone. [START_REF] Moertel | Levamisole and fluorouracil for adjuvant therapy of resected colon carcinoma[END_REF] Accurate lymph node staging is therefore crucial in terms of evaluating prognosis and deciding on further treatment. The International Union Against Cancer (UICC) recommends the examination of a minimum of 12 lymph nodes in colorectal cancer specimens for an accurate assessment of lymph node status. However, recommendations in the literature range all the way from 6 examined lymph nodes to as many as possible with no particular minimum number. [START_REF] Caplin | For patients with Dukes' B (TNM Stage II) colorectal carcinoma, examination of six or fewer lymph nodes is related to poor prognosis[END_REF][START_REF] Cianchi | Lymph node recovery from colorectal tumor specimens: recommendation for a minimum number of lymph nodes to be examined[END_REF][START_REF] Cserni | Is there a minimum number of lymph nodes that should be histologically assessed for a reliable nodal staging of T3N0M0 colorectal carcinomas?[END_REF][START_REF] Luna-Perez | Prognostic significance of retrieved lymph nodes per specimen in resected rectal adenocarcinoma after preoperative chemoradiation therapy[END_REF][START_REF] Swanson | The prognosis of T3N0 colon cancer is dependent on the number of lymph nodes examined[END_REF][START_REF] Tepper | Impact of number of nodes retrieved on outcome in patients with rectal cancer[END_REF][START_REF] Tsai | The prognostic significance of total lymph node harvest in patients with T2-4N0M0 colorectal cancer[END_REF][START_REF] Vather | Lymph node examination as a predictor of long-term outcome in Dukes B colon cancer[END_REF][START_REF] Wong | Number of nodes examined and staging accuracy in colorectal carcinoma[END_REF][START_REF] Törnroos | The number of identified lymph node metastases increases continuously with increased total lymph node recovery in pT3 colon cancer[END_REF] Studies have indicated that both stage II (T3-4, N0, M0) and stage III cancers are in fact very heterogeneous groups regarding prognosis and outcome. Part of this heterogeneity is thought to depend on the number of lymph nodes recovered, which is believed to affect the accuracy of the pathological diagnosis. [START_REF] Caplin | For patients with Dukes' B (TNM Stage II) colorectal carcinoma, examination of six or fewer lymph nodes is related to poor prognosis[END_REF][START_REF] Cianchi | Lymph node recovery from colorectal tumor specimens: recommendation for a minimum number of lymph nodes to be examined[END_REF][START_REF] Swanson | The prognosis of T3N0 colon cancer is dependent on the number of lymph nodes examined[END_REF][START_REF] Tsai | The prognostic significance of total lymph node harvest in patients with T2-4N0M0 colorectal cancer[END_REF][START_REF] Vather | Lymph node examination as a predictor of long-term outcome in Dukes B colon cancer[END_REF][START_REF] Wong | Number of nodes examined and staging accuracy in colorectal carcinoma[END_REF][START_REF] Sarli | Number of lymph nodes examined and prognosis of TNM stage II colorectal cancer[END_REF] Swanson et al. examined patients with stage IIA (T3, N0, M0) cancers and found a positive association between the number of lymph nodes recovered and prognosis. Stratifying the patients into groups depending on the number of recovered lymph nodes (1-7, 8-12, or ≥13 lymph nodes) yielded 5-year relative survival rates of 69.3%, 77.5% and 84.5%, respectively, which all differed significantly from each other [START_REF] Swanson | The prognosis of T3N0 colon cancer is dependent on the number of lymph nodes examined[END_REF] . This heterogeneity within TNM stage groups emphasizes the importance of adequate examination of lymph nodes to ensure accurate pathological diagnosis.

Understaging in colorectal cancer is suggested to result from an insufficient number of recovered lymph nodes at pathological examination. This hypothesis is known as the stage migration theory. [START_REF] Johnson | Increasing negative lymph node count is independently associated with improved long-term survival in stage IIIB and IIIC colon cancer[END_REF][START_REF] Sarli | Number of lymph nodes examined and prognosis of TNM stage II colorectal cancer[END_REF] An insufficient assessment of lymph nodes may leave undetected metastases. Patients diagnosed with stage II cancer and with few examined lymph nodes may in fact have stage III cancer. This may cause incorrect oncological treatment postoperatively and give the patient an unfavourable prognosis.

Intra-arterial methylene blue injection was recently introduced by Märkl et al. for improvement of lymph node recovery in colon and rectal cancer specimens. [START_REF] Märkl | Methylene blue-assisted lymph node dissection in colon specimens: a prospective, randomized study[END_REF] By postoperative injection of methylene blue into the specimen arteries, Märkl et al. significantly improved lymph node recovery compared to a control group: mean 35 ± 8 and 17 ± 10 lymph nodes per specimen, respectively. The technique was fairly simple, efficient and adaptable as a routine procedure for pathological examination of colorectal cancer specimens. The additional lymph nodes recovered in the stained group were mainly small nodes measuring 1-4 mm in diameter. [START_REF] Märkl | Methylene blue-assisted lymph node dissection in colon specimens: a prospective, randomized study[END_REF] Several studies have shown that smaller lymph nodes (of ≤ 5 mm in diameter) contain metastases just as frequently as larger nodes. [START_REF] Brown | Efficacy of manual dissection of lymph nodes in colon cancer resections[END_REF][START_REF] Haboubi | The importance of combining xylene clearance and immunohistochemistry in the accurate staging of colorectal carcinoma[END_REF][START_REF] Herrera | Incidence of metastases from rectal adenocarcinoma in small lymph nodes detected by a clearing technique[END_REF][START_REF] Herrera-Ornelas | Metastases in small lymph nodes from colon cancer[END_REF] Accurate specimen preparation and examination is important for detection of lymph nodes and to avoid understaging of colorectal cancer. We assumed that more nodal metastases can be identified and that understaging may be avoided when more lymph nodes are harvested and examined. The aim of this study was to investigate whether postoperative intra-arterial methylene blue injection of colorectal cancer specimens would improve lymph node recovery at pathological examination.

Material and Methods

Between September 2008 and January 2009, patients undergoing intended curative surgery for colorectal cancer at the Departments of Surgery at Norrköping County Hospital and Linköping University Hospital, Sweden were randomly assigned into two groups, either to Immediately postoperatively, the specimens in the staining group had their main artery or arteries cannulated with intravenous catheters of appropriate sizes (16-20 G). The proximal ends of the arteries were marked by the surgeon with sutures after the resection. The catheters were inserted using only the plastic tube portion, as there was a risk that the cannula would perforate the artery. The arteries were injected with 10-30 ml of methylene blue dye (50 mg methylene blue in 5 ml distilled water, diluted with 0.9% NaCl solution in the ratio 1:3) until the dye was visible on the surface of the specimen. The amount of dye refers to the amount used for the entire specimen. The number of arteries injected with dye per specimen varied between one and three. The specimens in both the unstained and the stained groups were fixed in formaldehyde for a minimum of 24 hours according to the standard protocol, and were dissected in the same manner and by the same person (AT). After having located the primary tumour and secured representative pieces for histological examination, the mesenterial fatty tissue was sliced and visually examined and palpated (Figure 1). All harvested lymph nodes were examined histopathologically by the same pathologist (HO) according to routine protocol.

All statistical analyses were performed using Statistica software version 7. The significance of differences was calculated using Fisher's exact t-test. P-values ≤ 0.05 were assumed to be statistically significant.

"Figure 1"

Results

Of 37 specimens, 20 were randomly assigned to the methylene blue staining group and 17 to the no staining group. Three specimens were excluded in the staining group because of failure to inject them with the methylene blue dye, and one specimen from each group was excluded because later histopathological examination showed adenoma. Sixteen specimens remained in each group for further analysis (Table 1 and2).

Metastases were found in 6 stained specimens and 9 unstained specimens. The median number of lymph nodes recovered was 61 (range 35-161) and 22 (range 13-51) in the stained and unstained groups, respectively. The median number of harvested lymph nodes was significantly higher in the stained specimen group (P < 0.0001) (Figure 2). There was no significant difference regarding the number of lymph node metastases found in the two groups. All specimens in both groups had lymph node recoveries that were better than the UICC-recommended minimum number of 12 nodes.

"Figure 2 Table 1-2"

Discussion

Our results show that intra-arterial injection of methylene blue into colorectal cancer specimens significantly improves the number of lymph nodes recovered compared to the traditional pathological method. The difference between the specimen groups is highly significant and although the study is fairly small we are confident that this difference would remain even if more specimens were examined.

The number of lymph nodes required for an accurate staging of colorectal cancer specimens is unclear, and the recommendation of at least 12 lymph nodes by the UICC is controversial. Some authors have reported that the benefit of finding additional lymph nodes increases in a linear fashion. [START_REF] Törnroos | The number of identified lymph node metastases increases continuously with increased total lymph node recovery in pT3 colon cancer[END_REF][START_REF] Goldstein | Lymph node recoveries from 2427 pT3 colorectal resection specimens spanning 45 years: recommendations for a minimum number of recovered lymph nodes based on predictive probabilities[END_REF] Much of the controversy is based on studies that have found that the prognosis for patients with metastasis-free (stage II) colorectal cancer is dependent on recovery of lymph nodes, there being a positive correlation between the number of lymph nodes recovered and better prognosis. [START_REF] Swanson | The prognosis of T3N0 colon cancer is dependent on the number of lymph nodes examined[END_REF][START_REF] Tsai | The prognostic significance of total lymph node harvest in patients with T2-4N0M0 colorectal cancer[END_REF][START_REF] Vather | Lymph node examination as a predictor of long-term outcome in Dukes B colon cancer[END_REF][START_REF] Sarli | Number of lymph nodes examined and prognosis of TNM stage II colorectal cancer[END_REF] Based on these data, various techniques for recovering lymph nodes have emerged such as simple fat-clearing techniques, specialised fixatives such as GEWF (glacial acetic acid, ethanol, distilled water and formaldehyde) and lymphatic system dye techniques. [START_REF] Märkl | Methylene blue-assisted lymph node dissection in colon specimens: a prospective, randomized study[END_REF][START_REF] Richter | Acetone treatment of lymph node preparations in staging colorectal specimens[END_REF][START_REF] Iversen | Increased lymph node harvest from colorectal cancer resections using GEWF solution: a randomized study[END_REF] The methylene blue injection method can provide some procedural difficulties. Difficulties were experienced with arterial catheterization, as the latter proved fragile, and the risk of perforating the arteries therefore proved to be a problem. However, as a failed staining does not appear to have any deleterious effect on specimen dissection, the method can be regarded as safe. No formal measurements were made regarding time spent on dissection, however in our experience it did not differ between the two specimen groups as the time spent removing the extra lymph nodes in the methylene blue specimens was compensated for by having to spend less time searching for them. The cost effectiveness of the methylene blue technique is completely dependent on whether or not it assists in the detection of lymph node metastases.

Our results show that it is possible to significantly improve the number of lymph nodes recovered by using a supplementary staining method; however, we cannot make any conclusions about the benefit of finding these additional lymph nodes. The study material was too small for us to examine any correlation between the number of lymph nodes recovered and the frequency of metastases. Richter et al. demonstrated this phenomenon when using an acetone fat clearing method for examining colorectal cancer specimens. They first manually dissected the specimens for lymph nodes and then used their clearing method on the same specimens. Of 188 specimens, 15% were found to have previously undetected metastases and 8% of the specimens had to have their N-stages upgraded. Nine specimens were upgraded from N0 to N1 or N2 (from stage II to stage III). [START_REF] Richter | Acetone treatment of lymph node preparations in staging colorectal specimens[END_REF] Lymph node size is another important issue, since the methylene blue technique mainly improves the recovery of very small lymph nodes (≤ 4 mm in diameter). [START_REF] Märkl | Methylene blue-assisted lymph node dissection in colon specimens: a prospective, randomized study[END_REF] Several authors have reported that even small lymph nodes (≤ 5 mm in diameter) carry metastases. [START_REF] Brown | Efficacy of manual dissection of lymph nodes in colon cancer resections[END_REF][START_REF] Haboubi | The importance of combining xylene clearance and immunohistochemistry in the accurate staging of colorectal carcinoma[END_REF][START_REF] Herrera | Incidence of metastases from rectal adenocarcinoma in small lymph nodes detected by a clearing technique[END_REF][START_REF] Herrera-Ornelas | Metastases in small lymph nodes from colon cancer[END_REF] However, it is not proven that metastases in small lymph nodes have the same clinical implications as metastases in larger lymph nodes. Care should be taken when referring to lymph node metastases without any reference to the size or nature of those metastases.

There may be different reasons for the great variation in the number of lymph nodes recovered from colorectal cancer specimens. For instance, a strong immune response against the tumour might enlarge lymph nodes, making them easier to find on examination. Surgical technique is a debatable subject. [START_REF] West | Complete mesocolic excision with central vascular ligation produces an oncologically superior specimen compared with standard surgery for carcinoma of the colon[END_REF] The number of lymph nodes recovered is a measure of quality, both from a surgical point of view but also for the pathologists performing the postoperative specimen investigation. Use of the staining method described would eliminate many of the pathologist's difficulties, thus highlighting the role of the surgeon.

In conclusion, intra-arterial methylene blue staining of colorectal cancer specimens significantly improves lymph node recovery. However, a possible correlation between the number of lymph nodes recovered and the frequency of metastases could not be shown in this fairly small study more focusing on pathological laboratory techniques.

F

  methylene blue or no staining. The exclusion criteria were concomitant inflammatory bowel disease and massively invasive tumours necessitating a surgical resection technique, rendering subsequent specimen staining impossible. The specimens were analysed at the Department of Clinical Pathology and Genetics at Linköping University Hospital, Sweden.

  Figures

Figure

  Figure 2

2

 2 Figure 2

Figure legends

  Figure legends

Figure 1 .

 1 Figure 1. Stained lymph nodes, marked with arrows.

Figure 2 .

 2 Figure 2. Median number of lymph nodes recovered (y-axis) in the stained and unstained groups, respectively.

  West et al. compared colon cancer specimens resected using complete mesocolic excision (CME) and central vascular ligation (CVL) with specimens resected using a more conservative and less radical technique. The CME/CVL operations in comparison to the alternative operating technique yielded a significantly larger area of mesentery removed, 19,7 versus 11,8 mm 2 , and a significantly higher specimen lymph node versus 18 lymph nodes, thus demonstrating the important influence of surgical technique on the outcome of the pathologists specimen dissection.

	recovery, 30
	F o r
	P
	e
	e r
	R
	e
	v i e
	w
	6

Table 1 .

 1 Specimen raw data.

	o r
	P
	e
	e r
	R
	e
	v i e
	w

Table 2 .

 2 Characteristics of patients and specimens.

	Stained group	Unstained group	P-
	(n = 16)	(n = 16)	value