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The AFIP (Armed Forces Institute of Pathology) Atlases (Fascicles) have been in continuous publication for nearly 75 years and have enjoyed a highly regarded reputation for their excellence. Throughout this time period, more than 130 volumes, encompassing the 1st to 4th series have been published. Since their inception in the 1940’s, the Fascicles have evolved from loose-leafed atlases illustrated with black and white images, to hardbound monographs with full color images and expansion of scope, including relevant clinical information, cytopathology, and the most recent advances in immunohistochemistry and molecular diagnostics. Each of the volumes undergoes a rigorous review process by the Editor-in-Chief, Associate Editors, members of the Editorial Advisory Board, and external reviewers. The 5th series, under the editorial direction of Drs. Elizabeth Montgomery and Jason Hornick, is well underway and will include an Epub version and a virtual slide box, in addition to the hardbound book. The Atlases of Nontumor Pathology will also continue to be published. With the closure of the AFIP in 2011, the American Registry of Pathology (ARP) has assumed full responsibility for the publication of both the Tumor and Nontumor Fascicles.

Medullary thyroid carcinoma (MTC) is a thyroid neoplasm with evidence of C-cell differentiation that accounts for approximately 2% to 3% of all thyroid malignancies. Approximately 70% of MTCs are sporadic, whereas the remainder are heritable and occur as a component of the multiple endocrine neoplasia type 2 syndromes. Multifocal primary C-cell hyperplasia is the precursor of the familial cases. In the past several decades, there have been remarkable advances in the understanding of this tumor type and its precursor lesions at the clinical, histopathologic, and molecular genetic levels. This article presents an update on MTC and C-cell hyperplasia in the context of the 2017 World Health Organization Classification of Endocrine Tumors.

Expression of S100A4has been associated with progression and poor clinical outcome in a variety of malignancies including those of the breast, pancreas, bladder, and thyroid. To date, the expression of S100A4 protein in renal epithelial neoplasms is poorly understood. In this study, we evaluated the expression of S100A4 protein and mRNA in the nontumoral kidney and renal epithelial neoplasms of different types and correlated its expression with patient outcome. The study population included 155 clear cell renal cell carcinomas (cRCC), 22 papillary renal cell carcinomas (pRCC), 13 chromophobe renal cell carcinomas and 13 oncocytomas. In nontumoral kidney, nuclear and cytoplasmic S100A4 staining was detected in the glomerular epithelium and endothelium, distal tubules and collecting ducts, and loops of Henle. A different expression pattern was noted in the various neoplasms. S100A4 expression was significantly increased in the stromal cells in cRCC (83) and pRCC (73) compared with paired nontumoral kidney tissue (P<0.001). There was no increased stromal cell expression of S100A4 in oncocytomas and chromophobe carcinomas. Positive epithelial staining was more common in pRCC (58) than cRCC (11) (P=0.01). The level of mRNA detected by reverse transcription-polymerase chain reaction was significantly higher in the tumor as opposed to normal tissue in cRCC but not in the other neoplasms (P=0.03). Multivariate analysis revealed that epithelial S100A4 protein expression is an independent poor prognostic factor along with grade and stage only in cRCC (P<0.01). Although S100A4 protein was expressed in a minority of cRCC, its expression was associated with shorter overall patient survival.

Spindle cell proliferations of the thyroid gland are uncommon lesions that encompass a wide spectrum of reactive, hyperplastic, and neoplastic processes. Spindle cells may occur in subsets of papillary carcinomas and follicular adenomas where they are thought to represent metaplastic foci. The goals of the present study are to further characterize the metaplastic nature of spindle cell foci of the thyroid (SCFT), to define their immunohistochemical profiles and to review their differential diagnoses. The study group included: multinodular goiter (2), follicular adenoma (2), and minimally invasive follicular carcinoma (2). SCFTs were composed of elongate cells with thin or slightly plump nuclei with finely granular chromatin and inconspicuous nucleoli. Rare mitotic figures were present but there was no necrosis or inflammation. All cases were positive for thyroglobulin, thyroid transcription factor (TTF)-1, and TTF-2. TTF-1 and TTF-2 had a characteristic nuclear localization although the intensity of staining for TTF-1 was consistently greater than that of TTF-2. Each of the 6 cases was positive for vimentin whereas 5 of the 6 cases were positive for broad-spectrum cytokeratins. None of the cases was positive for high molecular weight cytokeratin, cytokeratin-19, smooth muscle actin, desmin, calcitonin, chromogranin, or synaptophysin. The proliferative rate was less than 1 in all cases. Staining for TTF-1 and TTF-2 provided high specificity for identification of SCFT since these markers were not subject to the same diffusion artifact inherent in thyroglobulin-stained sections. The results of this study further support the hypothesis that SCFT result from metaplastic transformation of follicular cells.

Primary hyperparathyroidism (P-HPT) is a common endocrine disorder that occurs as a result of adenomas (80–85%), hyperplasias (10–15%) or carcinomas (<1%) of the parathyroid glands. Molecular genetic analyses of heritable P-HPT syndromes have provided considerable insight into the understanding of sporadic parathyroid tumors and hyperplasias. This review will focus on the criteria for classification of parathyroid proliferative disorders and will highlight our understanding of these lesions at the molecular level. Advances in radiological imaging techniques together with the rapid intraoperative parathyroid hormone assay will be reviewed with respect to current treatment approaches for P-HPT.

Primary hyperparathyroidism (P-HPT) is a common endocrine disorder that occurs as a result of adenomas (80–85%), hyperplasias (10–15%) or carcinomas (<1%) of the parathyroid glands. Molecular genetic analyses of heritable P-HPT syndromes have provided considerable insight into the understanding of sporadic parathyroid tumors and hyperplasias. This review will focus on the criteria for classification of parathyroid proliferative disorders and will highlight our understanding of these lesions at the molecular level. Advances in radiological imaging techniques together with the rapid intraoperative parathyroid hormone assay will be reviewed with respect to current treatment approaches for P-HPT.

This study evaluated the expression patterns of claudins 1, 3, 4, 7, and 8 in human renal cell carcinomas and oncocytomas and correlated expression with patient prognosis. Tissue microarrays were created from paraffin-embedded tissue samples from 141 patients with renal cell carcinomas or oncocytoma (90 clear cell, 22 papillary, 17 chromophobe renal cell carcinomas, and 12 oncocytomas). The staining pattern for claudins 3, 4, 7, and 8 was membranous and/or cytoplasmic, whereas claudin 1 was predominantly membranous in both nonneoplastic renal tissue and tumors. Negative to weak claudin 3 staining was predominantly detected in Fuhrman's grade 1 and 2 clear cell renal cell carcinomas (78%; P=0.016), suggesting that upregulation of claudin 3 potentially occurs concomitantly with increasing grade of clear cell renal cell carcinomas. In addition, Kaplan–Meier univariate analysis showed a significant inverse correlation between moderate to strong claudin 3 and 4 expression with overall survival in clear cell renal cell carcinomas (P=0.038 and P=0.031). Moderate to strong claudin 7 expression was significantly more common in chromophobe renal cell carcinomas (94%) than in oncocytomas (55%; P=0.041). Claudin 8 staining was moderate to strong in 92% of oncocytomas, which differentiated them from papillary and clear cell renal cell carcinomas (14 and 12%; both P<0.0001). Only negative to weak claudin 8 staining was detected in all chromophobe renal cell carcinomas, whereas there were no claudin 8 negative oncocytomas and 8% exhibited a weak staining pattern (P<0.0001). Due to their distinctive expression patterns, claudins 7 and 8 can be used as useful immunohistochemical markers for the separation of chromophobe renal cell carcinomas from oncocytomas, whereas claudins 3 and 4 may serve as indicators of prognosis in clear cell renal cell carcinomas.

Identification of clonal lymphocytic populations by polymerase chain reaction may be difficult in cases with scant cellular infiltrates or those with a heterogeneous population of cells. Here, we assessed the diagnostic utility of laser capture microdissection (LCM) and high-resolution microcapillary electrophoresis in the analysis of clonality of small biopsy specimens. Clonality was determined in 24 cases: five reactive tonsils, five reactive lymph nodes, six inflammatory skin lesions, and eight T-cell lymphomas. CD3-positive T lymphocytes were captured by LCM from paraffinized immunohistochemically stained sections. Genomic DNA was analyzed for T-cell receptor-γ gene rearrangement by polymerase chain reaction followed by high-resolution microcapillary electrophoresis with the DNA 500 LabChip and the Agilent Bioanalyzer. In the reactive specimens, T-cell receptor-γ polymerase chain reaction revealed monoclonal bands when 10 to 1000 cells were captured. This pattern changed to polyclonal when higher numbers of cells were microdissected (2000 to 10,000 cells). In contrast, lymphoma cells were consistently monoclonal whether low or high numbers were microdissected. Microcapillary electrophoresis coupled with LCM facilitated clonality analysis in equivocal cases. In two of eight lymphoma cases, LCM revealed diagnostic monoclonal bands, whereas routine T-cell receptor-γ assessment of whole tissue sections with 10% polyacrylamide gel electrophoresis demonstrated only minor clonal bands. We conclude that clonality determined by LCM is cell number-dependent. Biopsy specimens containing low numbers of reactive polyclonal T cells may produce pseudomonoclonal bands and therefore should be interpreted with great caution.

Neuroendocrine (NE) carcinomas may originate in a wide variety of tissues and organs, including those that do not normally contain NE cells. These tumors may occur in pure forms or in association with conventional adenocarcinomas or squamous cell carcinomas, and they may also be associated with the paraneoplastic production of adrenocorticotropin, antidiuretic hormone, or other hormones. The 6 cases presented in this paper highlight the morphologic and functional characteristics of well- and poorly differentiated NE carcinomas arising in the kidney, thymus, prostate, esophagus, cervix, and skin. Recognition of NE carcinomas in these sites is critical both for therapeutic and for prognostic purposes.

Neuroendocrine (NE) tumors include a morphologically and functionally diverse family of neoplasms that can arise in virtually all tissues and organs. Although early studies suggested a neural crest origin for all NE tumors, the term NEsimply describes a shared phenotype notable for the expression of multiple genes encoding a wide spectrum of neural and NE traits. NE tumors can be subdivided into epithelial and neural types with both types expressing a set of common NE markers, including synaptophysin. Epithelial NE tumors are further defined on the basis of expression of cytokeratins (CK), with variable expression of neurofilaments (NF), while neural NE tumors, such as neuroblastomas and paragangliomas, are negative for CKs but are positive for NFs. Epithelial NE tumors exhibit a broad range of cytologic and histologic features, ranging from those that have a well-defined nesting or ribbonlike growth pattern with low mitotic rates to those that have a small- or large-cell morphology with high mitotic rates and diffuse growth patterns. There is general agreement that the generic term carcinoidas proposed in the 1980 World Health Organization classification has become increasingly more inappropriate to describe the full range of low- and intermediate-grade neoplasms with NE features. The value of the term NE, modified by the inclusion of the grade of the neoplasm, lies in the fact that it defines a particular phenotype that may respond to specific forms of targeted therapy. Additional studies, including gene expression profiling, will be essential for further classifying tumors with NE phenotypes and for elucidating their interrelationships.

Medullary thyroid carcinomas (MTCs) are currently defined as malignant tumors with evidence of C-cell differentiation. They comprise 5%-10% of all thyroid malignancies and occur sporadically (75%) or as a manifestation of the type 2 multiple endocrine neoplasia syndromes (25%). Results of calcitonin screening in patients with nodules thyroid disease have revealed a mean prevalence of tumors of 0.61% (range, 0.24% to 2.85%), most of which represent microscopic or incidental MTCs measuring less than 1 cm. While heritable and sporadic tumors contain amyloid and have a lobular to solid growth pattern and polyhedral- to spindle-shaped cells, numerous variants of these tumors also occur. C-cell hyperplasia (CCH) has been regarded as the precursor of heritable MTCs, although recent studies suggest that CCH is a clonal process and should be considered a form of intraepithelial C-cell neoplasia. Activating germline mutations of RET, primarily affecting exons 10 and 11, with less frequent involvement of exons 13, 14, 15, and 16, represent the molecular basis for the development of heritable MTCs. Somatic RET mutations, primarily affecting codon 918, occur in a substantial proportion of sporadic MTCs. Five-year and 10-year survivals for sporadic MTCs are 65% and 50%, respectively, with major risk factors for unfavorable outcome including tumor stage, male sex, and age greater than 60 years. With the development of molecular testing for RET mutations, the prognosis for patients with heritable MTCs is generally excellent.

Cirrhosis occurs in 20-50% of patients with hepatitis C and is thought to be irreversible. We describe two patients with cirrhosis secondary to hepatitis C in whom the extensive fibrosis and cirrhosis appeared to regress in response to treatment with interferon-α (IFN-α). Both patients were in the early stages of cirrhosis, class A in the Child-Pugh classification, total score 5 for each patient. Both responded fully to IFN-α and had normalization of all liver function tests and disappearance of hepatitis C viral RNA. Liver biopsies, performed before and after treatment, were coded unpaired by patient, combined with 21 liver biopsies from eight other patients with chronic hepatitis, and read independently by two pathologists using the Knodell scoring system. Knodell scores decreased from 14 to 3.5 and from 13.5 to 4 in these two patients. Cirrhosis and extensive fibrosis, present at baseline, were not present on follow-up liver biopsies, which were of sufficient size to reduce the likelihood of sampling error. We conclude that hepatic fibrosis and clinically early cirrhosis may be reversible in some patients with hepatitis C who respond to treatment with IFN-α.

The development of assay techniques for the analysis of tumor markers in tissue extracts and in the peripheral circulation has had a major impact on the diagnosis and management of patients with endocrine neoplasms. In addition to eutopic and ectopic hormones, a variety of other tumor-derived products, including endocrine secretory proteins, enzymes, and oncodevelopmental antigens, can be assayed. The development of stimulatory tests to evaluate secretory reserve has been of particular value for the early detection of endocrine tumors and preneoplastic lesions in the multiple endocrine neoplasia syndromes. In addition, analysis of tumor markers has been of considerable importance for estimating total tumor burden and assessing responses to various therapeutic modalities. Recent advances in molecular biologic and cytogenetic technologies have the potential for prenatal detection of some forms of the inherited multiple endocrine neoplasia syndromes.

Patients with lymph node negative invasive breast carcinomas ≤1 cm in size have a low recurrence rate and may be spared adjuvant therapy. Reliable prognostic features will help physicians design appropriate treatment for these patients. The clinicopathologic features, prognostic marker profiles, and clinical outcomes of 88 T1a,bN0M0 carcinomas in 87 patients who presented between 1975 and 1990 were studied. The size of each tumor was determined by direct measurement of histologic sections. The median follow-up was 7.8 years (range, 4-15 years). The characteristics of tumors diagnosed between 1975 and 1983 and between 1984 and 1990 were also compared. Before 1984, the majority of patients presented with palpable mass lesions, whereas from 1984 on, more patients presented with mammographic abnormalities. However, no significant differences in the pathologic features of tumors were observed between the two periods. There were only 3 locoregional recurrences (3%) and 4 distant recurrences (5%). Palpable tumors had worse prognoses than mammographically detected lesions (P = 0.02). Histologic grade, lymphatic invasion, hormone receptors, Ki-67 antigen, and bcl-2 expression were significant univariate prognostic indicators. The small number of patients in the series precluded multivariate analysis. None of the 43 patients (49%) with tumors ≤0.5 cm, or of histologic and nuclear Grade 1, or of favorable histologic types developed recurrences; and their outcomes were significantly better than those of other patients (P = 0.013). Tumors originally classified as T1b, but which exceeded 1 cm on review and were excluded from the study, had a significantly higher distant recurrence rate (23%) than bona fide T1a,b carcinomas (P = 0.03). T1a,bN0M0 carcinomas have a low recurrence rate, especially those tumors ≤0.5 cm, or of low histologic or nuclear grade, or of favorable histologic type. The high recurrence among patients with tumors initially understaged as T1a,b carcinoma underscores the importance of accurately determining tumor size. Cancer 1997; 79:761-71. © 1997 American Cancer Socitey.

Argyrophil cells were identified by the single-impregnation Grimelius technique in 11 of 28 (39) Brenner tumors, accounting for < 1 of the tumor cell population in all the cases. All tumors with argyrophil cells were stained to demonstrate calcitonin, somatostatin, gastrin, adrenocorticotropic hormone, neurotensin, insulin, glucagon, and serotonin; and four of them (three benign and one borderline) were also stained for chromogranins with the monoclonal antibody LK2H10. Serotonin was present in nine of the 11 cases with argyrophil cells. Neurotensin and somatostatin were found in one borderline tumor, which also contained serotonin. Chromogranin reactivity was demonstrated in all four cases in which it was examined. Ultrastructural examination of one tumor revealed that the argyrophil cells contained secretory granules, 80 nm in diameter, and had elongated cytoplasmic processes that extended between the more numerous nonargyrophil tumor cells. The argyrophil cells of Brenner tumors are similar to those of urothelium in the frequency with which they are immunoreactive for serotonin and the rarity with which they are reactive for peptide hormones. These cells differ from those of mucinous ovarian tumors, which often contain both serotonin and peptide hormones. The findings of this study lend additional support to the close similarity of the epithelial components of Brenner tumors and urothelium.

This case report describes a patient with pancreatic cholera caused by a vasoactive intestinal polypeptide-producing pancreatic tumor. The case presents several unusual characteristics of this disease. The primary tumor was a mucinous adenocarcinoma of the pancreas. The serum vasoactive intestinal polypeptide level of 2400 pmol/L is the highest reported. At this vasoactive intestinal polypeptide level, the somatostatin analogue SMS 201-995 at doses up to 2 mg/24 h did not control the 21 L/24 h stool output. Fecal incontinence due to a manometrically documented hypotonic internal anal sphincter occurred. Using surgically created stomas, the segmental gastrointestinal fluid and sodium losses were shown to be greatest from the jejunum, whereas potassium losses from the colon and small intestine were equal. The cellular mechanism for the small intestinal potassium secretion is not known.

The chief cells of paraganglionic tissues have morphological and functional similarities to adrenal chromaffin cells, and both cell types are derived from the neural crest. In the present investigation cells from two glomus jugulare paragangliomas were studied in culture. Approximately 50% of the cells from one tumor, and 7% from the other spontaneously formed neurite-like processes. Numerous granular and agranular synaptic-like vesicles also appeared in the process-forming cells. In contrast to findings with normal and neoplastic adrenal chromaffin cells, addition of nerve growth factor (NGF) to the culture medium had no major effects on proportion of cells with processes. Dexamethasone caused only a small decrease in process length. Culturing of the tumors also appeared to promote production of material with VIP-like immunoreactivity. It is concluded that the phenotype of paraganglioma as well as pheochromocytoma cells may be altered in vitro. Responsiveness to specific factors such as NGF or steroids, however, may vary for related tumor cell types in different anatomic locations.

Normal postnatal rat chromaffin cells and rat pheochromocytoma cells are known to show extensive Nerve Growth Factor (NGF)-induced process outgrowth in culture, and this outgrowth from the postnatal chromaffin cells is abolished by the corticosteroid dexamethasone. To determine whether adult rat chromaffin cells respond to NGF and dexamethasone, dissociated adrenal medullary cells from 3-month-old rats were cultured for 30 days in the presence or absence of these agents. Such cultures contained typical chromaffin cells, chromaffin cells with processes, and neurons. Fewer than 2 % of normal adult chromaffin cells formed processes under any of the conditions studied, and statistically significant changes in this proportion were not detectable in the presence of NGF or dexamethasone. Adrenal medullary neurons, however, were observed only in the presence of NGF, in cultures with or without dexamethasone, and thus appear to be previously unreported NGF targets which require NGF for survival or process outgrowth. Dexamethasone markedly increased total catecholamine content, total content of epinephrine, and tyrosine hydroxylase activity in cultures with or without NGF. In contrast, postnatal rat chromaffin and rat pheochromocytoma cells which have been studied in culture do not produce epinephrine under any of these conditions. It is concluded that rat adrenal chromaffin cells undergo age-related changes in both structural and functional plasticity. The in vitro characteristics of rat pheochromocytoma cells more closely resemble those of postnatal than of adult rat chromaffin cells, but may not entirely reflect the properties of the majority of chromaffin cells in either age group.

PC 12 pheochromocytoma cells treated with nerve growth factor (NGF) in combination with high concentrations of the activators of adenylate cyclase, forskolin or cholera toxin, become more neuron-like in size than cells treated with NGF or with activators of adenylate cyclase alone. Cells treated simultaneously with NGF plus forskolin or cholera toxin paradoxically show less process outgrowth than cells treated with NGF alone. Addition of forskolin or cholera toxin to cells pretreated with NGF, however, produces enlarged cells with intact processes that are indistinguishable from cultured neurons. One possible implication of these findings is that NGF might act in concert with agents that increase intracellular cyclic AMP to cause neuronal maturation during embryogenesis, and that the proper sequence of exposure to these signals is necessary for normal development. Specific activity of acetylcholinesterase is increased by NGF but is unaffected or slightly decreased by forskolin, suggesting that individual aspects of the developing neuronal phenotype are subject to different types of control.

We have examined an unselected series of 72 lymphomas of diverse histologies with a panel of mouse monoclonal antibodies specific for human B-lymphoma-derived Ig idiotypes (anti-ids) to determine the nature and extent of id/anti-id crossreactivity. The anti-id antibodies were prepared from Ig isolated from seven follicular center cell lymphomas by heterohybridoma technique. Thirty-six of 75 individual anti-ids obtained in this manner were further selected based on their reactivity with highly restricted or private idiotypic determinants. Twelve of the 72 (17%) lymphoma biopsies reacted with one or more of the 36 anti-ids that detect private determinants. A pool consisting of five individual antibodies would have detected 11 of the 12 crossreacting tumors. Staining of tumor cell populations was homogeneous in the positive cases, suggesting uniform idiotype expression. If there was a segregated staining pattern, it was generally related to the presence of CD3+ T cells in the section. These follicular center cell-derived anti-ids crossre-acted with follicular center cell tumors of all histologic grades with frequencies ranging from 13% to 50%. The structural basis for the crossreactivity of our lymphoma-derived private anti-ids is as yet not known. However, the reactivity of certain anti-ids with both kappa- and lambda-expressing tumors suggests a biased use of V gene segments in these crossreactive clones that is probably related to the VH gene. These data suggest the possibility that lymphoma may develop in a highly restricted pool of normal differentiated B cells or in B-cell subsets that express a limited repertoire of unmutated VH gene segments.© 1992 by The American Society of Hematology.0006–4971.92/8004-0030$3.00/0

A patient with watery diarrhea, hypokalemia, hypochlorhydria, and a non-beta islet cell carcinoma of the pancreas (Verner-Morrison syndrome) was found to have an elevated vasoactive intestinal peptide (VIP) concentration in the plasma as well as in the tumor. Treatment with streptozocin resulted in a dramatic subjective and objective tumor response in this patient. Plasma VIP concentration fell into the normal range after four courses of treatment, diarrhea ceased after the third course of therapy, and measurable tumor mass markedly decreased during that same period of time. The patient remains in clinical remission with no evidence of tumor regrowth 18 months after the beginning of treatment. In this patient, plasma VIP measurements were an excellent marker of tumor activity and correlated well with objective disease measurements and clinical response.(Arch Intern Med 136:1429-1435, 1976)

Acid hydrolases are not exclusively lysosomal enzymes as indicated by the specific case of β-glucuronidase. This enzyme is a membrane protein found in the endoplasmic reticulum and in the lysosomes of the cell.

After 20 days of treatment with propylthiouracil, a two-fold increase in the amount of ß-glucuronidase per gram of rat thyroid was noted. This change was manifested cytochemically by both an increase in the number of ß-glucuronidase containing granules and an enhancement of the generalized cytoplasmic activity. The results are discussed in relation to the dual localization of ß-glucuronidase.

THE localization of beta-glucuronidase in populations of free cells has already been reported1from this laboratory. Thus, formaldehyde–chloral hydrate fixation of the sedimented cells followed by embedding in Knox gelatine1made it possible to cut thin sections of the frozen gelatine block on the freezing microtome and the sections so obtained were incubated while floating freely in Fishman–Baker substrate medium containing 8-hydroxyquinoline β-D-glucosiduronic acid and ferric ions. The enzymatically liberated 8-hydroxyquinoline became insoluble as the brownish-black ferric chelate at the sites rich in enzyme, and the ion was later converted to Prussian blue. The whole process required a minimum of 24 h from beginning to end and included many steps2. Efforts to use dried smears with this technique were usually unsuccessful (except for one report)3because of loss of cells from the slide during the various steps in the staining process.

Correction to: Modern Pathology advance online publication, 27 June 2008; doi:10.1038/modpathol.2008.116 Following the online publication of this article, the author found an error in Figure 3, panel C; the label should be 'IHC Staining for claudin 4' instead of 'IHC Staining for claudin 3.'