Supplemental Table 1. Characteristics and Summary of Results of Eligible Studies for the Current Systematic Review

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Supplemental Table 1. Characteristics and Summary of Results of Eligible Studies for the Current Systematic Review

Author, year

Study Design/

Location

Sample size

Patients Preservation Quantification Method

Primary Antibody-IHC/

RNA-isolation-PCR

Detection/Diagnostic Results

Prognostic Results

Covariates

THYROID CANCER Belge, 2008

8

Cross- sectional (Germany)

64 44 female/17 male 19-adenomas 28-PTC; 9-FTC;

3-ATC

FFPE IHC Goat anti-HMGA2 (Santa

Cruz Biotech., Santa Cruz, CA); Dilution: N/A

100% Age, sex,

histology, size, TNM

classification and grading RT-PCR

(Ct method)

Proteinase K & DNase I (Roche, Penzberg, Germany) Chiappetta,

2008 ⁹

Cross- sectional (Italy)

128 12-hyperplastic lesions;

31-adenomas;

21-FTC; 45-PTC;

12-ATC

FFPE IHC

(Cell count)

Goat anti-rabbit/anti- mouse (Dako LSAB2 System); Dilution: 2g/mL

Papillary carcinomas:

(30/45=75.5%

Follicular carcinomas:

4/21=19.1%

Anaplastic carcinoma:

11/12=91.6%

NA

RT-PCR (Ct method)

Trizol reagent (Invitrogen) Adenomas: 1/7=14.3%

Papillary carcinomas:

34/37=91.9%

Follicular carcinomas:

13/16=81.2%

Anaplastic carcinoma:

4/4=100%

Prasad 2008, ¹⁰

Cross- sectional (USA)

125 70-benign tumors (20-adenomatoid nodules; 20- adenomas; 17-HA;

13 lymphocytic nodules) 55-malignant tumors (19-PTC;

16-FVPTC; 14- FTC; 6-HC)

FFPE IHC

(Cell count)

Goat polyclonal anti- HMGA2; Dilution: N/A

PTC: 26/30=87%;

FVPTC: 13/16=81%);

FTC: 11/14=79%

NA

Trizol reagent (Invitrogen) Very high in malignant tumor

Arora 2009,

11

90 16-PTC, 22-

FVPTC, 15- hyperplastic nodules, 22-FA;

15-borderline

Gene chip (Array Assist Ver. 5.2.2, Strategene Inc., La Jolla, CA)

RNeasy Mini kit (Qiagen, Valencia, CA)

Tumor: 3.56 fold-change (P=0.02)

NA

Lappinga 2010, ¹²

115 71-benign (1- Goiter, 12- adenomatoid nodules, 37-FA, 21-HA) 44-malignant (13-

Proteinase K (Roche, Diagnostics Corp., Indianapolis, IN)

FTC (11/13= 85%) HC: 3/9=33%

PTC: 17/22=33%

Sex, age

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FTC, 22-PTC, 9- HC)

Jin 2011, ¹³ Cross- sectional (USA)

396 (170 FFPE &

226 FNA)

34 FA, 10 HA, 6 hyperplastic nodules, 4 atypical adenomas, 44 PTC, 29 FVPTC, 23 FTC, 17 HC, and 3 ATC

FFPE (170) FNA (226)

TRIzol (Invitrogene, Carlsbad, CA)

Overall:

71.6-79.8%

FNA: 79.8-88.6%

NA

Prasad 2012, ¹⁴

87+86+

95=228

FFPE:

36-PTC; 20- FVPTC; 17-FTC;

30-FA; 18- Lymphocytic thyroid nodule; 18- adenomatoid nodule; 11-HA; 4- HC; 30-Normal FNA:

67-benign; 28- malignant

FFPE (193) FNA (95)

IHC (Cell count)

Goat polyclonal antibody (Santa Cruz

Biotech, Santa Cruz, CA);

Dilution: 1:300

Follicular carcinoma:

6/17=35%

FVPTC : 12/20=60%

Papillary thyroid carcinoma: 26/36=72%

NA

miRCURY RNA isolation kit (Exiqon,

Woburn, MA) Klemke

2014, ¹⁵

37 14-FA, 11-PTC, 4- FVPTC, 8=FTC

FFPE RT-PCR

(Ct method)

RNeasy FFPE kit (Qiagen, Hilden, Germany)

FVPTC: 23.9-156.9 PTC: 128.2-1207.5

NA

Nagar 2014,

16

52 21-FA, 12-FTC, 19-FVPTC

FNA RT-PCR

(Ct method)

RNeasy Mini Kit (Qiagen, Valencia, CA)

FVPTC 28-fold higher (P<0.01)

Age, sex, history of cancer, history of hypo- hyperthyroidism Jin 2015, ¹⁷ Cross-

sectional (USA)

200 FFPE: 48 benign lesions-32 carcinoma (120 FNA (56-benign, 64-carcinoma)

FFPE FNA

TRIzol Reagents (Invitrogen, Carlsbad, CA)

PTC (23/24=95.8%) FVPTC (8/10=80%) FC (15/17=88.2%) HC (4/13=30.8%) Jang 2015,

18

Cross- sectional (South Korea)

192 41-Goiter, 72-FA, 79- FTC

FFPE IHC (Cell

count)

Polyclonal antibody (Biocheck,

Foster city, CA, USA)- Dilution: 1:200

FA (30/72= 41.7%

FTC: (44/79=55.7%) (P=0.05)

Age, sex, location, T- category, N category, tumor size, capsular invasion, vascular invasion, follow- up time, distant metastasis, local recurrence OVARIAN CANCER

Mahajan 2009,

19

Case only study (USA)

115 30 HG-PSC; 10 SBT; 15 MMMT 30 EOC; 15 MOC 15 CCOC;

Paraffin IHC (Cell count)

HMGA2 (BioCheck Inc, Foster City, CA, USA)- Dilution: N/A

HG-PSC: 18/30=64.3%

MMMT: 9/15= 64.3%

SBT: 1/10=10%

EOC: 2/15=7.1%

MOC: 1/15=6.7%

CCOC: 3/15=23.1%

Age, tumor size, FIGO grade, FIGO stage, Lymph node metastasis

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Hetland 2012, ⁷

Retrospective cohort (Norway)

199 199-effusion samples 50 Primary tumors 50 solid metastasis

FFPE IHC (Cell

count)

HMGA2 mAb (GenWay Biotech Inc, San Diego, CA, USA)-Dilution: 1:500

Effusion: 188/199=94.5%

Primary tumors:

48/50=96%

Solid metastasis:

45/50=90%

No association seen between HMGA2 expression and PFS and OS in effusions (P=0.5 &

0.9, respectively), primary tumors (P=0.7 & 0.5, respectively) or metastatic (P=0.1

& 0.5, respectively) samples

Grade, FIGO, Residual disease, Chemoresponse

Califano 2014, ²⁰

Retrospective cohort (Italy)

117 117 patients with primary advanced ovarian cancer

Paraffin IHC (Cell count)

Anti-HMGA2 rabbit polyclonal antibody (BioCheck Inc, Foster City, CA, USA)-Dilution:

1:300

62/117=53.0% Free-disease Survival;:

HR, 95% CI:

HMGA2 only:

0.83 (0.38-1.82) HMGA2+BMI 3.17 (1.25-8.03)

Age, grade, FIGO stage, BMI, HMGA2 expression, BMI- HMGA2 score

Kim 2015,

21

Retrospective cohort (South Korea)

74 74 post-surgical ovarian carcinoma treated additionally with Paclitaxel and Cisplatin or Carboplatin

35- Liquid nitrogen 39- FFPE

IHC (Cell count)

Mouse anti-HMGA2 monoclonal antibody (Novus Biologicals, Littleton, CO, USA)- Dilution: 1:100

30/74=40.5% in cancer tissue samples

HMGA2(+) expression was significantly correlated with shorter OS 5-year OS rate:

78% vs. 35%

(P=0.02)

Age, FIGO stage, Lymph node involvement, Distant metastasis, Recurrence

RT-PCR (Ct

method)

TRIzol Reagents (Invitrogen, Carlsbad, CA) Wu 2015 ²² Cross-

sectional (US- China)

Training (n=278) Validatio n (n=150)

Training:

Serous: 80%, mucinous: 20%

Validation:

Serous: 72%

Mucinous: 28%

FFPE IHC (Cell

count)

Rabbit polyclonal anti- HMGA2 (Bio-Check, Inc.

Foster City, CA)- Dilution:

1:100

Training (P<0.001) Serous: 130/222=58.6%

Mucinous: 15/56=26.8%

Validation (P=0.001) Serous: 56/108=54.6%

Mucinous: 10/42=23.8%

Age, stage, grade, lymph node, distant metastasis, histologic type GASTRIC CANCER

Motoyama 2008, ²³

Cohort (Japan)

110 110 gastric carcinoma

Liquid nitrogen, Paraffin

IHC (Cell count)

Goat polyclonal antibody (R&D Systems)- Dilution:

10g/mL

83/110=75.4% Overall Survival:

HR=2.00 (95% CI: 1.32- 3.15)

Sex, tumor size, type, serosal invasion, lymph node metastasis, Lymphatic invasion, venous invasion, liver metastasis, peritoneal dissemination, stage RT-PCR (Ct

method)

Lightsycler faststart DNA master SYBR Green I kit (Roche diagnostics)

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Kong 2014,

24

Cohort (China)

212 158-cancer tissues, 30- peritumoral tissues, 24-normal, control gastric tissues

FFPE IHC (Cell

count)

Rabbit antibody (Cell Signaling Technologies, Danver, MA, USA);

Dilution: 1:100

68/118=43.0% Overall Survival Multivariate:

HMGA2-only:

HR=0.98 (0.34-2.33) HMGA2 & Oct4(+) HR=2.89 (1.02-5.14)

Age, sex, tumor size, type, stage, serosal invasion lymph node metastasis, vascular invasion, liver

metastasis,HMG A2+, Oct4+, HMGA2+ and Oct4+

RT-PCR (Ct

method)

PrimeScript RT reagents kit (TaKaRa, Dalian, China)

Lee 2015, ²⁵ Cohort (South Korea)

170 170 gastric cancer FFPE IHC (Cell count) & 76- RT-PCR

Rabbit polyclonal antibody (Abcom, Cambridge, MA, USA); Dilution: 1:100

39/170=22.9% 5-year OS rate:

54.2% vs. 43.6%

(P=0.028)

Age, sex, tumor size, type, Lauren classification, lymphatic invasion, vascular invasion, perineural invasion, depth of invasion, lymph node metastasis, stage

RT-PCR RNeasey mini kit (Qiagen) Jun 2015, ²⁶ Retrospective

cohort (South Korea)

169 110 gastric cancer 29 adenoma 30 non-cancerous gastric tissues

FFPE IHC (Cell

count)

Rabbit or mouse antibodies (DAKO ChemMate^TM system, Peroxidase/

DAB kit (DAKO, Glostrup, Denmark); Dilution: 1:100

72/110= 65.5%

(P<0.001)

Recurrence-free survival HR: 3.20 (95% CI: 1.50- 6.79)

Age, sex, histologic type, TNM stage, lymph node, vascular invasion, perineural invasion, recurrence COLORECTAL CANCER

Huang 2009,

27

Cross- sectional (China)

62 6-A colorectal; 2-S colon; 3-D colon;

20-rectum

RT-PCR (Ct

method)

47/62=76% Age, sex, Dukes

stage, tumor stage, node stage, metastasis, chemo-therapy, tumor sites Wang 2011,

28

Cohort (USA- China)

280 89-training set (USA)

191-validation set (China); 66/191 had adjuvant chemotherapy after surgery

FFPE IHC (Cell

count)

& validated by RT-PCR

Rabbit antibody (Alexis BioCheck Co.); Dilution:

1:1,000

Training set:

32/89=35.9&

Validation set:

70/191=36.6%

Overall Survival Stage I+II vs.

Stage III+IV Training set:

HR=2.38 (95% CI: 1.30- 4.34) Validation set:

HR=2.14 (95% CI: 1.21- 3.79)

Age, sex, diagnostic date, date of operation, type chemo., date chemo, type radio, date radio, TNM stage, relapse/metastasi s status, date relapse /metastasis, date last follow-up,

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vital status last follow-up Helmke

2012,

29

38 38 colon cancer tissue samples expressing HGMA2

FFPE IHC (Cell

count)

Biotechnology, Santa Cruz); Dilution: 1:1,200

19/38=50% Age, sex,

localization, TNM, grade

RT-PCR (Ct

method)

RNeasy FFPE kit (Qiagen, Hilden, Germany) Rizzi 2013,

30

Retrospective cohort/Italy

103 103 patients diagnosed with colorectal cancer

FFPE IHC (Cell

count)

Rabbit polyclonal antibody; Dilution: N/A

90/103=87.4% No difference of OS between HMGA2-negative vs. HMGA2- positive tumors (P=0.59)

Sex, age, TNM, Dukes, differential grading, therapy after surgery

LIVER CANCER Wu 2012,

31

Cohort/China 23 pairs=46 FFPE=1 07

23- Hepatocellular carcinoma

FFPE IHC (Cell

count)

Rabbit polyclonal antibody (Abcom, Cambridge, MA, USA); Dilution: 1:500

51/107=47.7% Overall Survival HR=1.97 (95% CI: 1.17- 3.33)

Sex, age, HBsAg status, cirrhosis, AFP, tumor number, tumor size, capsule invasion, vascular invasion, Edmondson grading RT-PCR (Ct

method)

HGMA2 mRNA expression was higher in tumor cells than normal tissue (38.70 vs 8.40, p

<0.01) Lee 2013, ³² Cross-

sectional study (USA- China)

86 15-fibrolamella hepatocellular carcinoma 15-

hepatoblastomas 34- hepatocellular carcinoma 22- hepatic adenomas

FFPE IHC (Cell

count)

HMGA2 Antibody (Biocheck, Foster City, CA, USA); Dilution:

1:1,000

15/15=100% Sex, age,

etiology, AFP, tomor size, cirrhosis, vascular invasion

Lee 2014 ³³ Retrospective (USA)

68 FFPE IHC (Cell

count)

DAKO system (DAKO, Denmark); Dilution:

1:1,000

18/55=33% Overall survival HR: 2.28 (95% CI: 1.08- 4.81)

Age, sex, tumor size,

desmoplasia, satellite, grade, stage, vascular invasion, Perineural invasion, lymph node, etc.

BREAST CANCER

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Rogalla 1998, ³⁴

57 Breast cancer: 44 Non-malignant adjacent tissue: 13

RT-PCR TRIzol reagent (Gibco BRL, Eggenstein, Germany)

20/44=45.45% Histologic grade

Jones 2008,

35

Cross- sectional study (UK)

23 12- benign phylloides tumors 11- borderline.

malignant phylloides tumors

RT-PCR (Ct

method)

GenElute mammalian total RNA kit (Sigma)

Test set:

Microarray data: 4 fold change

RT-PCR: 6 fold change

N/A

LUNG CANCER Sarhadi 2006,

36

Cohort (Finland)

152 152 mainly small cell lung carcinoma

FFPE IHC (Cell

count)

Biotechnology, Santa Cruz, CA, USA); Dilution:

1:20

Overall: 130/144=90%

Squamous Cell Carcinoma:

60/62=96.8%

HGMA2 expression in AC was significantly associated with poor survival (P=0.05)

Tumor type, Age, sex, stage, 5-years survival status, smoking, asbestos exposure RT-PCR (Ct

method)

Roche Diagnostics GmbH, Germany

High expression of HMGA2 were detected compared to normal lung tissue

Meyer 2007,

37

Cross- sectional/Ger many

68 (34 pairs)

17- AC 17- SCC

HOPE-FFPE IHC (Cell count)

Biotechnology, Santa Cruz, CA, USA); Dilution:

1:50

10-50%: AC 80%: SCC

Tumor type, sex, age, grade, TNM, stage

RT-PCR (Ct

method)

RNeasy FFPE kit (Qiagen, Hilden, Germany)

AC: Mean: 158.41 fold (Range: 1.02-911.02) SCC: Mean: 336.26 fold (Range: 4.34-2,503.68) ORAL CANCER

Miyazawa 2004, ³⁸

Retrospective cohort/USA- Japan

42 42 primary oral squamous cell carcinomas

FFPE IHC (Cell

count)

Rabbit anti-HMGA2;

Dilution: 20 g/ml

31/42= 73.8% Disease-Free Survival:

HR: 3.48 (95% CI: 1.40- 8.69)

Age, sex, TNM, clinical stage, histologic differentiation RT-PCR (Ct

method)

SuperScript II (Life Technologies Inc)

Expression level:

163.4±90.4 (P<0.05) Chang 2015,

39

Cohort (Taiwan)

215 215 oral squamous cell carcinoma

FFPE IHC (Cell

count)

Rabbit monoclonal antibody (Cell Signaling, Danvers, MA, USA);

Dilution: 1:30

HMGA2 was significantly elevated in cases compared to adjacent tissue (p<0.001).

5-year OS rate:

75.6% vs. 57.7%

(P=0.007) 5-year DSS rate:

78.1% vs. 59.1%

(P=0.006) 5-year DFS rate:

72.7% vs. 53.1%

(P=0.002)

Sex, age, tumor status, lymph node metastasis, cell

differentiation, perineural invasion, tumor depth RT-PCR (Ct

method)

RNAzol B (Tel-Test, Friendswood, TX, USA)

48±75 vs. 1± 1.5 copy/

10⁵ GAPDH copy, P <

0.001 NASOPHARYNGEAL CANCER

Liu 2015, ⁴⁰ Cohort (China)

145 116-

nasopharyngeal carcinoma

FFPE IHC (Cell

count)

Rabbit antibody (Abcam, MA, USA); Dilution: 1:100

62/116=52.6% Overall Survival HR: 1.72 (95% CI: 1.02- 2.91)

Age, gender, stage, tumor size, lymph node, distant

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29 – non- cancerous NP tissues

metastasis, pathologic classification Xia 2015, ⁴¹ Retrospective

cohort (China)

144 124 –

nasopharyngeal carcinoma 20- non-tumoral inflammatory nasopharynx

FFPE IHC (Cell

count)

Rabbit polyclonal antibody (Abcam ltd, Cambridge, UK); Dilution: 1:60

54/124=43.55%(p<0.001 )

Overall Survival HR=2.68 (95% CI: 1.18- 6.08)

Sex, age, histological type, T-stage, N-stage, TNM stage, metastasis PANCREATIC CANCER

Piscuoglio 2012, ⁴²

Retrospective cohort (Switzerland)

210 PAD 40 PanIN-3 40 Normal control

210 pancreatic ductal

adenocarcinoma

FFPE IHC (Cell

count)

HGMA2-goat antibody ((Dako Cytomation, Carpinteria, CA, USA);

Dilution: 1:250

197/210 = 93.8% (PAD) 37/40 = 92.5% (PanIN-3)

Overall survival was not significantly different between HMGA2 positive cases and control Median Survival Time (months) Positive: 15 (95%

CI: 10-25) Negative: 14 (12- 18)

(P=0.20)

Sex, tumor grade, TNM, tumor diameter, survival time

MELANOMA Raskin 2013,

43

Retrospective cohort (USA)

127

330- validatio n

67- primary melanoma 20- metastatic melanoma 40 – normal skin tissue

FFPE IHC (Cell

count)

Goat polyclonal antibodies (Santa Cruz, CA, USA);

Dilution: 1:200

Primary

melanoma:26/46=57%

Melanoma metastasis:

10/12=

Training set:

Overall survival HR=6.30 (95% CI: 1.80- 22.30) Distant metastasis-free survival HR=6.40 (95% CI: 1.4-29.7) Validation set:

Overall survival HR=1.72 (95% CI: 1.09- 2.73)

Age, Breslow thickness, site, melanoma type, AJCC stage, ulceration, mitotic rate,

RT-PCR (Ct

method)

TRIzol reagent (Invitrogen, Carlsbad, CA, USA) BLADDER CANCER

Yang 2011,

44

Retrospective cohort (USA)

148 148 – urothelial bladder cancer

FFPE IHC (Cell

count)

Rabbit polyclonal antibody (R&D System, USA);

Dilution: 1:500

77/148=52% Recurrence-free

survival HR=3.83 (95% CI: 2.19- 6.71) Progression- free

survival HR=3.47 (95% CI: 1.43- 8.45)

Sex, age, tumor size, tumor number, grade, T- stage

RT-PCR (Ct

method)

RNAzol B (Tel-Test, Friendswood, TX, USA)

HGMA2 expression levels: 121 ± 31.13

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(cancer) vs. 1.74 ± 0.42 (normal) p<0.001) BILE DUCT CARCINOMA

Zakharov 2013, ⁴⁵

48 22

adenocarcinoma 12 adenoma 14 reactive atypia

FFPE IHC (Cell

count)

Polyclonal rabbit anti- human antibody (Biocheck,

Forst City, CA, USA);

Dilution: 1:200

41/48=86% Age, sex,

presentation, location, sub-type

GALLBLADDER CANCER Zou 2015 ⁴⁶ Retrospective

cohort (China)

204 108

adenocarcinoma 45: adjacent tissue 15: polyp 35: chronic cholecystitis

FFPE IHC (Cell

count)

Mouse anti-human HMGA2 monoclonal antibodies, Abcam Ltd., (Cambridge, UK); Dilution:

0.01mol/L

Adenocarcinoma:

64/108=59.3%

(P<0.01)

Overall survival:

HR, 95% CI:

3.02 (1.58-5.78)

Age, gender, pathologic type, differentiation, tumor size, lymph node, invasion to surrounding tissues, gallbladder stones GLIOMA

Liu 2014, ⁴⁷ Retrospective cohort (China- Japan)

85 78 - gliomas FFPE IHC (Cell

count)

Rabbit polyclonal antibody (Santa Cruz Biotecnology, Dallas, TX, USA); Dilution:

1:100

67.9% (53/78)

RT-PCR (Ct

method)

TRIzol Reagents (Takara Bio, Shiga, Japan) OESOPHAGEAL CANCER

Liu 2014, ⁴⁸ Cross- sectional study (China)

226 113 oesophageal squamous cell carcinomas

FFPE IHC (Cell

count)

Rabbit polyclonal antibody (Santa Cruz

Biotechnology Inc, Santa Cruz, CA, USA); Dilution:

N/A

98/113=86.7% Sex, age,

ethnicity, gross type,

differentiation, lymph node metastasis, TNM stage

RT-PCR (Ct

method)

TRIzol Reagents (Invitrogen)

AC: Adenocarcinoma; AFP: serum alpha-fetoprotein; AJCC: American Joint Committee on Cancer; AUC: area-under-curve; ATC: anaplastic thyroid carcinoma;

BMI: Body mass index; CCOC: Clear cell ovarian carcinoma; chemo: chemotherapy; EOC: Endometroid ovarian carcinoma; FA-follicular adenoma;

FFPE: formalin-fixed paraffin-embedded; FIGO: International Federation of Gynecology & Obstetrics; FNA: fine needle aspiration; FTC: follicular carcinoma;

FVPTC: follicular variant of papillary thyroid carcinomas; GBM – Glioblastoma multiforme; HA- Hürthle cell adenoma; HC-Hürthle cell carcinoma; HMGA2: High mobility group A2 protein HG-PSC: High-grade papillary serous carcinoma; IHC: Immunohistochemistry; MMMT: Malignant miixed Mullerian tumor; MOC: Mucinous ovarian carcinoma; PTC: papillary thyroid carcinomas; radio: radiotherapy; RT-PCR: Real time polymerase chain reaction; SBT: Serous borderline tumor; SCC: Squamous cell carcinoma; Sen: sensitivity; Spe: specificity