[CANCER RESEARCH 48, 7319-7322, December 15, 1988)
`A Novel Monoclonal Antibody, SCCL 175, with Specificity for Small Cell
`Neuroendocrine Carcinoma of the Lung1
`Vincent A. Memoli, 2 Andrea G. Jordan, 3 and Edward D. Ball
`Dtpartmtnts of Pathology {JI. A. M., A. G. J.) and Medicine and Microbiology {E. D. B.), Dartmouth-Hitchcock Medical Center, Hanover, New Hampshi� 037$$
`
`ABSTRACT
`The murine monoclonal antibody SCCL 175, which is one of several
`monoclonal antibodies directed against small cell neuroendocrfne carci­
`noma developed by one of us (E. D. B.), was studied for Its lmmunohfs­
`tochemical reactivity against normal human tissues and a spectrum of
`bronchopulmonary and metastatic carcinomas using the avldln-blotin
`complex technique. SCCL 175 reacted with 40 of 44 small cell carcinomas
`Including both primary and metastatic sites and was distributed both on
`the cell surface and intracytoplasmlcally. Staining was seen In fresh
`frozen tissues, cytology preparations, and In a llmlted number or paraflln­
`embedded tissue sections after trypsin pretreatment. It was nonreactive
`with all non-small cell lung carcinomas, neuroendocrine carcinomas from
`other primary sites, and nonpulmonary carclnomata studied to date. Its
`distribution in normal adult human tissues was llmlted to some hypothal­
`amic neurons and the apical membranes of renal proximal tubular epi­
`thelium. Cytotrophoblastic and syncytotrophoblastic cells from placental
`tissue demonstrated variable SCCL 175 lmmunoreactivity. or choriocar­
`cinomas studied, one of three demonstrated focal staining. These findings
`demonstrate the diagnostic utility of SCCL 175 In phenotyping small cell
`carcinoma or lung, and its specificity suggests a potential role in the
`therapy of this disease.
`
`erful role of this approach in tumor classification (8-18).
`The IgM murine MoAb, SCCL 175, prepared in one of our
`laboratories (E. D. B.) recognizes an antigen associated with
`SCCL in cells obtained from tissue samples and cultured cell
`lines (8). SCCL 175 is one of several MoAbs prepared by
`immunizing with fresh SCCL tumor tissue which we have
`shown by flow cytometry to be reactive with seven of seven
`patient-derived SCCL cells and nine of ten SCCL cell lines. It
`is nonreactive with a number of non-SCCL lung tumor cell
`lines, common blood group antigens, histocompatibility anti­
`gens, the terminal pentasaccharide lacto-N-fucopentaose III,
`and lipid extracts of SCCL tumor cells. SCCL 175 precipitates
`two polypeptides with molecular masses of 155 and 115 kDa,
`respectively, under reducing conditions from SCCL cell lines.
`Further characterization of this antigen is in progress. The
`purpose of this paper is to report the reactivity of MoAb SCCL
`175 with normal human tissues and carcinomata obtained from
`surgical biopsy, cytology, and autopsy and to demonstrate its
`clinical utility in the diagnosis of SCCL.
`
`MATERIALS AND METIIODS
`
`Normal Tissues. Fresh normal human tissue blocks were obtained
`from either surgically resected specimens or at autopsy performed
`within 4 h of death from patients with no history or evidence of
`carcinoma. All tissues were snap-froz.en and stored at -70"C prior to
`their use in immunohistochemical studies. Normal tissues are listed in
`Table 1 and include lung, heart, liver, kidney, pancreas, esophagus,
`stomach, small bowel, colon, thyroid, parathyroid, adrenal, pituitary,
`bone marrow, spleen, tonsil, lymph node, cerebral cortex, cerebellum,
`and hypothalamus. Normal fetal lung tissue from 8 autopsy cases and
`paratrm sections from 12 normal placentae including first, second. and
`third trimester cases were studied. Cryostat-froz.en, 8- to 10-µm sections
`were mounted on poly-L-lysintH:Oated slices (19), fixed for 5 min in
`cold acetone, and air dried for 10 min prior to immunostaining.
`Tumor Specimens. All human tumor tissues were obtained from
`surgical biopsies, resections, cytological specimens, and at autopsy
`performed within 4 h of death. Fresh human tumor tissue obtained
`from ten patients with SCCL and seven with non-SCCL was prepared
`according to the method outlined above for normal human tissues.
`Routine, formalin-fixed, paraffin-embedded. tissue blocks from nine
`cases of SCCL were obtained at flexible fiberoptic bronchoscopy, sec­
`tioned at 8 to 10 µm, and mounted on poly-L-lysine-coated glass slides
`for immunohistochemical studies. Pap-stained cytology smears from
`25 cases of SCCL, 65 cases of non-SCCL bronchopulmonary cancers,
`and 10 pulmonary metastases were decoverslipped and rehydrated in
`graded alcohols prior to immunohistochemical staining. A series of
`neuroendocrine and nonneuroendocrine carcinomas from other pri­
`mary sites was studied and listed in Table 2.
`lmmunohistochemistry Method. A modification of the ABC tech­
`nique (20) was used for all tissue sections and cytology smears. Tissues
`were treated with normal blocking serum at 37"C for 15 min. Sections
`were incubated with MoAb SCCL 175 at 37"C, for 60 min, washed,
`and then incubated with biotinylated anti-mouse lgG antibody at 37"C,
`for 30 inin. Endogenous peroxidase activity was blocked with 0.6%
`H202 in absolute methanol for 15 min at 25"C. ABC was applied to
`sections at 37"C, for 30 min. Sections were incubated with 3',3-
`diaminobenzidine substrate at 25"C for 5 min, washed, counterstained
`with hematoxylin, and subsequently dehydrated and coverslipped.
`7319
`
`INTRODUCTION
`Neuroendocrine SCCL 4 is a distinct clinical and pathological
`type of bronchopulmonary neoplasm, the recognition of which
`plays an important role in determining the management and
`outcome of patients with this diagnosis (1, 2). Modern diag­
`nostic techniques such as aspiration cytology and flexible fiber­
`optic bronchoscopy have provided the pathologist with a chal­
`lenge, requiring diagnostic precision from ever sinaller tissue
`and cell samples. Morphological heterogeneity within the var­
`ious subtypes of bronchopulmonary neoplasms has further com­
`plicated the task of the pathologist, requiring new diagnostic
`methodologies (3-6). Immunohistochemistry ·has become a
`powerful tool for diagnosis, allowing classification of a great
`variety of tumors via the recognition of tumor- and tissue­
`specific antigens in small biopsy and cytological samples. How­
`ever, its role in the classification of bronchopulmonary neo­
`plasms has been limited by the unavailability of suitable markers
`for distinguishing the various subtypes of these carcinomata.
`Previous work has demonstrated the applicability of immuno­
`histochemical analyses to the classification of pulmonary neu­
`roendocrine neoplasms using antibodies recognizing a variety
`of neuroendocrine marker substances and different classes of
`intermediate filament proteins (6, 7). Recent studies with a
`number of MoAbs having specificity for specific histological
`subtypes of bronchopulmonary cancers demonstrate the pow-
`
`Received 1/22/87; revised 10/1/87, 8/29/88; accepted 9/8/88.
`The costs of publication of this article were defrayed in pan by the payment
`or page charges. This article must therefore be hereby marked advntisement in
`accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
`1 Supported in part by Grant CA 37868 from the National Cancer Institute.
`1 To whom requests for reprints should be addressed.
`3 Present address: Department of Patholo11Y, Jefferson Medical College, Phil­
`
`adelphia, PA.
`•The abbreviations used are: SCCL, small cell carcinoma or the lung; MoAb,
`monoclonal antibody; ABC, avidin-biotin complex; GI, gastrointestinal; CNS,
`central nervous system.
`
`nnwnln::irlP.rl frnm r.;:mr.P.rrP.s ::i::ir.rint 1rn::ils nrn nn . h 1lv 1 n ?n 111 © 1 !=!RR AmP.rir.::m Assnr.i::itinn fnr r.::inr.P.r RP.sP.::irr.h
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`MoAb SCCL 17S AND SPECIFICITY FOR NEUROENDOCRINE LUNG CARCINOMA
`
`Table 1 MoAb SCCL I 75 imm11noreactivity with normal tissues
`MoAb SCCL 175
`
`Normal tissues Total Negative Positive
`
`Cell type
`
`Adrenal
`Bone marrow
`Cerebrum
`Cerebellum
`GllnlCt
`Heart
`Liver
`Lung
`Lymph node
`Kidney
`Pancreas
`Pituiwy
`Placenta
`Spleen
`Thyroid
`
`8
`12
`5
`s
`18
`5
`5
`20
`10
`12
`8
`5
`12
`8
`10
`
`8
`12
`4
`s
`18
`s
`5
`20
`10
`
`8
`5
`
`8
`10
`
`Hypothalamic neurons
`
`12
`
`Proximal tubules
`
`12
`
`Trophoblasts
`
`Table 2 MoAb SCCL 175 imm11norractivity in tllmor ti.ss�
`Reactivi�
`Negative + ++
`
`No. of cases
`(total)
`
`Tumor type
`
`+++
`
`Primary pulmonary
`Squamous carcinoma
`Adenocarcinoma
`Large cell carcinoma
`Undifferentiated carcinoma
`Carcinoid
`SCCL (primary)
`SCCL (metastatic)
`
`Nonneuroendocrine
`Colonic adenocarcinoma
`Prostatic adenocarcinoma
`Breast carcinoma
`Neuroblastoma
`Choriocarcinoma
`
`20
`23
`22
`13
`3
`34
`10
`
`17
`3
`3
`3
`3
`
`20
`23
`22
`13
`3
`4
`
`17
`3
`3
`1
`2
`
`6
`
`13
`4
`
`11
`6
`
`2
`1
`
`Neuroendocrine
`IS
`Thyroid medullary carcinoma
`15
`5
`5
`Adrenal pheochromocytoma
`S
`Merkel cell carcinoma
`5
`GI careinoid
`S
`5
`• Semiquantitative analysis of the percentage of positively stained tumor cells
`per case: neptive, no detectable positive cells; +, <20% positive; ++, 20 to 60%
`positive: +++, >60% positive.
`
`Fig. I. Section of human kidney showing apical staining of renal proximal
`tubular epithelial cells with SCCL 175. ABC technique, counterstained with
`hematoxylin: x 450.
`
`_. ··. ·:·�-7: :.: ·:··� . . :Tc.:!·._:;�-·�.
`·-��:;�·
`.: �.
`. ,:·r.>>
`�:�·7:�;:�_:_-{�: �'=·�� ::·:·.·�
`
`·;�:�p<;:•;
`:j,,:· ,:}/'·,
`:·· .
`. , . ._,
`--._· ..
`'·:· �-·.��:-·
`-.
`
`: ..
`
`."··:, ·.\=::;;;:, < +
`,, · . .:'. .. ;.'.".
`· ·'_.'·'_
`. �-�:.".�·_.�::·:.�):�.·:.:. ;-." .'· ·,' • · ..... _'. ·...
`''."�:./'.'.::\':.·1>·
`. . '": ��
`
`.·.
`
`.
`r�·��"/ .
`:: .... .
`..·
`._, .
`·�··
`- -
`- - --
`Fig. 2. A neuron with cytoplasmic SCCL 17S immunoreactivity in both cell
`body (single am>w) and dendritic process (doublt am>w). ABC technique, coun­
`terstained with hematoxylin; x 910.
`
`',···.
`
`...
`
`: .
`
`.. :·
`
`MoAb SCCL 175 used in these studies consisted of both hybridoma
`supernatant and mouse ascites fluid at a 1: 100 dilution. Blocking serum, .
`biotinylated linking antibody, and ABC peroxidase complex were ob­
`tained from Vector Labs, Burlingame, CA (Vectastain ABC kit) and
`prepared according to the manufacturer's instructions.
`Paraffin-embedded tissue sections were pretreated with 0.1 % trypsin
`in phosphate-buffered saline, pH 7.6, at 2S"C for S min prior to
`immunohistochemical staining.
`Positive controls for immunostaining were prepared from cytospin
`preparations of the OMS SCCL 153 cell line (8). Hybridoma superna­
`tant containing an lgM MoAb to an unknown irrelevant antigen served
`as a negative antibody control.
`
`RESULTS
`Normal Tissues. Table 1 summarizes the immunoreactivity
`of MoAb SCCL 175 with normal adult human tissues. Note­
`worthy is the absence of immunostaiiling of endocrine organs
`and neuroendocrine cells in the GI tract and lung. All 'levels of
`the GI tract were studied including esophagus, stomach, duo-
`denum, jejunum, ileum, and colon. Sections of normal lung
`20% of tumor cells demonstrated immunoreactive staining with
`from all lobes as well as fetal lung were entirely negative for
`SCCL 175. In the kidney, renal proximal tubular epithelium MoAb SCCL 175. Cases that were judged as negative had no
`showed apical staining with MoAb SCCL 175 (Fig. I). Scat-
`detectable immunostaining with MoAb SCCL 175 (Fig. 3).
`tered hypothalamic neurons also demonstrated cytoplasmic Table 2 summarizes the results of MoAb SCCL 175 immuno­
`immunoreactivity in one sample (Fig. 2).
`reactivity according to histological tumor type. Positive cases
`Tumor Samples. Cases were judged as positive when at least of SCCL demonstrated both surface and cytoplasmic immu-
`7320
`
`t�hnique, counterstained with hematoxylin; x 450.
`
`Fig. 3. Adenocarcinoma, cytology preparation, negative for SCCL 175. ABC
`
`nnwnln::irlP.rl frnm r.::inr.P.rrP.s ::i::ir.rirn1rn::ils nrn nn .h1lv 1Cl ?Cl1!i (c) 1�88 AmP.rir.::in Assnr.i::itinn fnr r.::inr.P.r RP.sP.::irr.h
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`MoAb SCCl. 175 AND SPEOFICITY FOR NEUROENDOCRINE LUNG CARCINOMA
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`Fig. 4. Cytological preparation of small cell neuroendocrine carcinoma with
`surface (single atTOW) and cytoplasmic (double IUTOW) SCCL 175 immunoreactiv­
`ity. ABC technique, counterstained with hematoxylin; x 450.
`
`strong and diffusely distributed throughout the tumor. Both
`surface and cytoplasmic immunoreactivity was detected on both
`cytology and tissue sections, showing that the antigen recog­
`nized ,,y this MoAb is distributed on both the cell surface and
`intracytoplasmically.
`All non-SCCL carcinomas thus far studied have been negative
`for binding with SCCL 175. Included in this group were carci­
`nomas that were his to logically classified as poorly differentiated
`in which a diagnosis of SCCL was initially considered in the
`differential diagnosis. The diagnostic dilemma posed by such
`cases has prompted the use of a variety of additional special
`techniques to aid in the classification of these tumors, including
`electron microscopy and immunohistochemistry (4-6). MoAb
`SCCL 175 provides a unique tool because of its specificity and
`sensitivity for SCCL and its ready applicability to routinely
`processed pathology specimens.
`Noteworthy is the absence of immunostaining of bronchial
`carcinoids and other neuroendocrine tumors, suggesting that
`MoAb SCCL 175 is not a general marker of neuroendocrine
`differentiation. Additional studies performed in this laboratory
`have demonstrated that this MoAb can distinguish SCCL from
`well-differentiated neuroendocrine carcinoma, a subtype of pul­
`monary neuroendocrine carcinoma having a morphology and
`clinical course intermediate between typical bronchial carcinoid
`and the highly aggressive SCCL (21, 22).
`Although SCCL 175 was reactive with a colonic carcinoma
`cell line DLD-1 (8), it does not appear to be reactive with the
`colonic carcinomas studied to date. Additionally, SCCL 175
`demonstrated only weak reactivity with one of three cases of
`choriocarcinoma despite its reactivity with the BeWo cell line
`(8). The studies performed on normal tissues indicate that the
`antigen recognized by MoAb SCCL 175 has a limited distri­
`bution in normal human tissues. The antigen does not appear
`to be expressed in normal or neoplastic adult neuroendocrine
`cells, suggesting that it is not a general marker of neuroendo­
`crine differentiation. The antigen recognized by MoAb SCCL
`175 may be expressed in a limited fashion in the CNS; however,
`given its limited distribution thus far in the CNS and its absence
`in normal neuroendocrine cells, it does not appear to recognize
`a common structural antigen in these systems like synaptophy­
`sin (23).
`SCCL 175 is variably reactive with cytotrophoblastic and
`syncytotrophoblastic cells of normal placenta. SCCL 175 re­
`acted only weakly with one choriocarcinoma; however, further
`studies are required to determine if this antibody has any utility
`in the characterization of germ cell tumors. Current work is
`under way to further assess the reactivity of SCCL 175 in
`nonpulmonary cancers.
`The antigen recognized by MoAb SCCL 175 appears to be
`preserved, although masked, in formalin-fixed, paraffin-em­
`bedded biopsy specimens obtained at fiberoptic bronchoscopy.
`Mild trypsin pretreatment can unmask immunoreactivity in at
`least some routinely processed cases of SCCL, although snap­
`frozen tissue sections ·and cytological preparations appear to
`provide more consistent results in this study. Additional studies
`are being performed on formalin-fixed, paraffin-embedded pul­
`monary cancers using trypsin pretreatment in order to deter­
`mine the pattern of immunostaining for routinely prepared
`DISCUSSION
`biopsy material.
`Based on these results, SCCL 175 provides an effective and
`This study demonstrates the utility of MoAb SCCL 175 as a
`selective and sensitive probe for SCCL. MoAb SCCL 175 objective tool in the diagnosis of SCCL and would serve as an
`excellent reagent in a panel of antibodies capable of phenotyp­
`immunoreactivity was identified in over 90% of the SCCL cases
`ing the spectrum of pulmonary cancers (9-18). In addition, its
`studied, and in most instances the intensity of staining was both
`7321
`
`noreactivity (Figs. 4 and 5). Of the material available for study,
`cytology preparations provided the best staining results, with
`tumor cells showing strong membrane immunoreactivity and
`less intense cytoplasmic immunoreactivity. In several SCCL
`preparations, scattered normal pulmonary reserve cells showed
`cytoplasmic immunoreactivity with SCCL 175; however, this
`staining was not seen in any other tumor samples or in normal
`lung tissue. Ten metastatic SCCL cases were positive with
`SCCL 175 including four hepatic, four lymph node, and two
`pleural fluid metastases. In two positive cases, both primary
`and metastatic sites were studied, and no difference in staining
`was observed. Two SCCL cases judged as negative were paraf­
`fin-embedded bronchial biopsy specimens, and the remaining
`two negative SCCL cases were bronchial washings. Prior to
`trypsin pretreatment, none of the paraffin-embedded SCCL
`cases demonstrated positive immunoreactivity with MoAb
`SCCL 175.
`
`Fig. 5. Paraffin-embedded bronchial biopsy specimen of small cell neuroen­
`docrine carcinoma with intense cytoplasmic SCCL 175 immunoreactivity. The
`intensity of the staining obscures the nuclear contours in this photomicrograph.
`
`ABC technique, counterstained with hematoxylin; x 450.
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`MoAb SCCI. 175 AND SPECIFICITY FOR NEUROENDOCRINE LUNG CARCINOMA
`
`specificity for SCCL suggests clinical utility in the treatment of
`this disease.
`
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`199, 1984.
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`175 distinguishes small .cell carcinoma from well-differentiated neuroendo­
`crine carcinomL Lab. Invest., 58: 63A, 1988.
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`
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`Cancer Research
`
`I The American
`
`The Journal of Cancer Research (1916-1930)
`Journal of Cancer (1931-1940)
`
`AA/""-_D American Association
`\... -n for Cancer Research
`
`A Novel Monoclonal Antibody, SCCL 175, with Specificity for
`Small Cell Neuroendocrine Carcinoma of the Lung
`Vincent A. Memoli, Andrea G. Jordan and Edward D. Ball
`Cancer Res 1988;48:7319-7322.
`
`Updated version
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