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Fig. 2: Increased expression of some components of the ER/UPR pathway in the SAT of obese subjects. Conclusions Increased expression of the UPR and HSR concomitant with the major inflammatory markers- IL6 and TNFα was observed in obese subjects. Physical exercise reduced expression of UPR pathway-GRP78 and its downstream targets along with the major HSPs in obese. Increased GRP78 expression and reduced HSP60 levels were observed with diabetes, reflecting the difference in the adaptive response by HSR and UPR systems to diabetes. Enhancing HSR and UPR system through exercise might have a beneficial impact on future approaches to diabetes prevention and treatment [1]. Tiss A et al. (2014). Lipids in health and disease, 13:106 [2]. Khadir et al. (2016). Am J Physiol Endocrinol Metab, 308, E71-83. References Acknowledgement Kuwait Foundation for the Advancement of Sciences (KFAS) for financial support Results Dysregulated heat shock and unfolded protein responses in obese humans and their modulation through physical exercise Sina Kavalakatt , Dhanya Madhu, Maha Hammad, Abdelkrim Khadir, Ali Tiss * Dasman Diabetes Institute, Kuwait 0 0.5 1 1.5 2 2.5 CHOP IRE1α PERK ATF4 XBP1 ATF6 GRP78 Fold Changes in mRNA * * ** ** 0.0 0.5 1.0 1.5 2.0 2.5 CHOP IRE1α XBP1 ATF6 GRP78 Fold Changes in proteins * * ** Obese (n=10) Lean (n=10) Obese (n=10) Lean (n=10) *: P<0.05; **: P<0.01 Fig. 6: Upregulated expression of GRP78 and its downstream targets with obesity and their attenuation with physical exercise Materials And Methods Adult subjects consisting of 40 lean and 40 obese ( non-diabetic and diabetic ) were recruited and enrolled for a 3-month supervised exercise program. Subcutaneous adipose tissue (SAT) biopsies and venous peripheral blood were collected before and after exercise. Plasma inflammatory and metabolic markers were measured using Bioplex-200 system. The expression levels were studied using immunohistochemistry, confocal microscopy, western blotting and Real Time-PCR. Marker circulating levels were estimated using ELISA. All statistical analyses were done using SAS version 9.2. Introduction and Objectives Obesity-induced insulin resistance and Diabetes are characterized by low grade inflammation, impaired heat shock response (HSR) and endoplasmic reticulum (ER) stress. Perturbation in the ER homeostasis occurs due to failure in the adaptive unfolded protein response (UPR) to restore normal ER function under metabolic stress. However, HSR and UPR expression pattern in adipose tissue (AT) is yet to be elucidated. Physical activity is a non- pharmacological intervention known to improve overall health by modulating metabolic stress pathways along with improving HSR and antioxidant defense system. This effect of physical exercise on restoring HSR and ER homeostasis in human obese/diabetic subjects has not been reported. Here, we investigated the status of key UPR markers and heat shock proteins (HSP) in subcutaneous AT of lean and obese humans along with their modulation through physical exercise and diabetes. Fig 1: Increased expression of the major heat shock proteins in obese subjects B A Lean (n=9) Obese (n=9) HSP-90 HSP-60 HSC-70 Actin GRP-94 HSP-72 HSP-27 DNAJB3 Lean (n=10) Obese (n=10) HSP-60 HSP-72 DNAJB3 0 1 2 3 4 HSP-27 DNAJB3 HSP-60 HSC-70 HSP-72 HSP-90 GRP-94 Fold Changes in the proteins (Adipose tissue) ** * * * * ** 0 2 4 6 8 HSP-27 DNAJB3 HSP-60 HSC-70 HSP-72 HSP-90 GRP-94 Fold Changes in the proteins (PBMCs) * * * ** ** ** *: P<0.05; **: P<0.01 Lean (n=40) Obese (n=40) P-value Anthropometric and physical characteristics AGE (year) 38.75 ± 9.60 40.13 ± 8.68 0.55 Gender (M/F) 16/24 20/20 0.37 Body mass Index (kg/m 2 ) 22.72 ± 2.09 34.83 ± 2.99 <0.0001 Waist (cm) 79.45 ± 15.96 108.52 ± 13.71 <0.0001 Hip (cm) 92.07 ± 14.98 118.15 ± 8.29 <0.0001 Percent Body Fat (%) 27.50 ± 5.35 39.36 ± 5.12 <0.0001 Systolic BP (mmHg) 113.00 ± 10.81 127.50 ± 11.89 0.01 Diastolic BP (mmHg) 76.43 ± 6.33 82.00 ± 10.14 0.13 V O2, Max (ml/kg/min) 21.63 ± 3.76 17.48 ± 4.83 0.03 Metabolic markers Cholesterol (mmol/l) 5.22 ± 0.91 5.19 ± 1.01 0.74 HDL (mmol/l) 1.48 ± 0.53 1.10 ± 0.26 0.0001 LDL (mmol/l) 3.23 ± 0.91 3.39 ± 0.98 0.28 TG (mmol/l) 0.91 ± 0.42 1.47 ± 0.83 0.0008 Glucose (mmol/l) 4.98 ± 0.56 5.44 ± 0.86 0.013 HbA1c (%) 5.50 ± 0.44 5.93 ± 0.68 0.004 Insulin (ng/ml) 2.6 ± 1.3 4.2 ± 2.5 0.029 HOMA-IR 0.7 ± 0.5 1.1 ± 0.7 0.005 C-Reactive Protein (g/ml) 1.7 ± 1.2 5.3 ± 3.6 <0.0001 Table 1: Clinical & biochemical characteristics of the study population at baseline. Table 2: Clinical and biochemical characteristics of the obese after exercise. Obese before exercise Obese after exercise P-value Anthropometric and physical characteristics AGE (year) 40.13 ± 8.68 - - Gender (M/F) 20/20 - - Body Mass Index (kg/m 2 ) 34.83 ± 2.99 34.16 ± 2.59 0.18 Waist (cm) 108.52 ± 13.71 108.00 ± 9.31 0.10 Hip (cm) 118.15 ± 8.29 118.25 ± 6.61 0.67 Percent Body Fat (%) 39.36 ± 5.12 38. 22 ± 4.57 0.04 Systolic BP (mmHg) 127.50 ± 11.89 119.29 ± 8.28 0.013 Diastolic BP (mmHg) 82.00 ± 10.14 78.86 ± 4.26 0.11 V O2, Max (ml/kg/min) 17.48 ± 4.83 19.66 ± 3.17 0.011 Metabolic markers Cholesterol (mmol/l) 5.19 ± 1.01 5.09 ± 1.03 0.58 HDL (mmol/l) 1.10 ± 0.26 1.08 ± 0.26 0.31 LDL (mmol/l) 3.39 ± 0.98 3.34 ± 1.00 0.89 TG (mmol/l) 1.47 ± 0.83 1.43 ± 0.60 0.71 Glucose (mmol/l) 5.44 ± 0.86 5.65 ± 1.03 0.78 HbA1c (%) 5.93 ± 0.68 5.87 ± 0.61 0.43 Insulin (ng/ml) 4.2 ± 2.5 2.9 ± 1.3 0.039 HOMA-IR 1.1 ± 0.7 0.8 ± 0.2 0.047 C-ReactiveProtein (g/ml) 5.3 ± 3.6 5.1 ± 2.7 0.28 Fig. 3: Increased expression of inflammatory markers in obese SAT IL-6 Lean (n=10) Obese (n=10) A 0.0 0.5 1.0 1.5 2.0 2.5 Lean Obese Fold Changes in the proteins (Adipose tissue) * TNF-α 0.0 0.5 1.0 1.5 2.0 Lean Obese Fold Changes in the proteins (Adipose tissue) B * B 0 0.5 1 1.5 2 2.5 Obese ND Obese D Fold Changes in GRP78 protein (PBMCs) Obese ND Obese D GRP78 GAPDH * A 0.0 0.5 1.0 1.5 2.0 Obese ND Obese D Fold Changes in GRP78 protein (SAT) Obese GRP78-Alexa 488 Obese (after) HSP60-Alexa 488 A 0.0 1.0 2.0 3.0 4.0 5.0 HSP-27 DNAJB3 HSP-60 HSC-70 HSP-72 HSP-90 GRP-94 Fold Changes in the proteins (Adipose tissue) Obese before exercise (n=10) Obese after exercise (n=10) Obese before exercise (n=12) Obese after exercise (n=12) 0 1 2 3 DNAJB3 HSP-60 HSC-70 HSP-72 HSP-90 Fold Changes in the mRNA (PBMCs) B Fig. 4: Decreased expression of major heat shock proteins after exercise in obese A B 0.0 0.5 1.0 1.5 Obese (before) Obese (after) Fold Changes in the proteins (Adipose tissue) TNF-α 0.0 0.5 1.0 1.5 Obese (before) Obese (after) Fold Changes in the proteins (Adipose tissue) Obese (before exercise) (n=14) IL-6 Obese (after exercise) (n=14) Fig. 4:Decreased expression of inflammatory markers after exercise in obese SAT Obese (before exercise) (n=14) Obese (after exercise) (n=14) Fig. 7: Immunoflourescence microscopy of GRP78 and Hsp60 before and after exercise Fig. 8: Expression of GRP 78 and Hsp60 in Diabetic obese diabetic subjects Obese Diabetic Non-Diabetic Immuno-Fluorescence Microscopy of HSP60 in SAT Contact info: [email protected] Lean (n=10) Obese (n=10) 0 0.5 1 1.5 CHOP IRE1α PERK ATF4 XBP1 ATF6 GRP78 Fold changes in mRNA Obese (before) Obese (after) 0 0.2 0.4 0.6 0.8 1 1.2 CHOP IRE1 XBP1 ATF6 GRP78 Fold changes in protein Obese before Obese after ** * * * * * * ** ** **

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Page 1: Dysregulated heat shock and unfolded protein responses in ... Researcher, Resident Winners.pdf · markers and heat shock proteins (HSP) in subcutaneous AT of lean and obese humans

Fig. 2: Increased expression of some components of the ER/UPR pathway in the

SAT of obese subjects. Conclusions

Increased expression of the UPR and HSR

concomitant with the major inflammatory markers-

IL6 and TNFα was observed in obese subjects.

Physical exercise reduced expression of UPR

pathway-GRP78 and its downstream targets

along with the major HSPs in obese.

Increased GRP78 expression and reduced

HSP60 levels were observed with diabetes,

reflecting the difference in the adaptive response

by HSR and UPR systems to diabetes.

Enhancing HSR and UPR system through

exercise might have a beneficial impact on future

approaches to diabetes prevention and treatment

[1]. Tiss A et al. (2014). Lipids in health and disease, 13:106

[2]. Khadir et al. (2016). Am J Physiol Endocrinol Metab, 308, E71-83.

References

Acknowledgement

Kuwait Foundation for the Advancement of Sciences (KFAS) for financial support

Results

Dysregulated heat shock and unfolded protein responses in obese

humans and their modulation through physical exercise

Sina Kavalakatt, Dhanya Madhu, Maha Hammad, Abdelkrim Khadir, Ali Tiss *

Dasman Diabetes Institute, Kuwait

0

0.5

1

1.5

2

2.5

CHOP IRE1α PERK ATF4 XBP1 ATF6 GRP78

Fo

ld C

ha

ng

es

in

mR

NA

* *

****

0.0

0.5

1.0

1.5

2.0

2.5

CHOP IRE1α XBP1 ATF6 GRP78

Fo

ld C

ha

ng

es

in

pro

tein

s

*

*

**

Obese (n=10)

Lean (n=10)

Obese (n=10)

Lean (n=10)

*: P<0.05; **: P<0.01

Fig. 6: Upregulated expression of GRP78 and its downstream targets with

obesity and their attenuation with physical exercise

Materials And Methods

Adult subjects consisting of 40 lean and 40 obese ( non-diabetic and diabetic ) were

recruited and enrolled for a 3-month supervised exercise program. Subcutaneous adipose

tissue (SAT) biopsies and venous peripheral blood were collected before and after

exercise. Plasma inflammatory and metabolic markers were measured using Bioplex-200

system. The expression levels were studied using immunohistochemistry, confocal

microscopy, western blotting and Real Time-PCR. Marker circulating levels were estimated

using ELISA. All statistical analyses were done using SAS version 9.2.

Introduction and ObjectivesObesity-induced insulin resistance and Diabetes are characterized by low grade inflammation, impaired heat shock

response (HSR) and endoplasmic reticulum (ER) stress. Perturbation in the ER homeostasis occurs due to failure

in the adaptive unfolded protein response (UPR) to restore normal ER function under metabolic stress. However,

HSR and UPR expression pattern in adipose tissue (AT) is yet to be elucidated. Physical activity is a non-

pharmacological intervention known to improve overall health by modulating metabolic stress pathways along with

improving HSR and antioxidant defense system. This effect of physical exercise on restoring HSR and ER

homeostasis in human obese/diabetic subjects has not been reported. Here, we investigated the status of key UPR

markers and heat shock proteins (HSP) in subcutaneous AT of lean and obese humans along with their modulation

through physical exercise and diabetes.

Fig 1: Increased expression of the major heat shock proteins in obese subjects

BA

Lean (n=9) Obese (n=9)

HSP-90

HSP-60

HSC-70

Actin

GRP-94

HSP-72

HSP-27

DNAJB3

Lean

(n=10)

Obese

(n=10)

HSP-60 HSP-72DNAJB3

0

1

2

3

4

HSP-27 DNAJB3 HSP-60 HSC-70 HSP-72 HSP-90 GRP-94Fo

ld C

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ng

es

in

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rote

ins

(Ad

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se

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e)

**

* ** *

**

0

2

4

6

8

HSP-27 DNAJB3 HSP-60 HSC-70 HSP-72 HSP-90 GRP-94Fo

ld C

ha

ng

es

in

th

e p

rote

ins

(PB

MC

s)

*

*

*

**

** **

*: P<0.05; **: P<0.01

Lean (n=40) Obese (n=40) P-value

Anthropometric and physical characteristics

AGE (year) 38.75 ± 9.60 40.13 ± 8.68 0.55

Gender (M/F) 16/24 20/20 0.37

Body mass Index (kg/m2) 22.72 ± 2.09 34.83 ± 2.99 <0.0001

Waist (cm) 79.45 ± 15.96 108.52 ± 13.71 <0.0001

Hip (cm) 92.07 ± 14.98 118.15 ± 8.29 <0.0001

Percent Body Fat (%) 27.50 ± 5.35 39.36 ± 5.12 <0.0001

Systolic BP (mmHg) 113.00 ± 10.81 127.50 ± 11.89 0.01

Diastolic BP (mmHg) 76.43 ± 6.33 82.00 ± 10.14 0.13

VO2, Max (ml/kg/min) 21.63 ± 3.76 17.48 ± 4.83 0.03

Metabolic markers

Cholesterol (mmol/l) 5.22 ± 0.91 5.19 ± 1.01 0.74

HDL (mmol/l) 1.48 ± 0.53 1.10 ± 0.26 0.0001

LDL (mmol/l) 3.23 ± 0.91 3.39 ± 0.98 0.28

TG (mmol/l) 0.91 ± 0.42 1.47 ± 0.83 0.0008

Glucose (mmol/l) 4.98 ± 0.56 5.44 ± 0.86 0.013

HbA1c (%) 5.50 ± 0.44 5.93 ± 0.68 0.004

Insulin (ng/ml) 2.6 ± 1.3 4.2 ± 2.5 0.029

HOMA-IR 0.7 ± 0.5 1.1 ± 0.7 0.005

C-Reactive Protein (𝜇g/ml) 1.7 ± 1.2 5.3 ± 3.6 <0.0001

Table 1: Clinical & biochemical characteristics of the study population at baseline. Table 2: Clinical and biochemical characteristics of the obese after exercise.

Obese before exercise Obese after exercise P-value

Anthropometric and physical characteristics

AGE (year) 40.13 ± 8.68 - -

Gender (M/F) 20/20 - -

Body Mass Index (kg/m2) 34.83 ± 2.99 34.16 ± 2.59 0.18

Waist (cm) 108.52 ± 13.71 108.00 ± 9.31 0.10

Hip (cm) 118.15 ± 8.29 118.25 ± 6.61 0.67

Percent Body Fat (%) 39.36 ± 5.12 38. 22 ± 4.57 0.04

Systolic BP (mmHg) 127.50 ± 11.89 119.29 ± 8.28 0.013

Diastolic BP (mmHg) 82.00 ± 10.14 78.86 ± 4.26 0.11

VO2, Max (ml/kg/min) 17.48 ± 4.83 19.66 ± 3.17 0.011

Metabolic markers

Cholesterol (mmol/l) 5.19 ± 1.01 5.09 ± 1.03 0.58

HDL (mmol/l) 1.10 ± 0.26 1.08 ± 0.26 0.31

LDL (mmol/l) 3.39 ± 0.98 3.34 ± 1.00 0.89

TG (mmol/l) 1.47 ± 0.83 1.43 ± 0.60 0.71

Glucose (mmol/l) 5.44 ± 0.86 5.65 ± 1.03 0.78

HbA1c (%) 5.93 ± 0.68 5.87 ± 0.61 0.43

Insulin (ng/ml) 4.2 ± 2.5 2.9 ± 1.3 0.039

HOMA-IR 1.1 ± 0.7 0.8 ± 0.2 0.047

C-ReactiveProtein (𝜇g/ml) 5.3 ± 3.6 5.1 ± 2.7 0.28

Fig. 3: Increased expression of inflammatory markers in obese SAT

IL-6

Lean

(n=10)

Obese

(n=10)

A

0.0

0.5

1.0

1.5

2.0

2.5

Lean Obese

Fo

ld C

han

ge

s i

n t

he

pro

tein

s(A

dip

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*

TNF-α

0.0

0.5

1.0

1.5

2.0

Lean Obese

Fo

ld C

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ge

s i

n t

he

pro

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s(A

dip

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B

*

B

0

0.5

1

1.5

2

2.5

Obese ND Obese D

Fo

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GR

P7

8

pro

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(PB

MC

s)

Obese ND Obese D

GRP78

GAPDH

*

A

0.0

0.5

1.0

1.5

2.0

Obese ND Obese D

Fo

ld C

han

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s i

n G

RP

78 p

rote

in(S

AT

)

Obese

GRP78-Alexa 488

Obese

(after)

HSP60-Alexa 488

A

0.0

1.0

2.0

3.0

4.0

5.0

HSP-27 DNAJB3 HSP-60 HSC-70 HSP-72 HSP-90 GRP-94

Fo

ld C

han

ge

s i

n t

he p

rote

ins

(Ad

ipo

se

tis

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Obese before exercise (n=10)

Obese after exercise (n=10)

Obese before exercise (n=12)

Obese after exercise (n=12)

0

1

2

3

DNAJB3 HSP-60 HSC-70 HSP-72 HSP-90Fo

ld C

han

ge

s in

th

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RN

A(P

BM

Cs)

B

Fig. 4: Decreased expression of major heat shock proteins after exercise in obese

AB

0.0

0.5

1.0

1.5

Obese (before) Obese (after)

Fo

ld C

han

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s i

n t

he p

rote

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(Ad

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TNF-α

0.0

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1.0

1.5

Obese (before) Obese (after)

Fo

ld C

ha

ng

es

in

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rote

ins

(Ad

ipo

se

tis

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e)

Obese

(before exercise)

(n=14)

IL-6

Obese

(after exercise)

(n=14)

Fig. 4:Decreased expression of inflammatory markers after exercise in obese SAT

Obese

(before exercise)

(n=14)

Obese

(after exercise)

(n=14)

Fig. 7: Immunoflourescence microscopy of GRP78 and Hsp60 before and after

exercise

Fig. 8: Expression of GRP 78 and Hsp60 in Diabetic obese diabetic subjects

Obese

DiabeticNon-Diabetic

Immuno-Fluorescence Microscopy of HSP60 in SAT

Contact info: [email protected]

Lean

(n=10)

Obese

(n=10)

0

0.5

1

1.5

CHOP IRE1α PERK ATF4 XBP1 ATF6 GRP78

Fo

ld c

ha

ng

es

in

mR

NA

Obese (before)

Obese (after)

0

0.2

0.4

0.6

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1.2

CHOP IRE1 XBP1 ATF6 GRP78

Fo

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in

pro

tein

Obese before

Obese after

**

* * * *

* *

**

** **

Page 2: Dysregulated heat shock and unfolded protein responses in ... Researcher, Resident Winners.pdf · markers and heat shock proteins (HSP) in subcutaneous AT of lean and obese humans

Linear Psoriasis: A Rare Presentation of PsoriasisZahraa Al-Hashemi1, Lulwa Ahmad1

Department of Dermatology, Faculty of Medicine, Kuwait University1very itchy and unilateral across Blaschko’slines, whereas in LP, patients usually present later in life and it is only occasionally itchy and more widespread than ILVEN. Seasonal remissions are usually present in LP, but absent in ILVEN. As opposed to ILVEN, LP is characterized by its responsiveness to antipsoriatic treatment. Characteristic histopathology findings as well as immunohistochemistry markers are helpful in confirming the diagnosis and differentiating between the two conditions. Conclusion:In patients with late-onset linear erythematousand scaly plaques along the lines of Blaschko, the diagnosis of LP should be considered. ILVEN is the main disease to be considered in the differential diagnosis. LP lesions usually respond more poorly to topical and systemic antipsoriatic treatment than lesions of common plaque-type psoriasis but the response is more favourable than ILVEN to the treatment.

Key References:1. Nasimi, M., et al. “Isolated linear blaschkoidpsoriasis.” Clinical and Experimental Dermatology, vol. 41, no. 7, 2016, pp. 775–778.2. Kim, Sang Jin, and You Chan Kim. “Linear Psoriasis along Blaschkos Lines.” Annals of Dermatology, vol. 29, no. 1, 2017, p. 106.3. Sengupta, Dipayan, et al. “Diagnostic dilemma between linear psoriasis and inflammatory linear verrucousepidermal nevus.” Indian Journal of PaediatricDermatology, vol. 17, no. 2, 2016, p. 159.4. Saraswat, Abir, et al. “Unilateral Linear Psoriasis with Palmoplantar, Nail, and Scalp Involvement.” Pediatric Dermatology, vol. 21, no. 1, 2004, pp. 70–73.

Background:Linear psoriasis (LP) is an exceedingly rare clinical presentation of psoriasis. It is characterized by linear lesions along the lines of Blaschko. LP may occur as the only manifestation of psoriasis (the isolated type) or it may coexist with nonsegmental plaques of psoriasis vulgaris(Superimposed LP). A limited number of cases have been reported in the literature.Case Summary:A 36-year-old male presented with sudden eruption of linear red scaly plaques on his right arm and right half of the back. The patient has a history of psoriasis five years ago. Past medical history was not contributory and similar or other types of psoriasis were not observed in his family. Cutaneous examination showed erythematous, scaly, raised, confluent and discrete papules and plaques localized to a linear band-like area on the right side of the back. The plaques were distributed along the lines of Blaschko, with a marked midline demarcation. Auspitz sign was positive.

Histopathological examination of the skin biopsy revealed an acanthotic epidermis composed of pale keratinocytes with absence of the granular layer. Parakeratosis and microabscesses of Munro were observed in the stratum corneum. The dermis showed elongated papillae with only a thin suprapapillaryplate covering the tips of these papillae.

Discussion:Linear psoriasis (LP) is an uncommon variant of psoriasis that presents as a linear distribution of psoriatic lesions along Blaschko’s lines. Affected areas may include the trunk, extremities, glutealregion, scalp, and nails..An important differential diagnosis to consider is inflammatory linear verrucous epidermal naevus (ILVEN). ILVEN classically occurs during the first months of life and is usually

Page 3: Dysregulated heat shock and unfolded protein responses in ... Researcher, Resident Winners.pdf · markers and heat shock proteins (HSP) in subcutaneous AT of lean and obese humans

www.postersession.com

Gastrointestinal stromal tumors (GIST) are the most common mesenchymal tumors of the gastrointestinal tract. They are derived from the interstitial cells of Cajal that serve as the electrical pacemakers of the gut. The majority of GISTs are driven by activating mutations of KIT (≈75%) or PDGFRA (≈8%) genes, while 10-15% lack such mutations and are designated as “KIT/PDGFRA wild-type”1. Type of mutation predicts response to tyrosine kinase inhibitors (TKIs) such as imatinib2. Very few original studies have examined the molecular characteristics of GISTs in our geographical region. Herein, we present a pilot study to molecularly characterize a cohort of GIST cases referred to Kuwait Cancer Control Center (KCCC) between 2005 and 2015.

References 1. Wada R, Arai H, Kure S, et al. "Wild type" GIST: Clinicopathological

features and clinical practice. Pathol Int. 2016 Aug;66(8):431-7. 2. Lasota J, Miettinen M. Clinical significance of oncogenic KIT and

PDGFRA mutations in gastrointestinal stromal tumours. Histopathology. 2008 Sep;53(3):245-66.

3. Rossi S, Gasparotto D, Miceli R, et al. KIT, PDGFRA, and BRAF mutational spectrum impacts on the natural history of imatinib-naive localized GIST: a population-based study. Am J Surg Pathol. 2015 Jul;39(7):922-30.

4. Miettinen M, Makhlouf H, Sobin LH, et al. Gastrointestinal stromal tumors of the jejunum and ileum: a clinicopathologic, immunohistochemical, and molecular genetic study of 906 cases before imatinib with long-term follow-up. Am J Surg Pathol. 2006 Apr;30(4):477-89.

5. Miettinen M, Sobin LH, Lasota J. Gastrointestinal stromal tumors of the stomach: a clinicopathologic, immunohistochemical, and molecular genetic study of 1765 cases with long-term follow-up. Am J Surg Pathol. 2005 Jan;29(1):52-68.

Molecular analysis of KIT and PDGFRA in GISTs serves multiple purposes: diagnostic (especially for tumors of unusual morphology or at unusual sites), prognostic (e.g. more aggressive behavior is associated with KIT exon 11 deletions), and predictive (e.g. tumors with KIT exon 9 mutations require a double dose of imatinib). It is recommended that conventional clinicopathological risk stratification be complemented by molecular data3. As expected, KIT mutations were the most frequent in this cohort, with exon 11 mutations being the most common4. These tumors could be found anywhere along the GI tract and were of spindle-cell histology. On the other hand, PDGFRA mutations occurred exclusively in gastric GISTs, were of epithelioid or mixed histology5, and showed reduced cKIT expression. Three of the KIT/PDGFRA wild-type GISTs were shown to harbor other gene mutations (e.g. PIK3CA, NF1, BRCA1/2), the significance of which is unknown. A larger study is needed to assess reproducibility of these results. No BRAF or KRAS mutations, which are known to occur in a subset of wild-type GISTs, were detected. In summary, this is the first study in Kuwait to evaluate GISTs at the molecular level which will serve as the foundation for larger future studies.

Background Discussion

Funding: This work was funded by Research Grant No. MG02/15, Research Sector, Kuwait University. Acknowledgment: We would like to thank the Molecular Lab staff at KCCC for performing the sequencing part of the study.

Tissue Samples A total of 114 GISTs from 100 patients, diagnosed between 2005-2015, were identified from the pathology archive at KCCC. The pathology reports were reviewed for demographic and pathological parameters: tumor location, size, histological type, mitotic count, and risk assessment. H&E slides were reviewed by 2 pathologists for confirmation. Clinical data were obtained through medical record review when possible. TMA Construction and Immunohistochemistry Sixty eight specimens had available paraffin blocks. These were used to construct tissue microarray (TMA) blocks, by harvesting 1.5-mm-diameter tissue cores from donor blocks and arraying them on a recipient block in duplicates. After construction, 4  µm sections were cut and u s e d f o r c K i t ( C D 11 7 ) , D O G 1 , a n d C D 3 4 immunohistochemistry (IHC). IHC positivity was scored as weak or strong, irrespective of whether the reactivity was diffuse or focal. Tumors were scored negative when no staining could be detected. DNA Sequencing Genomic DNA was extracted from a total of 24 formalin-fixed paraffin-embedded tissue samples (using MagMax FFPE DNA/RNA Ultra Kit) and isolated on the KingFisher Duo Prime System. DNA concentrations were checked on a Qubit 3.0 Fluorometer (Thermo Fisher Scientific) and then adjusted to 10 ng. Library preparation was carried out manually using the Oncomine Comprehensive Assay v1, which covers 161 of the most relevant cancer driver genes. The libraries were then loaded onto an Ion Chef System (Thermo Fisher Scientific) to prepare them for sequencing. Finally, the samples were sequenced on an Ion Torrent S5 XL Semiconductor sequencer (Thermo Fisher Scientific) and the sequence data were analyzed by the Ion Reporter v5.2 Software.

Results

ID Age/Gender Tumor location

Size (cm)

Histology Mitosis (/50hpf)

Risk Mutation Tumor status

Imatinib (400mg)

Follow-up (months)

1i 58/M Small bowel 11.5 Spindle >5 High KIT Exon 11 del WK557-558 Primary unknown - 1ii 61/M Peritoneal 15 Spindle >5 High KIT Exon 11 del WK557-558 Recurrent unknown - 3 46/M Stomach 2.7 Spindle >5 Mod KIT Exon 11 del WK557-558 Primary yes Alive (32.1) 9 70/M jejunum 6 Spindle >5 High KIT Exon 13 (K642E) Primary yes Alive (82.9) 10 77/F Jejunum 2 Spindle ≤5 None KIT Exon 11 del WK557-558 Primary no Alive (1.7) 13 53/M Stomach 5.5 Spindle ≤5 Low KIT Exon 11 del WK557-558 Primary no Alive (14.9) 14 40/M Stomach 2.5 Spindle ≤5 Very low KIT Exon 11 (V559D) Primary unknown - 15i 52/F Rectum 5 Spindle ≤5 Low KIT Exon 11 (V559A) Primary unknown - 15ii 52/F Rectum 5 Spindle ≤5 Low KIT Exon 11 (V559A) Primary unknown - 19 59/F Stomach 14 Spindle >5 High KIT Exon 11 (K558R) Primary unknown - 55 84/M Mesentery NA Mixed >5 NA KIT Exon 13 (K642E) NA unknown - 58 69/M Stomach 10 Spindle ≤5 Mod KIT Exon 11 (V560D) Primary unknown - 8 33/M Stomach 15 Mixed ≤5 Mod PDGFRA Exon 18 (D842V) Primary yes Alive (100.9) 12 48/M Stomach NA Epithelioid ≤5 NA PDGFRA Exon 18 (D842V) Primary unknown - 17 55/M Stomach 6 Epithelioid ≤5 low PDGFRA Exon 18 (D842V) Primary unknown - 48 56/F Stomach 6.5 Mixed ≤5 Low PDGFRA Exon 12 (V561D) Primary yes Alive (37) 2 58/F Duodenum 4 Spindle ≤5 Low DNMT3A, NFE2L2, SF3B1, VHL, BAP1, TET2,

FBXW7, PIK3R1, EGFR, PAX5, PTCH1, ABL1, TSC1, PTEN, ATM, FLT3, BRCA2, RB1, TSC2, BRCA1

Primary unknown -

4 44/M Small bowel 3.8 Spindle ≤5 Low NRAS, NFE2L2, PIK3CA, TET2, FBXW7, APC, BRAF, PTCH1, NOTCH1, ATM, HNF1A, RB1, NF1, BRCA1

Primary no Alive (27.5)

16 54/M Stomach 3 Mixed ≤5 Very low PIK3CA Primary unknown - 18 54/F Stomach 0.7 Spindle ≤5 None None Primary unknown - 23ii 50/F Small bowel 6 Spindle ≤5 Mod None Primary unknown - 24 67/F Jejunum 4.5 Spindle ≤5 Low None Primary no Alive (2.4) 49 62/M Small bowel 3 Epithelioid ≤5 Low None Primary unknown Alive (23.7) 59 41/F Colon 6 Spindle >5 High None Primary unknown -

Methods

Table 1: Clinicopathological characteristics and mutation types of 24 GIST tumors subjected to sequencing.

Of the 100 patients, 63(63%) were males and 37(37%) females. Age range was 5-84 years (mean 54.7). Most GISTs were primary tumors localized to the gut (82.9%). Forty seven (42.7%) were located in the stomach, 44(40%) small bowel, 11(10%) colon/rectum, 5(4.6%) peritoneal cavity, and 3(2.7%) in a metastatic site. Size ranged between 0.4-22 cm, with a mean of 7.2 cm. Pathological “risk-of-progression” assessment was: none in 10(8.8%) tumors, very low in 7(6%), low in 18(15.8%), moderate in 14(12.3%), high in 25(21.9%), & indeterminate in 40(35%). The morphology was spindled in 73(66.4%), mixed in 26(23.6%) cases, and epithelioid in 11(10%). See Figure 1. On IHC, although cKIT(CD117) expression was detected in all 65 tumors tested, 3 were weak (found to be PDGFRA-mutated). DOG1 expression was seen in 63/65 (97%), while CD34 was seen in 39 (60%). The observed mutations & associated clinicopathological features are summarized in Table 1. Of 24 tumors tested, 12 (50%) showed KIT mutations; 10(42%) in exon 11, 2(8%) in exon 13, and none in exon 9 or 17. PDGFRA mutations were identified in 4 (16.7%) tumors; 3 in exon 18 (D842V) and 1 in exon 12. Eight GISTs (33.3%) were wild-type, 3 of which showed other mutations that are of unknown significance (case ID 2 and 4 in Table 1).

Figure 1: A GIST harboring a KIT Exon 11 del WK557-558 (ID 1i & 1ii). A) Gross appearance of the recurrent tumor in the peritoneal cavity 3 years after primary surgery. The tumor originally arose in small bowel, measured 11.5cm, and was of high-risk class. B) Microscopic appearance. Both the primary and recurrent tumors showed a spindle cell morphology with mitosis >5/50 hpf. CD117 and DOG1 were positive.

A

DOG1 CD117 Recurrence Primary B

Molecular Alterations in Gastrointestinal Stromal Tumors (GISTs) Using Targeted Next-Generation Sequencing

Sulaiman Alrushaidan1, Ahmed Mohieldin2, Abeer Alabdallah3, Hiba Mehdawi3, Fahad Elenezi2, Shakir Bahzad4, Rola H. Ali3,4 1Histopathology Laboratory, KCCC; 2Medical Oncology, KCCC; 3Department of Pathology, Faculty of Medicine, Kuwait University; 4Molecular Laboratory, KCCC