Supplemental Data For:

Mutter GL, Ince TA, Baak JPA, Kust G, Zhou X, Eng C. Molecular identification of latent precancers in histologically normal endometrium. Cancer Res 2001; 61(8).

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PE Mutant Seq.jpg (31038 bytes)  Supplemental Figure 1: Sequence confirmed PTEN mutation in proliferative endometrium. Single-plex PCR and denaturing gradient gel electrophoresis of the exon 5 of PTEN gene. DNA from PTEN-null glands (NULL) have aberrantly migrating species (arrowheads) compared to PTEN expressing (POS) glands. Bidirectional (for, forward; rev, reverse) direct sequence confirmation of mutations is shown in panels below. PTEN immunohistochemistry of these two proliferative endometria is shown in Figure 1, left of the published paper.

 

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PE Mutant Model.gif (5030 bytes)Supplemental Figure 2: Model of endometrial cancer pathways. Endometrioid endometrial adenocarcinoma (excluding papillary serous and non-endometrioid cancers) can arise through a PTEN dependent (left) or independent (right) pathway(7). Three stages of EIN-mediated endometrial carcinogenesis are shown, top to bottom, as tissue compartments proportionately shaded to show that fraction (% PTEN defect) of endometrial glands at each stage  which have PTEN mutation and/or deletion (black) or normal PTEN (white). Normal proliferative endometria contains a small fraction (42% of women have 2-3% PTEN mutated/deleted glands, for overall PTEN null rate estimated here as 1%) of PTEN-defective cells. EIN and cancer (CA) are usually homogenous regarding PTEN expression, so defective and non-defective fractions are reported as published PTEN mutation/deletion rates for these diagnoses in cases with a documented premalignant phase {4636}. Latent PTEN mutant clones within proliferative endometria are, individually, at least 50 times more susceptible to EIN conversion than glands with normal PTEN genotype.

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Supplemental Table 1. PTEN mutation, deletion, and expression status in Normal Proliferative Endometria.
Laser capture microdissection was directed by PTEN immunohistochemistry of an adjacent serial section to enable DNA isolation from PTEN expressing (+) and non-expressing (null, -) endometrial glands.  All mutations listed are based upon sequence confirmed changes screened by Denaturaing Gradient Gel Electrophoresis.  Examples of Sequence Confirmation appear in Supplemental Figure 1, above.

         PTEN protein Null (-) and Positive (+) glands from individual biopsies.
          LOH, Loss of Heterozygosity (NI=not informative, ROH=retention of heterozygosity;
            LOH=loss of heterozygosity). 

Patient #

Expression*

Mutation

LOH Status at Markers†:

D10S579

D10S2491

D10S541

704

+

None

NI

NI

ROH

704

-

None

NI

NI

LOH

339

+

None

ROH

ROH

NI

339

-

Y88S

LOH

ROH

NI

521

+

None

ROH

NI

ROH

521

-

None

LOH

NI

LOH

366

+

None

NI

NI

ROH

366

-

IVS4-1G>T

NI

NI

LOH

932

+

None

NI

ROH

NI

932

-

c963-8InsA

NI

ROH

NI

603

+

None

ROH

ROH

ROH

603

-

c462-473del12

LOH

LOH

LOH

471

+

None

ROH

ROH

ROH

471

-

C643-5delT

LOH

ROH

ROH

253

+

None

ROH

NI

NI

253

-

None

LOH

NI

NI

479

+

None

NI

ROH

ROH

479

-

None

NI

ROH

LOH

071

+

None

NI

ROH

NI

071

-

None

NI

ROH

NI

075

+

None

ROH

NI

NI

075

-

c462-473del12

LOH

NI

NI

908

+

None

ROH

ROH

ROH

908

-

None

LOH

LOH

LOH

106

+

None

ROH

ROH

NI

106

-

None

LOH

LOH

NI

613

+

None

NI

ROH

ROH

613

-

None

NI

ROH

ROH

039

+

None

NI

ROH

ROH

039

-

None

NI

ROH

LOH

229

+

None

ROH

ROH

ROH

229

-

C988-90InsA

ROH

ROH

ROH

421

+

None

ROH

ROH

ROH

421

-

IVS4-1G>A

ROH

ROH

ROH

717

+

None

ROH

ROH

ROH

717

-

None

ROH

ROH

LOH

469

+

None

ROH

ROH

ROH

469

-

None

ROH

ROH

ROH

 

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Supplemental Table 2: Morphometric Characteristics of PTEN-null Glands, by Diagnosis within the Morphometry Window.  This is a tabular format of the same data which appears in published Figure 2.
*          two-tailed ANOVA, significance

PTEN Parameter

Abbrev.

Units

Proliferative

mean (SD)

Persistent Proliferative

mean (SD)

EIN

mean (SD)

p*

Sample

n

patients

20

20

17

 

Volume % Stroma

VPS

percentage

78.3 (10.2)

65.2 (13.7)

35.0 (7.9)

<0.001

Volume % Null Glands

VPNULL

percentage

13.0 (10.1)

25.6 (17.7)

63.9 (8.7)

<0.001

Volume % Positive glands

VPPOS

percentage

8.7 (5.4)

9.2 (1.1)

1.1 (1.9)

0.001

Density of Null Glands

DENNULL

# glands/mm2

8.4 (5.1)

9.8 (6.6)

26.2 (8.0)

<0.001

Density of Positive Glands

DENPOS

# glands/mm2

10.5 (8.7)

3.5 (3.3)

0.9 (1.7)

<0.001

Size of Null Glands

SZNULL

mm2 per gland

0.016(0.010)

0.030 (0.017)

0.026 (0.007)

<0.004

Size of Positive Glands

SZPOS

mm2 per gland

0.014(0.015)

0.024 (0.012)

0.018 (0.015)

<0.090

 

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Supplemental Table 3: PTEN status in repeat biopsies of proliferative endometria in POSTMENOPAUSAL women taking sequential replacement hormones.  
* Initial (1st sample) and repeat (2nd sample) endometrial samples scored as PTEN-nonexpressing (null) or having only PTEN-expressing glands (positive).

 

2nd Sample PTEN positive

2nd Sample PTEN null

Total

1st  Sample PTEN positive

8 1 9

1st Sample PTEN null

0 2 2

Total

8 3 11

 

 

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