Endocr Pathol DOI 10.1007/s12022-014-9322-y

In Search of a Prognostic Classification of Endocrine Pituitary Tumors Jacqueline Trouillas

# Springer Science+Business Media New York 2014

Abstract Pituitary tumors, the most frequent intracranial tumors, are historically considered benign. However, various pieces of clinical evidence and recent advances in pathological and molecular data suggest the need to consider that these tumors are more than an endocrinological disease despite the low incidence of metastasis. We present here a historical and critical review of the classifications of pituitary tumors, including a new prognostic clinicopathological classification based on tumor size, immunohistological subtype (prolactin (PRL), growth hormone (GH), follicle-stimulating hormone and luteinizing hormone (FSH-LH), adrenocorticotropic hormone (ACTH), and thyroid-stimulating hormone (TSH)), and five grades, which take into account invasion and proliferation of the tumors. We also present a brief review of the main markers of tumor behavior. We believe that a better classification of these tumors and the identification of prognostic markers will help the clinician to choose the appropriate therapeutic management.

This paper is dedicated to my friend Professor Carlo Capella, one of the pioneers of neuroendocrinology and a member of the “International Pituitary Pathology Club” founded in 1980 by Jules Hardy, Françoise Robert (deceased 1995), Kalman Kovacs and me. J. Trouillas Neuro-oncology and Neuro-inflammation team, Lyon Neuroscience Research Center, INSERM U1028, CNRS UMR5292, 69372 Lyon, France J. Trouillas Université de Lyon, Université Lyon 1, 69372 Lyon, France J. Trouillas (*) Laboratoire d’Histologie et Embryologie Moléculaires, Faculté de Médecine Lyon Est, Site Laennec, and Centre de Pathologie Est, Groupement Hospitalier Est, Hospices Civils de Lyon, Rue Guillaume Paradin, 69372 Lyon, Cedex 8, France e-mail: [email protected]

Keywords Classification . Pituitary tumor . Pituitary adenoma . Prognostic marker

Introduction Endocrine pituitary tumors are the most frequent intracranial neoplasm affecting 1/1,000 of the population [1, 2]. They arise from adenohypophyseal cells and are clinically classified into functioning (mainly growth hormone (GH) with acromegaly; prolactin (PRL) with amenorrhea-galactorrhea; adrenocorticotropic hormone (ACTH) with Cushing’s disease) and nonfunctioning (mainly follicle-stimulating hormone and luteinizing hormone (FSH-LH) tumors. Over the years, the increase of technology and knowledge has allowed the evolution of the pathological classification of pituitary endocrine tumors from a tinctorial classification (three types: acidophilic, basophilic, and chromophobic) to an immunohistochemical classification (five types) plus a dozen ultrastructural subtypes. These tumors were considered as benign. But, 30 to 45 % of them are invasive for the cavernous or the sphenoid sinus [3, 4], and a significant number are considered as aggressive based on recurrence during follow-up [5, 6]. Some of them are named “atypical” adenoma. This new subtype with uncertain malignancy has been identified in the World Health Organization (WHO) 2004 classification [7]. Some of these tumors develop metastasis during the follow-up and are considered as carcinomas [8–10]. While pituitary carcinoma with metastasis is rare, recent evidence suggests that the benign status of the pituitary tumors must be revised. The major hurdles in the management of pituitary tumors are the lack of prognostic markers and also of prognostic classification. I will present a historical and critical review of the pathological classifications of pituitary tumors to date and propose a new prognostic clinicopathological classification [11]. I will

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list some pathological, molecular, and genetic markers associated with tumor behavior (not tumorigenesis) which allow personalized management of patients with these tumors.

The Endocrine Pituitary Tumors’ Classifications: 1970–2010 From the Tinctorial Classification to the 2004 WHO Classification Until the 1980s, pituitary tumors were classified based on their tinctorial properties with hematoxylin-eosin-safran/hematoxylin-phloxine-safran (HES or HPS) and were correlated with clinical disease into eosinophilic or acidophilic with acromegaly, basophilic with Cushing’s disease, and chromophobic adenomas. Later, with the development of electron microscopy (EM) and immunohistochemistry (IHC), the tumors were classified based on the appearance of their organelles (granulations and mitochondria) and their hormonal secretion [12–15]. Taking into account the clinical signs and the ultrastructural and IHC data, many subtypes were described: the PRL adenoma or prolactinoma, the GH adenoma with both the sparsely and densely granulated ultrastructural subtypes, the gonadotroph adenoma with hypersecretion of FSH-LH (rev in the study of Snyder [16]) or with normal plasma values [17, 18], the null cell adenoma [19] or immunonegative adenoma [17], the thyroid-stimulating hormone (TSH) adenoma (rev in the study of Beck-Peccoz et al. and Bertholon-Gregoire et al. [20, 21], the “silent” corticotroph subtypes 1 and 2 [22], and the acidophilic stem cell adenomas [23]. In 1996, Kovacs et al. [24] proposed the WHO classification of adenohypophysial neoplasms using a five-tier scheme on the basis of functional, imaging/surgical, histological, immunohistochemical, and ultrastructural findings. While this detailed classification was very interesting, it is highly complex and can only be used by specialized pathologists. For 17 years (1970–1987), I systematically performed EM, but I am convinced that IHC is more powerful than EM and with the promise of molecular biology, I decided to freeze the pieces of tumors rather than keep fragments for EM. Nowadays, expensive and time-consuming EM is only rarely performed by some specialists. The more powerful IHC technique is now used on a routine basis, and tumor fragments are frozen for molecular analyses when possible. Tumors are currently classified into five main IHC types PRL, GH, ACTH, TSH, and FSH-LH which can be monohormonal or plurihormonal, with or without (silent) signs of hypersecretion (Table 1). Almost all the ultrastuctural subtypes were found to be morphological variants identifiable by IHC, and with the improvement of the IHC technique (automation and new antibodies), some disappeared. An

Table 1 Immunohistochemical classification of endocrine pituitary tumors Tumor types

Immunoprofiles

PRL tumor Densely granulateda Sparsely granulated GH tumor

PRL PRL, α-SU

Monohormonal Densely granulated Sparsely granulated Plurihormonal Mixed GH-PRL Mammosomatotroph ACTH tumor Densely granulated Sparsely granulated TSH tumor Monohormonala Plurihormonal FSH-LH tumor Immunonegative tumora “Silent tumors Mono/plurihormonal ACTH GH TSH

GH, (α-SU), Ck, (CgA) GH, (α-SU), Ck, (CgA) GH, PRL, (α-SU, β-TSH) GH, PRL, (α-SU, β-TSH) ACTH, β-endorphin, β-LPH, Ck, (CgA) ACTH, β-endorphin, β-LPH, Ck, (CgA) β-TSH, α-SU β-TSH, α-SU, GH, PRL β-FSH, β-LH, (α-SU), CgA (CgA)

ACTH, β-endorphin, β-LPH GH, PRL (β-TSH) β-TSH, α-SU, GH, PRL

PRL prolactin, SU sub unit, GH growth hormone, FSH-LH folliclestimulating hormone and luteinizing hormone, ACTH adrenocorticotropic hormone, TSH thyroid-stimulating hormone, LPH lipotropin, CgA chromogranin A, CK cytokeratin a

Rare subtype

example is the null cell adenoma [19], which corresponds to FSH-LH tumors with low intracellular hormonal content, undetected by some antibodies yet identified in culture or with a more sensitive IHC technique [25]. We prefer the term of nonimmunoreactive or immunonegative tumors which are now very rare (from 10 % in 1992 to 1 % in 2012 in Lyon’s pathological series). The oncocytoma [26, 27] is not an entity per se but rather a tumor, most often of FSH-LH type with numerous mitochondria. The ultrastructurally densely and sparsely granulated GH tumors are most easily identified using the IHC detection of low-molecular-weight keratins (CAM 5.2 or cytokeratin 8). The juxtanuclear dots, known as “fibrous bodies,” are characteristic of this latter subtype. The expression of cytokeratin is also helpful when identifying ACTH tumors which are usually strongly positive, especially those undergoing Crooke’s changes, although there can be weak staining in silent ACTH tumors which are also positive with galectin-3 [28]. The detection of chromogranin A, characteristic of gonadotroph tumors [29], is also very useful to

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establish pituitary origin, especially in the cases of negative immunostaining of the pituitary hormones. Some other subtypes must be considered because they may have a particular behavior. For example, silent corticotroph subtypes 1 and 2 [22] are clinical or cytological variants of ACTH tumors. As recently reported [30, 31], a number of patients with a silent ACTH tumor may present with clinical symptoms of Cushing’s disease at some stage during their follow-up; evidence also exists of a continuum from the macroadenoma with Cushing’s to the silent ACTH tumor [31]. We propose therefore renaming such silent tumors as “ACTH tumor without signs of Cushing’s” similar to the renaming of silent GH tumors [32] as GH adenoma without acromegaly [33]. Acidophilic stem cell adenomas [23] are either GH-PRL or PRL tumors with giant and dilated mitochondria. I believe that it is necessary to provide such precision to clarify the current terminology since some of these subtypes, e.g., the sparsely granulated GH subtype [34], the silent ACTH, silent GH-PRL, or subtype 3 adenomas, may be considered as poorly differentiated tumors with potentially aggressive behavior. Such subtypes must be taken into account for postoperative management, especially in cases of residual tumor. These clinically nonfunctioning tumors should not be confused with the silent FSH-LH tumors, which have a less aggressive course (rev in the study of Cooper and Melmed [30]). The WHO 2004 Classification and a New Prognostic Clinicopathological Classification In the decade after the millenium, it appeared that some tumors without metastasis behaved differently from benign tumors. MRI indicated that some of these tumors invaded the skull base or both cavernous sinuses, but crucially, because the pathologist had no proof of the invasion and metastasis, these tumors continued to be named adenoma and considered benign. However, in 2004, the WHO classification [7] classified all benign tumors as typical adenomas (ICD-0 8272/0) but identified atypical adenomas showing “borderline or uncertain behavior” (ICD-0 8272/1). Such tumors have atypical morphologic features suggestive of aggressive behavior such as invasive growth. Other features include an elevated mitotic index and a Ki-67 labeling index greater than 3 %, as well as extensive nuclear staining for p53 immunoreactivity. The term of pituitary carcinomas (ICD-0 8272/2) continues to be restricted to the pituitary tumor with systemic or cerebrospinal metastases [35, 36]. Although markers of proliferation, p53 detection, and invasion were all mentioned as criteria of the “atypical adenoma,” invasion has not been systematically considered, as illustrated by the two following papers. In Laws’ series of 121 consecutive patients [4], the frequency of atypical adenoma was 15 % compared to 2.5 % in the Saeger et al.’s series of 241 tumors from the German registry [37]. This discrepancy is explained by the differences in

criteria taken into account for the diagnosis: a Ki-67 > 3 %, p53 positivity, and increased mitotic activity without a cutoff value in the Laws’ series [4] and invasion associated with Ki67 > 3 % and p53 > 5 % in the Saeger’s series [37]. Thus, as advised by Wolfsberger and Knosp [38], “the definition of invasiveness is needed and should be included in this classification as in a previous classification” [39], and the proliferation must be evaluated by markers of the cell cycle with welldefined thresholds. Moreover, as some experts [40–42] have pointed out, this classification needs to be correlated to clinical evolution with postoperative results, progression, and recurrence. Thus, we initiated a multicentric collaborative study in 2006 to validate the clinical prognostic value of a clinicopathological classification which has been recently published [11]. This classification takes into account both tumor size and the five IHC types (PRL, GH, FSH-LH, ACTH, and TSH). We set up a grading system, such as that used for other endocrine tumors of the foregut, [43] based on invasion and proliferation status (Table 2). MRI is needed to evaluate the invasion since the histological proof of invasion is rare (9 % of invasive tumors in this series). Only invasion into the sphenoid sinus, confirmed by the infiltrated respiratory mucosae on histology, and unequivocal invasion of the cavernous sinus were considered [44]. Indeed, recent anatomical studies have shown that the medial wall of the cavernous sinus is composed of dura [45], and moreover, the intracavernous invasion can be assessed peroperatively by an endoscopic technique. Using multivariate statistical analysis and a ROC curve [11], we

Table 2 Clinicopathological classification of the endocrine pituitary tumors The classification is based on the three following characteristics: 1. Tumor size into micro (40 mm) by MRI 2. Tumor type into GH, PRL, ACTH, FSH-LH, and TSH by IHC 3. Tumor grade based on the following criteria: Invasion defined as histological and/or radiological (MRI) signs of cavernous or sphenoid sinus invasion Proliferation considered on the presence of at least two of the three criteria: Mitoses: n>2/10 HPF; Ki-67: ≥3 %; p53: positive (>10 strongly positive nuclei/10 HPF) The five grades are the following: Grade 1a: noninvasive tumor Grade 1b: noninvasive and proliferative tumor Grade 2a: invasive tumor Grade 2b: invasive and proliferative tumor Grade 3: metastatic tumor (cerebrospinal or systemic metastases) From Trouillas J et al. [11]

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confirmed that invasion is a major prognostic factor in predicting both the disease-free status following surgical removal of pituitary tumors [46] and recurrence/progression [47]. In our opinion, invasion must be added to the synoptic checklist for pituitary lesions recently published by a group of experts [48], especially considering that data from pituitary imaging and surgical findings are included in the patient database, which is now available to pathologists. Tumor proliferation is evaluated by the two most commonly used cell cycle markers in oncology (Ki-67 index, and mitotic count) as well as p53. Considering the controversial value of these markers, which will be discussed later, and the lack of methodological standards and validated cutoff for p53, we defined proliferation as the presence of at least two of these markers with cutoff values of 3 % for Ki-67 index with formol fixative and 1 % for Bouin-Hollande fixative and the number of mitoses n>2/10 high power fields (HPFs), as for endocrine pancreatic tumors [43] and the positivity for p53 if at least 10 nuclei/10 HPFs were strongly positive. With this grading, nearly half of the tumors were grade 1a (47.3 %), 10 % were grade 1b, 27.6 % were grade 2a, and 15.1 % were grade 2b. Eight tumors (2.0 %), four PRL, and four ACTH, classified as grade 1b (n=1), 2a (n=1), and 2b (n=6) at the time of initial surgery, were later classified as carcinomas (grade 3) based on the occurrence of metastases during the 8-year follow-up. The series of 410 patients in our case-control study is representative of pituitary tumor behavior with a similar percentage of invasive tumors (43 %) to that in other series [3] and a percentage of grade 2b (15 %) similar to the percentage of the atypical adenoma in the Laws’ surgical series [4]. However, prospective studies must now be performed to evaluate the exact percentage of each grade for each IHC type. By multivariate analysis, we confirmed the poor prognostic value of young age in predicting disease-free outcome and recurrence/progression status at 8 years (rev in the study of Roelfsema et al. [49]). However, while tumor size was not associated with the recurrence/progression status, it was with disease-free outcome [50]. Patient sex had no prognostic value, as suggested previously [49]. Our proposed classification displayed highly significant prognostic value for predicting postoperative disease-free outcome or recurrence/ progression status, across all tumors and for each type of tumor. At 8-year follow-up, the probability of a patient showing evidence of disease or tumor progression was 25and 12-fold higher, respectively, if they had an invasive and proliferative tumor (grade 2b) as compared to if they had a noninvasive and nonproliferative tumor (grade 1a). These results confirm those obtained in our preliminary study on 94 PRL tumors [51] that were classified into three groups, i.e., noninvasive, invasive, and aggressiveinvasive, corresponding respectively to the grades 1a, 2a, and 2b of the present classification.

Challenges of Pituitary Tumors As there are no obvious signs of malignancy, pituitary carcinoma is only defined when the tumor has metastasized. It is rare and accounts for about 0.2 % of pituitary tumors with only 132 cases reported in the literature from 1961 to 2009 [9, 10, 36]. Most pituitary carcinomas are functioning, secreting PRL (36 % cases) (rev in the study of Kars et al. [52]) or ACTH hormone (30 % cases) (rev in the study of Landman et al. [53]). Nonfunctioning tumors are less frequent (23 % cases), especially the FSH-LH carcinoma [54]. Previously, histological signs of malignancy were thought to be absent; however, based on the frequency of certain signs in previous pathological series of pituitary carcinomas [55–57], a potential malignancy could reasonably be suspected based on the association of the following pathological signs: invasion, neoangiogenesis, vascular invasion, abnormal mitoses, very high index of Ki-67> 10 %, and p53>5 % and genomic alteration (chromosome 11 for PRL tumors), which if combined might be considered as criteria of malignancy. However, these cutoff values were not validated, and some of the above signs were found to be absent in certain metastatic pituitary carcinomas [56, 57]. The observation that six out of the eight carcinomas of our series were grade 2b at the first surgery [11], together with the comparison of human PRL tumors with our animal model (SMtTW model) [58], led us to postulate that grade 2b or aggressive-invasive tumors are in fact malignant tumors without metastasis [59–61]. In oncology, the term “adenoma” signifies that a tumor is benign. I propose that the generic term should be “pituitary tumor” instead of “pituitary adenoma” because this tumor can be benign or malignant. Furthermore, we propose that, instead of atypical adenoma or “aggressive adenoma,” the term “tumor suspected of malignancy” (tumor grade 2b) be used. The characteristics of the tumors classified according to malignancy are summarized in Table 3 [55, 62].

Molecular Markers Associated with Tumor Behavior While the expression of several biological markers has been investigated by IHC and correlated to invasiveness and/or aggressive behavior, no single marker has been found to predict the tumor behavior, and until now, their detection is only performed at the research level. As some exhaustive reviews have been published on this subject [63–66], here, I will focus on biomarkers that appear to correlate with invasive and aggressive behavior of pituitary tumors.

Endocr Pathol Table 3 Classification of endocrine pituitary tumors according to malignancy

From Pernicone PJ et al. [55], Trouillas J et al. [62] HPF high power field, LI labeling index

Tumor characteristics

Benign tumor/adenoma

Tumor suspected of malignancy/grade 2b

Malignant tumor/carcinoma

Tumor size (MRI) Invasion Metastasis Mitoses (n/10 HPF) Ki-67 LI p53 positivity Neoangiogenesis Vascular invasion

< or >10 mm No No 0 or 2 >3 % Variable 0 0

>10 mm Yes Yes 2–10 (abnormal) >10 % or low Usually positive Variable Rare

Cell Cycle Markers Ki-67 Nuclear proteins, such as Ki-67, are also used to assess proliferation. Ki-67, which is expressed throughout the cell cycle, is now detected in routine by a monoclonal antibody (MIB-1). The Ki-67 labeling index (LI), defined as a percentage of positive nuclei, is a very easy reproducible and reliable method. Although the Ki-67 LI varied according to the fixative (it is lower in Bouin-Hollande than in formol fixative), its correlation with tumor growth and invasion is conflicting [46, 47, 67–72]. Ki-67 LI >3 % is considered as a good sign of proliferation, but it varies according to IHC type [47], and it is higher in the functioning tumors than in nonfunctioning ones [73], and so by itself, it does not predict the tumor behavior, and the use of other makers is recommended. Mitoses The presence and the number of mitoses are important predictive factors of a rapid growth. However, given the low proliferative rate of the majority of pituitary tumors, only scarce mitoses can be identified, more easily with Ki-67 immunostaining than with HPS staining. As for endocrine pancreatic tumors [43], I consider that a tumor is proliferative if the number of mitoses is higher than 2/10 HPFs. p53 and Other Cell Cycle Markers The expression of p53 gene product is an important marker in many cancers. But, p53 mutations have not been described in pituitary tumors, and the value of its detection is controversial. Indeed, its evaluation may differ from one laboratory to another and from one pathologist to another, and its correlation with invasion and aggressiveness/malignancy was found in some series [71] and not in others [74]. Since p53 has been found elevated in almost all pituitary carcinomas [71] and “an

extensive p53 positivity” is one of the criteria of the atypical adenoma [7], we decided to consider p53. In our classification, we considered its positivity and not its precise percentage. I propose to stipulate its percentage for future studies. Compared to their noninvasive counterparts, invasive pituitary tumors also express higher levels of pituitary tumor transforming gene (PTTG) which is a member of the securin family that regulates sister chromatid separation during mitosis [75]. Other cell cycle markers have also been studied (topoisomerase IIα, cyclins, cyclin-dependant kinases, etc.) but are not used routinely.

Growth Factors, Metalloproteinases, and Angiogenesis Of the fibroblast growth factors (FGFs) and their receptors (FGFRs), which regulate growth, differentiation, migration, and angiogenesis, FGF2 and FGF4 are expressed in the pituitary. Tumors show a loss of FGFR2, with a resultant upregulation of MAGEA3, and a truncated isoform of FGFR4, known as pituitary tumor-derived (ptd-FGFR4). The expression of the latter induces invasive growth of pituitary tumor cells in vivo with loss of membranous N-cadherin expression [76]. Moreover, ptd-FGFR4 interacts with a polysialated form of neuronal cell adhesion molecule (PSA-NCAM), which correlates with invasiveness [72, 77]. The matrix metalloproteinase (MMP) family of proteolytic enzymes cleaves extracellular matrix molecules and plays a role in invasiveness of many neoplasms. The expression of MMP9 is significantly higher in invasive pituitary tumors [78] and may be correlated with the activation of protein kinase C, which is also known to be involved in the invasion of pituitary tumors. Although the tumors are less vascular than the normal pituitary, a significantly increased microvessel density has been found in invasive PRL tumors than in the noninvasive ones. Moreover, the expression of endocan, a proteoglycan secreted by endothelial cells, has also been associated with size and progression of pituitary tumors [79].

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Transcription Factors

Table 4 Checklist for pathological diagnosis of pituitary endocrine tumors

Transcription factors involved in pituitary cell differentiation can also be examined and may be useful to confirm the IHC diagnosis, particularly of immunonegative or “silent tumors.” Pit-1 is expressed in GH, PRL, and TSH tumors [80], T-pit in ACTH tumors with and without Cushing’s disease [81], and SF-1 in FSH-LH tumors [82]. Moreover, the detection of somatostatin receptors type 2 (SSTR2) and 5 (SSTR5) could be helpful in GH, TSH, ACTH, and FSH-LH tumors to identify those with low SSTR2 expression, shown to represent a higher risk of resistance to octreotide/lanreotide, or those with high SSTR5R expression more likely to respond to pasireotide treatment. This detection is always negative in PRL tumors [83]. My pathological diagnosis of endocrine pituitary tumors uses this checklist, which is different from the recent published one [48], in that it takes into account the MRI and the peroperative data. It also includes freezing tumor samples for future molecular biology analyses (Table 4).

I - Preoperative pituitary MRI Tumor size: Micro, Macro, Giant tumor Invasion: No yes localization: cavernous sinus sphenoidal sinus other Peroperative invasion: yes no ND II - Histology 1) Description 2) Invasion ND No yes localization: Juxtatumoral pituitary: 3) Immunohistochemistry (Note the fixative and the clones of the tested antibodies)

Genetic Markers Recent progress in molecular and genetic techniques (transcriptome, comparative genetic hybridization, and epigenetic) allowed the identification of variations in gene expression (microRNA and miRNA), loss of heterozygosity (LOH), and gene mutation in pituitary tumors (rev in the study of Raverot et al. [66]). These techniques need quickly frozen tissue in sufficient quantity and a control of the frozen samples by the pathologist to avoid contamination by normal pituitary. Moreover, the molecular data must be correlated to precise clinical and pathological data. Using rigorous conditions, some of these markers were related to invasion and aggressiveness in gonadotroph tumors [75, 84] and PRL tumors [51, 59, 60]. Fifteen years ago, Bates et al. identified a significantly higher frequency of LOH in invasive tumors compared to noninvasive tumors [85], and this has been confirmed in other publications. We recently demonstrated that the 11p region was commonly deleted in PRL aggressive tumors (grade 2b) [61] and a loss of the 11q arm and a gain in the 1q arm in the three aggressive tumors that developed metastasis during follow-up and so became malignant. These results suggested that accumulation of new chromosomal alterations (11q loss and 1q gain) may transform an “aggressive” pituitary tumor into a pituitary carcinoma. Pituitary tumors are mostly sporadic, but some familial cases have been identified leading to the characterization of genetic forms of pituitary adenoma. The search for MEN1 or AIP mutation is necessary in patients with a familial history [86], or in young patients with a large macroadenoma [87, 88],

Antibodies to be tested: a) For the tumor typage Chromogranine A, βFSH, βLH, αSU → FSH or FSH-LH or LH or αSU tumor PRL, GH, CK → GH or GH-PRL or PRL tumor, silent GH or GH-PRL tumor ACTH, βend, CK → ACTH tumor, silent ACTH tumor -βTSH, CK → TSH tumor -SSTR 2-5 for GH, TSH, FSH-LH and ACTH tumors If all the antibodies negative: immunonegative tumor b) For evaluation of proliferation (count for 10 HPF) Index of Ki-67:(cutoff >3 %); Number of mitoses: (cutoff n > 2) Expression of p53: + or − (cutoff n > 10 positive nuclei); Index: c) Grading: taking into account invasion (MRI) and proliferation (two or three markers > cutoff) Grade 1a, Grade 1b, Grade 2a, Grade 2b, Grade 3 (if metastasis) III - Molecular biology Frozen samples of tumor after control by smears or frozen sections. Keep at −80° or in liquid nitrogen for microarrays, PCR, and CGH.

or in the case of unusual plurihormonality or double adenomas [89]. Recent advances in pituitary tumor classification now allow the early identification of pituitary tumors with high risk of recurrence, associated with a malignant potential. In such cases (grade 2b), after discussion by a designated team of neurosurgeon, endocrinologist, pathologist, and oncologist, an optimized therapeutic strategy should be proposed taking into account new therapeutic options [6] in addition to conventional therapies associating surgery and radiotherapy [90].

Conclusion The evidence against pituitary tumors being considered only as benign tumors inducing hormonal disease is mounting. Besides the rare carcinomas and the frequent noninvasive, nonproliferative benign pituitary tumors, a group of tumors

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representing almost 15 % of all pituitary tumors should be individualized. We propose to name such grade 2b tumors with high risk of recurrence as tumor suspected of malignancy. Indeed, clinical, pathological, and molecular evidence suggests that some of these tumors may develop metastasis during follow-up. Recent genomic studies identifying new molecular markers associated with tumor behavior may help to classify these tumors. Until markers of malignancy were discovered, comprehensive classification of pituitary tumors remains the best indicator of clinical behavior in patients with pituitary tumors, but their prognostic value has to be proved with multivariate statistic methods in prospective studies. Acknowledgments I thank G Sassolas who, in 1970, suggested that I study pituitary tumors, which, at this time, were not considered as the most frequent cause of acromegaly and of amenorrhea-galactorrhea. I am delighted that G. Raverot, Prof. of endocrinology, now the leader of the research on pituitary tumors in Lyon, along with E. Jouanneau, Prof. of neurosurgery, and Dr A. Vasiljevic, neuropathologist, all cooperate in the diagnosis and patient management of pituitary tumors. Many thanks to P Chevallier, A. Reynaud, and M.P. Guigard for expert technical assistance during 10 to 30 years and to my secretary P. Gerardi for typing our manuscripts. At least, I thank all the members of HYPOPRONOS who gave data of their patients: R. Assaker, C. Auger, A. Barlier, M. Bernier, F. Bonnet, J. F. Bonneville, F. Borson-Chazot, G. Brassier, T. Brue, S. CauletMaugendre, O. Chabre, F. Chapuis, P. Chanson, A. Cornelius, C. Cortet-Rudelli, J. F. Cottier, E. Dantony, B. Delemer, E. Delgrange, L. Di Tommaso, H. Dufour, S. Eimer, D. Figarella-Branger, P. François, S. Gaillard, F. Galland, J. J. Girard, M. Jan, J. Lachuer, V. Lapras, H. Loiseau, C. A. Maurage, F. Mougel, J. G. Passagia, M. Patey, A. Penfornis, J. Y. Poirier, G. Perrin, P. Roy, N. Sturm, A. Tabarin, G. Viennet, L. Villeneuve, and A. Wierinckx. Conflict of Interest The authors have nothing to disclose. This work was supported by grants from the Ministère de la Santé (Programme Hospitalier de Recherche Clinique National no. 27-43, HYPOPRONOS) and research contracts with the Institut National de la Santé et de la Recherche Médicale and the Ligue Contre le Cancer Rhône-Alpes.

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