lines have altered large cell morphology,

shorter doubling times, higher cloning ef-

ficiencies in soft agarose, and very low

levels of L-dopa decarboxylase production

and bombesin-like inlmlnoreactivity. C-myc is

amplified and expressed in some small cell

lung cancer cell lines and all c-myc

amplified lines studied to date display the

variant phenotype. To investigate if c-myc

an~lification and expression is responsible

for the variant phenotype, a normal human c-

myc gene was transfected into a cloned clas-

sic small cell lung cancer cell line net

a~lified for or expressing detectable c-myc

messenger RNA (mRNA). Clones were isolated

with one to six copies of c-myc stably in-

tegrated into DNA that expressed c-myc mRNA.

In addition, one clone with an integrated

neo gene but a deleted c-myc gene was iso-

lated and in this case c-myc was not

expressed. C-myc expression in transfected

clones was associated with altered large

cell morphology, a shorter doubling time,

and increased cloning efficiency, but no

difference in L-dopa decarboxylase levels

and bombesin-like immunoreactivity. We con-

clude increased c-myc expression observed

here in transfected clones correlates with

some of the phenotypic properties distin-

guishing c-myc amplified variants form un-

amplified classic small cell lung cancer

lines.

Heterogeneity of Cell Surface Antigen Ex-

pression of Human Small Cell Lung Cancer

Detected by Monoclonal Antibodies.

Fargion, S., Carney, D., Mulshine, J. et al.

NCI-Navy Medical Oncology Branch, Division

of Cancer Treatment, National Cancer In-

stitute, Naval Hospital, Bethesda, MD 20814,

U.S.A. Cancer Res. 46: 2633-2638, 1986.

Using ~ohistochemistry, radiobind-

ing, and indirect imnmofluorescence assays,

seven distinct cell surface antigens,

detected by monoclonal antibodies, were

analyzed for the degree of homogeneity or

heterogeneity of antigen expression on a

panel of human small cell lung cancers. The

panel included 7 tumors taken directly from

patients, 21 established cell lines (9 of

which were derived from different metastatic

sites of 3 patients), and 33 clonal deriva-

tives of 3 lines. With all assays, con-

siderable heterogeneity of antigen expres-

sion between tumors from different patients

was observed. In both fresh tumors and in

cell lines, as well as in cell lines estab-

23

fished from different metastatic sites in an

individual patient, we observed intratumor

heterogeneity finding antigen positive and

negative cells and variation in antigenic

density, by im~mohistochemistry and in-

direct inlnunofluorescence assays. Antigenic

expression was not cell cycle dependent. In

addition, when cell lines or patient sables

expressing antigen positive and antigen

negative tumor cells were cloned,

heterogeneity of antigenic expression was

still present in the clonal lines. This sug-

gests that either the expression of the an-

tigen was not heritable and/or the ability

to regenerate antigenic heterogeneity is an

intrinsic property of the tumor cells. The

heterogeneity of antigen expression on lung

cancer cells has significant i~lications

for the use of these and other monoclonal

antibodies in the study and therapy of lung

cancer.

Antigens Associated with Human S~Lqmous Cell

Lung Carcinoma Defined by Murine Monoclonal

Antibodies.

Fernstein, P.D., Pekny, K.W., Reisfeld,

R.A., Walker, L.E. Department of Immunology,

Scripps Clinic and Research Foundation, La

Jolla, CA 92037, U.S.A. Cancer Res. 46:

2970-2977, 1986.

A panel of 12 monoclonal antibodies

that preferentially react with ~,,an

squamous lung carcinoma cells has been

produced. All are reactive with fresh frozen

sections of sq, mmous cell lung carcinoma

tissues in immnlnoperoxidase assays and are

unreactive with lymphoblastoid cells, red

blood cells, and fibroblasts in enzyme-

linked immunosorbent assay. At least eight

of these antibodies interact with cell sur-

face components. These reagents can be sub-

divided into four groups based upon their

reactivities. Groups 1 to 3 are unreactive

with normal liver, lung, kidney, colon,

spleen, and pancreas in inm~unoperoxidase as-

says. Group 1 antibodies (PFI/A, PFI/B,

PFI/C, PFI/d, and PFI/E) are all of IgG3

subclass and immunoprecipitate nonsulfated

glycoprotein components with molecular

weights of 80,000 and 180,000 and a non-

glycosylated polypeptide with a molecular

weight of 38,000. Group 1 antibodies are

also reactive with some lung adenocarcinomas

and, with the exception of PFI/E, stain cer-

tain differentiated strata within normal

adult plantar and fetal epidermis. Group 2

antibodies (PF2/A and PF2/B) react also with

24

breast, gastric, and colonic adenocarcinomas

and some tumors of neuroectodermal origin.

Group 2 antibodies, which are both of IgG3

subclass imnxnoprecipitate a nonglycosylated

MSr) 24,000 polypeptide. Group 3 antibodies

(PF3/A, an IgGl; PF3/B, and IgGM; and PF3/C,

an IgG2a) react additionally with certain

other tumors, as well as with normal adult

and fetal epidermis. Group 4 antibodies

(PF4/A, an IgG2a; and PF4/B, an IgGl) are

less specific than those of the preceding

groups, as they react with some normal

tissues, including pancreatic islets and

pneumocytes, as well as with a variety of

adenocarcinomas and tumors of neuroectoder-

mal origin. PF4/A and PF4/B im-

munoprecipitate (M(r) I00,000 and 95,000

glycoproteins, respectively.

Cytotoxic Murine Monoclonal Antibody LAM8

With Specificity for Human Small Cell Car-

cinoma of the Lung.

Stahel, R.A., O'Hara, C.J., Mabry, M. et al.

Division of Oncology, Department of

Medicine, University Hospital of Zurich, CH-

8091 Zurich, Switzerland. Cancer Res. 46:

2077-2084, 1986.

The reactivity of the murine im-

munoglobulin monoclonal antibody LAM8

directed against a membrane antigen of ~m~n

small cell carcinoma (SCC) of the lung was

investigated on human cell lines and

tissues. Indirect immmofluorescence stain-

ing, radioimmunoassays, and cytotoxicity as-

says showed LAM8 antibody to selectively

react with SCC but not with non-SCC lung

cancer cell lines and extrapulmonary tumor

cell lines. Unlike other SCC antibodies, in-

cluding those we have previously described,

highly preferential reactivity with SCC

tissues was also demonstrated by im-

munoperoxidase staining of deparaffinized

formalin-fixed tissue sections. Membrane and

cytoplasmic staining was seen in of 9 of 12

SCC tissues. No significant staining was

seen in non-SCC lung cancer and a wide range

of other tumors, including mesothelioma and

bronchial carcinoids. Significant LAM8 reac-

tivity was also absent in normal tissues of

all major organs. Few tumors and epithelial

tissues, including bronchial epithelium had

rare LAM8 positive cells which were always

less than 2% of the entire cell population.

In vitro treatment with antibody and ~,nnn

complement was highly cytotoxic to SCC

cells, but had no effect on bone marrow

progenitor cells. Immunoblotting of membrane

extracts separated on sodium dodecyl

sulfate-polyacrylamide gels showed the LAM8

antigen to have a band of an approximate

molecular weight of 135,000 and a cluster of

bands with approximate molecular weights of

90,000. This reactivity was lost after in-

cubation of the extracts with periodate.

LAM8 antibody shows a highly preferential

reactivity with SCC cell lines and formalin-

fixed paraffin-embedded SCC tissues and is

selectively cytotoxic to cells expressing

LAM8 antigen.

Anti-Neurofilament Antibodies in the Sera of

Patients with Small Cell Carcinoma of the

Lung and With Visual Paraneop lastic

Syndrome.

Kornguth, S.E., Kalinke, T., Grunwald, G.B.

et al. Department of Neurology, University

of Wisconsin, Madison, WI, U.S.A. Cancer

Res. 46: 2588-2595, 1986.

The sera of patients with small cell

caricnoma of the lung (SCCL) and an as-

sociated visual paraneoplastic syndrome

(VPNS) have higher titer ~oglobulins

that react with retinal ganglion cells and

with cloned lines of the SCCL. The im-

munoglobulins in the sera of two patients

with SCCL and VPNS reacted with at least one

common antigen shared by neural cells and

cloned lines of the SCCL. The molecular

weights of the predominant neural and tumor

antigens were 205,000, 145,000, 65,000, and

20,000-24,000 as determined by Western

blots. Three of the antigens from neural

tissue copurify and comigrate

electrophoretically with neurofilament

proteins. Polyclonal antibodies prepared

against authentic neurofilament proteins

react with antigens having molecular weights

identical to those of proteins t_hat react

with immunoglobulins form the SCCL-VPNS

patients. Polyclonal antibodies that were

prepared against isolated retinal ganglion

cells and that were shown previously to

cause the inmn/noablation of the ganglion

cells in vivo reacted most intensely with

the M(r) 205,000 antigen and weakly with the

M(r) 145,000 and M(r) 70,000 antigens.

Treatment of the Western blots with alkaline

phosphatase from Escherichia coli did not

affect the immunoreactivity between the im-

munoglobulins and the purified neurofilament

proteins. It is proposed t_hat the im-

munoglobulins in the sera of patients with

SCCL-VPNS may be involved etiologically in

the development of the VPNS.