J C E M O N L I N E

Hot Topics in Translational Endocrinology—Endocrine Care

Temozolomide Treatment for Aggressive Pituitary

Tumors: Correlation of Clinical Outcome with
O6-Methylguanine Methyltransferase (MGMT)
Promoter Methylation and Expression

Zachary M. Bush, Janina A. Longtine, Tracy Cunningham, David Schiff,

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John A. Jane, Jr., Mary Lee Vance, Michael O. Thorner, Edward R. Laws, Jr.,
and M. Beatriz S. Lopes
Division of Endocrinology and Metabolism (Z.M.B., M.L.V., M.O.T.), University of Virginia, Charlottesville,
Virginia 22908; Department of Pathology (J.A.L.), Brigham and Women’s Hospital, Boston,
Massachusetts 02115; School of Medicine (T.C.), Division of Neurooncology (D.S.), and Department of
Neurosurgery (J.A.J.), University of Virginia, Charlottesville, Virginia 22908; Department of Neurosurgery
(E.R.L.), Brigham and Women’s Hospital, Boston, Massachusetts 02115; and Department of Pathology
(M.B.S.L.), Division of Neuropathology, University of Virginia, Charlottesville, Virginia 22908

Context: The typically indolent behavior of pituitary tumors is juxtaposed with high rates of tumor
cell invasion into adjacent dural structures, and occasional aggressive behavior. Although clinically
significant invasion and malignant transformation remain uncommon, there are limited treatment
options available for the management of these aggressive tumors. Recently, case reports have
described efficacy of temozolomide for the treatment of aggressive pituitary tumors.

Design: Seven patients with aggressive pituitary tumors have been treated with temozolomide. We
compared O6-methylguanine methyltransferase (MGMT) promoter methylation and MGMT ex-
pression in 14 surgical specimens from these seven patients and correlated these molecular features
with the clinical response to temozolomide.

Results: Significant tumor regression was seen in two patients (29%), a 20% reduction in tumor
volume with subsequent stable tumor size was noted in one patient, arrest of tumor growth
occurred in three patients, and progressive metastatic disease developed during treatment in one
patient. The DNA promoter site for MGMT was unmethylated in all 14 adequate specimens, and
variable MGMT expression was seen in all 14 cases. There was no correlation between MGMT
expression and clinical outcomes.

Conclusions: We conclude that medical therapy with temozolomide can be helpful in the man-
agement of life-threatening pituitary tumors that have failed to respond to conventional treat-
ments. The optimal duration of treatment in patients with stabilization or reduction of tumor size
has not been established, and long-term follow up studies are needed. (J Clin Endocrinol Metab 95:
E280 –E290, 2010)

he natural history of pituitary adenomas varies widely.
T The typically indolent growth rate of these tumors is
juxtaposed with high rates of tumor cell invasion into ad-
jacent dural structures (1). Clinically significant invasion
and malignant transformation remain uncommon, but
morbidity and mortality are high when aggressive tumor
behavior occurs (2). With the exception of dopamine ag-
onist therapy for prolactinomas, surgery has long been the
first line treatment for pituitary tumors. Options for med-
ical treatment are limited, and none of the current thera-

ISSN Print 0021-972X ISSN Online 1945-7197
Printed in U.S.A.
Copyright © 2010 by The Endocrine Society
doi: 10.1210/jc.2010-0441 Received February 23, 2010. Accepted July 1, 2010.
First Published Online July 28, 2010
Abbreviations: CN, Cranial nerve; CSF, cerebrospinal fluid; GKR, GammaKnife stereotactic
radiosurgery; IHC, immunohistochemistry; MGMT, O6-methylguanine methyltransferase;
MRI, magnetic resonance imaging; MSP, methylation-specific PCR.

E280 jcem.endojournals.org J Clin Endocrinol Metab, November 2010, 95(11):E280 –E290

J Clin Endocrinol Metab, November 2010, 95(11):E280 –E290
pies is tumoricidal (3– 6). When invasive tumors occur,
repeat surgery and radiation therapy are second-line ther-
apies that have variable efficacy depending on the char-
acteristics of the tumor. Extrasellar tumor extension,
proximity to cranial nerves and critical blood vessels, and
the delayed therapeutic effects of radiotherapy can all limit
the efficacy of these treatment modalities (7, 8). These
therapeutic shortfalls are most critical in the management
of highly invasive adenomas and pituitary carcinomas.
Therapeutic efforts have been made with conventional
chemotherapeutic agents that cross the blood-brain bar-
rier to treat invasive and malignant pituitary tumors, but
efficacy has been poor, and partial responses are generally
short-lived (9 –13). Temozolomide is an oral alkylating
agent that readily crosses the blood-brain barrier and has
been shown to be efficacious in malignant gliomas. Several
recent case reports have reported efficacy of temozolo-
mide for aggressive pituitary tumors (14 –18). The mech-
anism by which temozolomide acts on pituitary tumors
has not been established, but in general, the alkylating
agents disrupt gene transcription by inducing DNA dam-
age by attaching a methyl group to the guanine base. Rap-
idly dividing tumor cells often lack competent DNA repair
mechanisms, and thus are more prone to cytotoxic DNA
disruption (19, 20). Epigenetic inactivation of the gene for
the DNA repair enzyme O6-methylguanine methyltrans-
ferase (MGMT) via promoter hypermethylation has been
show to predict therapeutic response to temozolomide in
glioblastomas (21, 22), and retrospective analyses of pi-
tuitary tumors have reported MGMT methylation and ex-
pression by immunohistochemistry (17, 23).
We report a series of seven patients with aggressive
pituitary tumors that have been treated with temozolo-
mide. We compare MGMT promoter methylation status,
as established by methylation-specific PCR (MSP), and
nuclear MGMT expression by immunohistochemistry
(IHC) and correlate these molecular features with the clin-
ical response.
Patients and Methods
From the records of the University of Virginia Pituitary Clinic, we
identified all of the patients with invasive pituitary tumors who
received temozolomide as adjuvant medical therapy. Medical
records were reviewed to establish duration of treatment, tumor
response, tolerability of temozolomide treatment, and adverse
events. Tumor response was established by review of serial mag-
netic resonance imaging (MRI) studies, and in the case of hor-
monally active tumors, the hormone response to treatment. The
chemotherapeutic dose, the calendar regimen employed, and the
number of cycles administered were established by review of
medical records. Tumor volume was calculated as elliptical vol-
ume—the product of the largest diameter in three planes from
coronal and sagittal postgadolinium T1 MRI images, multiplied
jcem.endojournals.org E281
by 0.5. The study was approved by the University of Virginia
Institutional Review Board.
The presence of tumor within selected specimen blocks and
confirmation of the cell lineage of each tumor were made by
review of the standard histological, IHC, and ultrastructural
studies. MGMT methylation was established by the MSP. The
intrapatient stability of MGMT promoter methylation was de-
termined in surgical specimens obtained before and after radia-
tion therapy in patients who underwent multiple operations dur-
ing the course of multimodality treatment; this includes pre- and
postradiation specimens in case 3. IHC was used to determine
nuclear expression of the MGMT DNA repair enzyme.
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Methylation-specific PCR
Genomic DNA was isolated from three to five 10-␮m paraffin
tissue sections containing at least 70% tumor (QIAamp DNA
Mini Kit; QIAGEN, Valencia, CA). DNA methylation patterns
in the CpG island of the MGMT gene (GenBank accession num-
ber AL355531, nucleotides 46931– 47011) was determined by
chemical (bisulfite) modification of unmethylated, but not meth-
ylated, cytosine to uracil (CpGenome Fast DNA Modification
Kit; from Chemicon, now part of Millipore, Billerica, MA). The
bisulfite-treated DNA was precipitated, rehydrated, and ampli-
fied by MSP using primers specific for either the methylated or
the modified unmethylated DNA (18). PCR amplification with
Taq-Gold (Applied Biosystems, Foster City, CA) was performed,
and PCR products were analyzed in duplicate parallel runs by
capillary gel electrophoresis (ABI 3130xl; Applied Biosystems)
with an expected product of 93 bp for methylated DNA and 80
bp for unmethylated DNA. Bisulfite-treated CPGenome Univer-
sal Unmethylated DNA (Millipore) and SssI methyltransferase
(New England Biolabs, Ipswich, MA)-treated human DNA were
used as negative and positive methylation controls, respectively.
No template controls were performed. The sensitivity of the as-
say based on DNA dilution studies is at least 1:1000. A dichot-
omous result of methylated or unmethylated MGMT promoter
was reported for each case.
Immunohistochemistry
IHC for MGMT was performed in formalin-fixed, paraffin-
embedded tissue using the avidin-biotin-peroxidase technique
(24). Antigen retrieval by boiling in a citrate solution (Target
Retrieval Solution, pH 6.1; Dako, Carpinteria, CA) was per-
formed using a pressure cooker (25). Tissue sections were incu-
bated with anti-MGMT monoclonal antibody (clone MT3.1;
1:50 dilution; Chemicon) using the automated staining sys-
tem (DakoCytomation Autostainer Plus; Dako, Glostrup, Den-
mark). Positive and negative controls were run concomitantly in
all analyses. Tissue sections from human glioblastomas in which
MGMT methylation status was known were used to establish
positive IHC control. In addition, normal tissues adjacent to the
adenoma specimens including blood vessels were used as positive
internal controls.
IHC scoring and statistical analysis
IHC specimens were analyzed independently by three review-
ers using a semiquantitative method to determine both the pro-
portion of MGMT-positive cells and the intensity of the immu-
noreactivity. Reviewers were blinded to clinical outcome.
Positivity was quantified as negative or 1 for tumors with less
than 10% of cells positive for MGMT; 2 for tumors with 10 –
 E282
Bush et al.

TABLE 1. Temozolomide treatment cohort

Response
Case Tumor Radiation MGMT
no. Surgery Cell lineage features therapy score Temozolomide Biochemical Imaging Side effects Outcome
1 TSR: 10/08 Null-cell Ki-67 ⬍ 3%; None 3 10 of 12 cycles N/A 20% reduction in Fatigue, headaches Clinically stable
prominent completed tumor volume;
nucleoli subsequent
stable tumor
volume
2 TSR: 07/04; Corticotroph-cell Ki-67 ⫽ 18%; GKRT 10/18/07; 1 11 completed ACTH: 221 pg/ml ⬎80% tumor Sensory neural Clinically improved
craniectomies: with clinical p53 ⫹; 5/02/08 decreased to reduction hearing loss, during
07/07; 06/09 Cushing’s large, 18 ng/ml; fatigue, treatment
disease multinucleated adrenally headaches
cells insufficient at
cycle 8/11
3 TSR: 01/92; 06/ FSH/TSH/␣-SU Ki-67 ⬍ 3%; Conventional 2 13 completed, N/A Stable tumor size Fatigue and Clinically stable
03; 12/06 positive; no atypical fractionated still on tx; 12 headaches,
clinically features radiation cycles of 21/7, lessened with
Temozolomide for Aggressive Pituitary Tumors

nonfunctional 1992 now on 18/10 dose reduction

4 TSR: 07/94; 02/00 Null-cell Ki-67 ⫽ 6%; None 3 10/12 completed N/A Stable tumor size Fatigue, dry mouth Clinically stable
mitotic
figures
present
5 TSR: 08/06; 12/06 Prolactinoma Ki-67 ⬎ 20%; GKRT 02/2007 1 11 completed Prolactin: 5702 ⬎80% tumor Well tolerated Clinically improved
prominent still on tx 21/7 ng/ml reduction during
nucleoli decreased to treatment
121 ng/ml
6 TSR: 06/94; 10/ Pituitary Ki-67 ⬎ 20%; Conventional 3 2 cycles, Apr/ N/A Stable tumor size Fatigue, severe Deceased; cause of
01; 04/03 carcinoma; brisk fractionated May 2005 during 2 dizziness death unrelated
null-cell mitotic radiation temodar/ months of to pituitary
activity 1994; GKRT thalidomide treatment carcinoma
02/05
7 TSR: 01/05; 03/05 Pituitary Ki-67 ⬎ 20%; IMRT 04/05 1 7 cycles N/A Progressive Fatigue, headaches Deceased; death
carcinoma; rare mitotic metastatic attributed to
null-cell figures disease on complications of
treatment pituitary
carcinoma

TSR, Transsphenoidal surgery; GKRT, GammaKnife radiation treatment; IMRT, intensity modulated radiation therapy; N/A, not available; tx, treatment; ␣-SU, ␣-subunit of glycoproteins. Median
MGMT IHC score reported for each case (1 ⫽ ⬍10% MGMT positive cells; 2 ⫽ 10%–50% MGMT positive cells; 3 ⫽ ⬎50% MGMT positive cells).
J Clin Endocrinol Metab, November 2010, 95(11):E280 –E290

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J Clin Endocrinol Metab, November 2010, 95(11):E280 –E290
FIG. 1. Serial T1, Postcontrast MRI images before and after temozolomide treatment. A and
B, Case 2, an invasive corticotroph tumor pretreatment (A) and after 11 cycles (B) (arrows
highlight tumor extension). C and D, Case 5, an invasive prolactinoma pretreatment (C) and
after 8 cycles (D) (arrows highlight substantial reduction of tumor).
50% positive cells; or 3 for tumors with more than 50% positive
cells. Intensity was scored on a scale where 1 ⫽ faint, 2 ⫽ mod-
erate, and 3 ⫽ distinct immunoreactivity equivalent to the in-
tensity seen in internal controls. Marginals were not known to
the reviewers a priori; thus, interoperator variability was deter-
mined using Randolph’s multirater variation on Fleiss’ free-mar-
ginal ␬ equation (26 –29). The free-marginal ␬ statistic for the
semiquantitative IHC analysis was determined for interoperator
agreement for both the whole integer score (IHC positivity score
0 vs. 1 vs. 2, etc.) and the dichotomous outcome measure of
positive (scores of 1, 2, or 3) vs. negative.
Results
We identified seven cases of invasive pituitary tumors that
had been treated with temozolomide between 2005 and
2009. All seven patients were initially treated with surgery.
Six of the seven patients had repeat surgery before receiving
adjuvant treatment. A total of 19 operations were performed
on the seven patients during multimodality management. We
eliminated redundant surgical specimens obtained in rapid
sequence within the same treatment phase (i.e. before or after
radiation). Fifteen specimens from the seven patients were
analyzed by IHC and MSP. One specimen was too necrotic
to yield results in our molecular studies.
jcem.endojournals.org E283
Five of the seven patients were
treated with conventional radiation or
GammaKnife stereotactic radiosurgery
(GKR) before temozolomide treatment.
The interval between the last radia-
tion dose and temozolomide treatment
ranged from 5 months to 17 yr (Table
1). The duration of temozolomide treat-
ment ranged from 2 to 18 months. The
initial treatment regimen for all seven pa-
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tients was a 28-d cycle of temozolomide
75 mg/m2䡠d for 21 d followed by 7 d off
therapy. One patient had a dose adjust-
ment after 12 standard cycles to 75 mg/
m2䡠d for 18 d, 10 d off therapy in an effort
to reduce side effects of fatigue and head-
ache. One patient with a null cell tumor
received octreotide concomitant with
seven cycles of temozolomide; progres-
sive metastatic tumor burden in this pa-
tient resulted in progressive clinical de-
cline and death. One patient with an
undifferentiated pituitary carcinoma was
treated with thalidomide concomitant
with temozolomide; the latter drug was
stopped after two cycles because of debil-
itating dizziness and headaches. There
was no evidence of tumor growth during
2 months of treatment.
Clinical response
Tumor regression of more than 80% tumor volume
resulted in rapid clinical improvement in two patients
(29%; Fig. 1), a 20% reduction in tumor size with subse-
quent stable tumor volume was noted in one patient, arrest
of tumor growth was seen in three patients (Fig. 2), and
progressive metastatic disease while on temozolomide oc-
curred in one patient with a null cell carcinoma that re-
sulted in death (Table 1).
In two patients, the aggressive pituitary tumor— one
prolactinoma (case 5, Table 1) and one corticotroph
tumor (case 2, Table 1)— had caused compression of
cranial nerves and the brain stem. Both tumors had
more than 80% reduction in tumor volume with chronic
temozolomide therapy, and clinical improvement was
evident within the first four cycles; serial MRI studies
demonstrated marked reduction in tumor size, but after
11 cycles of treatment some residual tumor remained in
both cases (Fig. 1). Biochemically, the prolactin levels in
case 5 decreased from 5702 ng/ml to 121 ng/ml over 11
cycles, but have not fully normalized. Symptoms of cra-
nial nerve (CN) dysfunction (partial 6th CN palsy and
E284 Bush et al. Temozolomide for Aggressive Pituitary Tumors
FIG. 2. Serial MRI images from case 4. A–E, Progressive tumor growth
in a patient with no history of radiation treatment. E, Immediately
before temozolomide; F, arrest of tumor growth during eight cycles of
temozolomide.
a painful 5th CN neuropathy) improved during the first
6 wk of treatment and resolved within four cycles of
temozolomide. This patient received GKR treatment 26
months before temozolomide, with evidence of rapidly
progressive disease up to the point of initiating temo-
zolomide treatment. We attribute her treatment re-
sponse to temozolomide. Case 2 had an ACTH-secret-
ing macroadenoma with clinical Cushing’s disease. The
marked reduction in tumor volume led to a cerebrospi-
nal fluid (CSF) leak during cycle 11 of temozolomide (a
phenomenon seen when prolactinomas rapidly respond
to dopamine agonist therapy). The ACTH level pre-
treatment was 221 pg/ml, which steadily declined dur-
ing 13 cycles, ultimately normalizing at 18 pg/ml, with
clinical adrenal insufficiency developing near the end of
the treatment period. Tissue specimens obtained during
transsphenoidal surgery to repair the CSF leak revealed
histological evidence of invasive corticotroph tumor
cells in dural structures; nonetheless, the patient has
been off treatment for 6 months without signs or symp-
toms of tumor regrowth or recurrence of Cushing’s dis-
ease, and there has been no MRI evidence of growth in
the residual tumor. Importantly, this patient had a sec-
ond GKR treatment 5 months before starting temozo-
lomide, so it is possible that the treatment response in
this patient is in part the result of radiosurgery (thera-
peutic response in Cushing’s patients has been shown to
be delayed 3– 48 months after GKR) (30).
J Clin Endocrinol Metab, November 2010, 95(11):E280 –E290
Side effects and adverse events
Side effects were common in our cohort. Fatigue was
experienced by six patients and prompted dose reduction
after 12 cycles in one patient with a stable tumor burden
(case 3, Table 1). Case 2 experienced fatigue throughout
treatment, and once biochemical remission was achieved
temozolomide was discontinued to relieve the persistent
side effect, given the unknown benefit of continued ther-
apy. Headache occurred in four patients, but was not
dose-limiting. Severe dizziness occurred in one patient
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(case 6) who was on concomitant thalidomide; the temo-
zolomide was stopped after two cycles, and symptoms
were relieved (thalidomide is also know to cause dizziness
and may have contributed to the severity of this side ef-
fect). One adverse event occurred during the first four
cycles of temozolomide treatment in case 2; the patient
developed progressive bilateral sensorineural hearing loss,
which necessitated hearing aids that were unable to cor-
rect the sensory deficit. We have not seen hearing loss in
the hundreds of patients receiving temozolomide mono-
therapy for malignant gliomas at our center, but we cannot
exclude temozolomide as the causative agent in this case.
Despite the adverse event, the patient and treating physi-
cians elected to continue treatment because of clinical and
radiological evidence of tumor response. There has been
no improvement in the patient’s hearing loss over the 6
months since temozolomide was stopped.
MSP and IHC
The MGMT promoter was unmethylated in all 14 ad-
equate specimens. The unmethylated state was stable over
time in case 3 in which both pre- and postradiation sur-
gical specimens were available (Table 2). The IHC analysis
demonstrated variable immunoreactivity for MGMT in a
nuclear pattern in all 14 adequate specimens (Table 2 and
Fig. 3). Four specimens were scored as negative for
MGMT immunoreactivity by at least one reviewer, but
there was not a consensus for negative MGMT immuno-
reactivity in any of the 14 specimens (Table 2). Median
MGMT scores for each case are reported in Table 1. Three
cases had less than 10% of cells immunoreactive for
MGMT; one case had 10 –50% MGMT immunoreactive
cells; three cases had MGMT immunoreactivity in more
than 50% of cells. Therefore, methylation status of the
MGMT gene promoter was not predictive of relative
MGMT expression by IHC in any of the cases. Marked
heterogeneity in MGMT expression was noted within
many of the tumors, some with expression predominantly
in single regions of the tumor, some with predominant
expression in the periphery of the tumor. Likewise, there
was significant heterogeneity of the intensity of MGMT
expression as demonstrated in Table 2 and Fig. 3.
TABLE 2. Fifteen surgical specimens from seven patients with invasive or metastatic pituitary tumors
MGMT IHC
PCR: MGMT promoter Reviewer 1 Reviewer 2 Reviewer 3
Patient MGMT MGMT MGMT
no. Specimen Methylated Unmethylated positive Intensity Total positive Intensity Total positive Intensity Total
1 A X 3 1 4 3 1 4 3 2 5
2 A X 1 2 3 Negative 0 0 1 1 2
J Clin Endocrinol Metab, November 2010, 95(11):E280 –E290

B X 1 1 2 2 3 5 Negative 0 0
3 A X 2 2 4 2 2 4 3 2 5
Ba X 2 2 4 2 1 3 1 1 2
Ca X 2 1 3 1 3 4 2 1 3
4 A X 3 2 5 3 1 4 3 2 5
B X 2 1 3 2 3 5 3 2 5
5 A X 1 1 2 Negative 0 0 1 1 2
B X 1 1 2 2 2 4 1 1 2
6 Aa X 3 1 4 3 1 4 3 2 5
Ba N/A; necrotic N/A; necrotic N/A N/A Necrotic N/A N/A Necrotic N/A N/A Necrotic
specimen specimen
Ca X 3 2 5 3 1 4 3 2 5
7 A X 1 1 2 1 2 3 Negative 0 0
B X 1 1 2 1 2 3 1 1 2
Methylation status of the MGMT promoter by MSP and MGMT immunoreactivity are shown. MGMT positive: 1 ⫽ ⬍10% of cells positive in tumor; 2 ⫽ 10%–50%; 3 ⫽ ⬎ 50%. MGMT intensity: 1,
faint; 2, moderate; 3, equivalent to positive controls. N/A, Not available.
a
Postradiation surgical specimen.
jcem.endojournals.org
E285

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E286 Bush et al. Temozolomide for Aggressive Pituitary Tumors J Clin Endocrinol Metab, November 2010, 95(11):E280 –E290

this tumor and thus predict temozo-

lomide responsiveness; however, the
patient developed spinal metastasis
and increasing parasellar tumor bur-
den during 7 months of temozolomide
treatment, which was discontinued 2
months before her death to alleviate the
side effects of severe fatigue and head-
aches. The two tumors with the most
significant response to temozolomide

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therapy were cases 2 and 5 described
in Clinical response, both of which
were locally invasive with encroach-
ment on cranial nerves, optic appara-
tus, and midbrain structures. Like
case 7, these tumors were MGMT
positive in less than 10% of cells.
Thus, clinical response was not reli-
ably predicted in our cohort by either
MGMT promoter methylation or
MGMT IHC immunoreactivity.

Discussion
To our knowledge, this is the largest
published cohort of patients treated
with temozolomide for aggressive pitu-
itary tumors (invasive adenomas and
carcinomas). The lack of clinical guide-
FIG. 3. IHC for MGMT. A and B, Diffuse pattern of stain (score 2); C, strong stain (score 3); lines for chemotherapy management of
D, example of heterogeneous stain in a single focus; E, internal positive control endothelial
cells; F, internal positive control sinus mucosa.
life-threatening pituitary tumors has
led to significant heterogeneity in the
clinical use of temozolomide for these
IHC statistical analysis rare tumors. The single-center aspect of this cohort has
The free-marginal ␬ statistic for the semiquantitative allowed for some uniformity in regard to the multimodal-
IHC analysis of MGMT positivity was 0.40, which sug- ity treatment algorithm and temozolomide dosing. None-
gests poor to moderate agreement by three reviewers on
theless, our data underscore the limitations of our current
the proportion of cells with MGMT immunoreactivity
understanding of aggressive pituitary tumors and the un-
within any given specimen (␬ ⬎ 0.70 indicates good in-
derlying molecular features that determine the heteroge-
teroperator reliability). With a dichotomous outcome
neity in tumor behavior and response to multimodality
measure of positive (scores of 1, 2, or 3) vs. negative, the
therapy. We believe that our data raise more questions
free-marginal ␬ score is 0.62, suggesting moderate inter-
than are answered, and we caution against any conclu-
operator agreement.
sions regarding what type of aggressive pituitary tumors
Correlation of clinical outcomes and molecular may or may not respond to temozolomide based on this
studies limited data set. Previously published case reports dem-
The MGMT promoter regions in the two postradiation onstrate at least a partial response to temozolomide in
surgical specimens from the most aggressive tumor (case 7, some aggressive tumors arising from all anterior pituitary
Table 1) in our cohort remained unmethylated, but IHC cell types including functioning and nonfunctioning pitu-
immunoreactivity for MGMT was less than 10% of cells itary adenomas and pituitary carcinomas.
in both specimens. The lack of MGMT expression would Most patients receiving treatment for aggressive pitu-
be expected to predict impaired DNA repair capacity in itary tumors in our center undergo at least two operations;
J Clin Endocrinol Metab, November 2010, 95(11):E280 –E290 jcem.endojournals.org E287

the second procedure is routinely undertaken to decom-
press compromised neural structures, reduce symptoms,
or create adequate space from the optic chiasm to allow
for focused radiation therapy. Radiation therapy with
GammaKnife is routinely performed for treating residual
tumor seen either intraoperatively or by MRI after a sec-
ond surgical procedure. In those cases in which there is
tumor adjacent to optic nerves or other barriers to GKR
treatment, conventional fractioned radiation therapy is
recommended. In cases 1 and 4, the patients refused ra-
contrast to other published findings (31, 32), our data
suggest that MGMT promoter methylation and MGMT
expression by IHC are not clinically useful in predicting
tumor response to temozolomide therapy. The intraop-
erator heterogeneity in MGMT IHC scores in our series is
in line with recent large studies that have evaluated
MGMT expression in gliomas; the intraoperator variabil-
ity seen across the various scoring methods used in these
studies reflects the significant intratumor variability of im-
munoreactivity patterns (multiple regions within a single

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diation. Temozolomide is rarely used to treat pituitary
tumors at our center and is only used as last-line treatment
for life-threatening pituitary tumors that have been refrac-
tory to standard treatment modalities. We use the 21 d on,
7 d off temozolomide regimen—a “dose-dense” sched-
ule—that delivers a higher dose of temozolomide per unit
time (75 mg delivered 21/28 d vs. 150 or 200 mg delivered
5/28 d). There is a theoretical advantage to the dose-dense
schedule in that it may deplete tumor cells of their MGMT
and thereby circumvent an important mechanism of re-
sistance in tumors that lack promoter methylation. This
regimen is associated with a greater risk of lymphopenia
than standard 5/28-d regimens. However, the association
with higher risk of other side effects or secondary malig-
nancies is unclear.
Our clinical results suggest that temozolomide induces
clinically significant tumor volume regression in a minor-
ity of invasive pituitary tumors [two of seven patients
(29%)], but stabilization of tumor growth occurred in
four additional cases [four of seven patients (57%)]. In
tumor can vastly differ in regard to the frequency and the
relative intensity of the immunoreactivity) (33–35). Fur-
thermore, our MSP data suggest that MGMT promoter
methylation is rare among invasive pituitary tumors and
does not explain the wide variability of IHC expression
seen in our cohort of tumors.
Clonality studies employing X-chromosome inactiva-
tion analyses have demonstrated that the majority of spo-
radic human pituitary adenomas are monoclonal (36).
This implies that these tumors arise from de novo somatic
genetic changes in a single pituitary cell. Our DNA meth-
ylation data suggest that the heterogeneity of MGMT ex-
pression within these monoclonal tumor cell populations
may result not from epigenetic modification of the MGMT
promoter but instead from a unique transcriptional mi-
croenvironment dictated by nuclear receptors and their
corepressors and coactivators or at the level of mRNA
translation.
The alkylating action of temozolomide can cause epi-
genetic modification of DNA by methylation of gene pro-

FIG. 4. Pre- and posttemozolomide treatment specimens from case 2. A and B, Surgical specimen before GammaKnife radiation and
temozolomide treatment showing extensive cellular atypia (A) and high Ki-67 labeling index (18%) (B). C–F, Surgical specimen after treatments
and collected during CSF leak correction showed focal islands of tumor cells entrapped on fibrous connective tissue with intense inflammatory
reaction (C). Extensive cellular pleomorphism was present (D), but adenoma cells were still immunoreactive for ACTH (E). Ki-67 labeling was
practically absent on tumor cells, but present in inflammatory cells seen on the specimen (F).
E288 Bush et al. Temozolomide for Aggressive Pituitary Tumors
moter sites, which disrupts protein synthesis. In vitro stud-
ies suggest that temozolomide arrests tumor growth
through cell cycle arrest rather than direct cytotoxic mech-
anisms. As such, complete remission is not an expected
outcome of temozolomide treatment; instead, growth ar-
rest, diminished tumor volume, and biochemical control
are the goals of temozolomide treatment. Tumor cells un-
dergoing cell replication are most vulnerable to alkylating
agents. This was demonstrated in the recent publication
from Kovacs et al. (17) in which Ki-67 staining of post-
temozolomide treatment specimen demonstrated a greater
proportion of cells in G0 (fewer Ki-67-positive cells) than
the pretreatment specimen without any evidence of apo-
ptosis or necrotic cell death. Our own pre- and posttemo-
zolomide surgical specimens from case 2 confirm these
findings, with Ki-67 labeling index in the pretreatment
specimen being very high at 18% and subsequently unde-
tectable in the posttreatment specimen (Fig. 4). A popu-
lation of apparently viable corticotroph tumor cells is ev-
ident in both pre- and posttemozolomide specimens.
Radiation therapy is known to exert some antitumor
effects via DNA methylation (37, 38); however, the
MGMT promoter in our five postradiation specimens re-
mained unmethylated (prior conventional radiation in
two specimens from case 3, and prior conventional and
GKR treatment in three specimens from case 6, Table 2),
and MGMT IHC does not suggest significant impact of
radiation on MGMT expression. This suggests that pre-
temozolomide radiation treatment may not have the same
therapeutic synergy with temozolomide that has been ob-
served in glioblastomas (22, 39, 40). A recent publication
by Su et al. (41) that evaluated MGMT promoter meth-
ylation in sputum samples of uranium miners showed in-
creasing promoter methylation with cumulative low-dose
radiation exposure. Multicenter investigation will be nec-
essary to establish the treatment effect of the combined
effects of radiation and temozolomide. Evaluation of dif-
ferential tumor response depending on interval between
radiation and temozolomide along with the study of the
molecular effects of single-dose radiation treatment vs.
chronic or intermittent low-dose radiation will be neces-
sary to establish definitive treatment guidelines.
We conclude that adjuvant medical therapy with temo-
zolomide may be considered for the management of life-
threatening invasive pituitary tumors that have failed to
respond to conventional treatments. Based on our cumu-
lative experience and previous reported data, we do not
believe that there are any molecular or clinical features of
these tumors that adequately predict tumor response;
therefore, we do not recommend that tumor cell lineage or
MGMT molecular analysis influence the clinical decision
for treatment with temozolomide in individual cases. Case
J Clin Endocrinol Metab, November 2010, 95(11):E280 –E290
5 in our series, a prolactinoma, was one of the two tumors
with significant tumor regression. There is a preponder-
ance of prolactinomas among those tumors reported in the
literature to be temozolomide-responsive (14 –17, 31, 42).
However, we believe this is indicative of the overrepre-
sentation of prolactinomas among functional tumors that
progress to a more malignant phenotype (13) rather
than a true molecular predisposition to temozolomide
responsiveness.
Complete remission is not an expected outcome of te-
Downloaded from https://academic.oup.com/jcem/article-abstract/95/11/E280/2835354 by guest on 16 June 2020
mozolomide therapy, but tumor stabilization or reduction
of tumor volume can improve clinical outcomes. Any ben-
efit of long-term therapy with a tumorstatic agent such as
temozolomide is typically limited by the increased likeli-
hood of adverse effects with cumulative doses. Cytopenias
are the most likely to occur, but myelodysplasia syndrome
and secondary malignancies including myeloid leukemia
have been observed with temozolomide (43). Thus, in our
opinion, temozolomide remains a last-line of defense for
life-threatening pituitary tumors, and the duration of ther-
apy must be determined on a patient-by-patient basis.
The lack of prospective clinical trial data for these rare
aggressive tumors necessitates multidisciplinary patient
care teams to provide patients with the most informed
treatment options and demands multicenter cooperation to
facilitate the future clinical studies that are needed to estab-
lish treatment guidelines for aggressive pituitary tumors.
Acknowledgments
Address all correspondence and requests for reprints to: M. Beatriz
S. Lopes, M.D., Ph.D., Division of Neuropathology, P.O. Box
800214, Charlottesville, Virginia 22908-0214. E-mail: msl2e@
virginia.edu.
Disclosure Summary: All authors have nothing to declare.
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