European Journal of Cardiovascular Medicine

Growth Factor

As we know Growth factors are cytokines acting through specific cell-surface receptors, which results in normal homeostasis by bringing intricately balanced interactions between cells and the network of secreted proteins ¹.

Growth factor activity is receptor mediated action which influence the expression of genes which can either promote entry of cells into the cell cycle, relieve blocks on cell cycle progression, prevent apoptosis or enhance biosynthesis of cellular components (nucleic acids, proteins, lipids, carbohydrates) required for a mother cell to give rise to two daughter cells.

Uncontrolled proliferation can result when the growth factor activity is dysregulated, or when its signaling pathways are altered to become constitutively active. Thus, many growth factor pathway genes are proto-oncogenes; gain-of-function mutations in these genes can convert them into oncogenes capable of driving unfettered cell proliferation and tumor formation².

Hepatocyte Growth Factor

Hepatocyte growth factor is one of the growth factors which have many functions. Hepatocyte growth factor (HGF) was first identified in 1984 and 1985 and purified as a potent mitogen of primary cultured hepatocytes.

Structural characteristics of HGF

Molecular cloning revealed that it is a heterodimeric molecule composed of a 69-kDa alpha-chain and a 34-kDa beta-chain. The alpha-chain contains an N-terminal hairpin domain and subsequent four-kringle domains, and the beta-chain contains a serine protease- like domain with no enzymatic activity.

HGF is synthesized and secreted as a biologically inactive single- chain precursor form; further processing by serine proteases into the two-chain form is coupled to its activation. Serine proteases responsible for the activation of HGF include HGF activator or HGF-converting enzyme and urokinase-type plasminogen activator (uPA).³

The receptor for HGF was identified as a c-Met proto-oncogene product. The c-Met receptor is composed of a 50-kDa a-chain and 145-kDa  b-chain. The a- chain is exposed extracellularly, while the b-chain is a transmembrane sub-unit containing an intracellular tyrosine kinase domain⁴.

Figure 1: Structure of HGF⁴ Mechanism of Action of HGF

Binding of HGF to the c-Met receptor induces activation of tyrosine kinase, an event that results in subsequent phosphorylation of C- terminally clustered tyrosine residues. Now with considerable evidence it is known that intracellular signalling pathways driven by HGF-c-Met receptor coupling lead to multiple biological responses. These include motogenic (enhancement of cell motility), morphogenic, neurite extension and anti-apoptotic activities.⁵

Figure 2: Schematic Representation and Mechanism of Action Of HGF And Its Pathways⁶.

In general, the c-Met oncogene is expressed in epithelial cells, whereas the ligand is expressed in the surrounding mesenchyme, providing a mechanism for the epithelial mesenchymal inductive processes during development. ⁷

Role of HGF in Morphogenesis

HGF was identified as the fibroblast- derived growth factor that was both necessary and sufficient to stimulate epithelial cells derived from a variety of different organs to form tubule-like extensions (so-called 'branching morphogenesis')⁸. In one in-vitro assay, HGF stimulates some cells in the cell clusters to rearrange their cell polarity and extend processes.⁹ For example in the mammary gland, elevated expression of endogenous c-Met and HGF correlates with stages of active tubulogenesis, expression being high through early pregnancy but virtually absent during late pregnancy and lactation when alveologenesis and gland differentiation take place.¹⁰

Role of HGF in Angiogenesis

HGF has angiogenic action and acts directly on endothelial cells, inducing proliferation, migration and on tumor cells, in which it triggers angiogenic switching by up-regulating the expression of the proangiogenic factor VEGF and down-regulating the expression of TSP-1, an angiogenesis inhibitor. Inhibitors of the HGF-cMet pathway would therefore have the potential to both block VEGF expression and boost TSP-1 levels while simultaneously interfering with invasion and metastasis.¹¹

Anti-apoptotic role of HGF.

HGF prohibits apoptotic signals via inhibition of caspase-3 activity or induction of anti-apoptotic molecules, such as Bcl-xL.¹² HGF also prohibits Fas-mediated apoptosis signals via sequestration of Fas and c-Met on cell surfaces.¹³

HGF and cMet regulate both morphogenic and tumorigenic phenotypes:

HGF is a potent inducer of EMT(epithelial mesenchymal transition) in many epithelial systems, like other growth factors eg., FGF and EGF.

Over-expression of HGF and Met occurs in many types of invasive cancers, including breast carcinomas. A shift from transient activation of Met to sustained high levels of c-Met activation and co-operativity with other RTKs can cause a switch in the HGF response from morphogenesis to tumorigenesis.¹⁴

Role of HGF in Breast Carcinoma:

HGF is synthesized in the mammary stroma, probably by fibroblasts, and acts on receptor-expressing ductal epithelial cells. This concept strongly suggests that HGF/SF c-Met signalling is a classical epithelial-mesenchymal inductive pathway that is important for ductal morphogenesis in the mammary gland. HGF may co-operate in the regulation of the migration of epithelial cells across the fatty stroma by altering integrin-matrix signaling locally. High levels of HGF and Met expression in breast carcinoma cells is a possible independent predictor of recurrence and shortened survival in breast cancer patients.¹⁵

In normal breast epithelium and ductal carcinoma in situ (DCIS), strong expression of E-cadherin with accentuation at cell-cell contacts is evident. In contrast, decreased expression of E-cadherin frequently occurs in invasive ductal carcinoma (IDC), while c-Met expression is relatively consistent throughout, and most intense in IDC. Sustained activation of HGF-cMet signalling is associated with dissociation of cadherin-based adherens junctions, followed by loss of cadherin expression. The modulation of adherens junctions by HGF-cMet involves phosphorylation of b- catenin, leading to its reduced affinity for the E-cadherin complex and subsequent degradation. HGF disrupts adherens junctions and promotes cell dispersal, so stimulating invasive capacity.^{16, 17}

HGF and Its Signalling Complex As Therapeutic Targets

Targeting HGF, HGF receptor, and signalling events has been an attractive option for cancer therapy. Therapeutic approaches have been attempted by developing tools against: HGF (neutralizing antibodies, antisense oligonucleotides, ribozyme, short interfering RNA (siRNA), and HGF regulators including HAls), cMet (HGF antagonists, antibodies, small molecules, antisense, ribozymes, siRNA, and non- specific inhibitors), cMet signalling events (coincident with anti-cMet methods), and HGF activation inhibitors. These therapeutic approaches are largely in the development phase, with a small number-mostly non-specific inhibitors to cMet- now in early clinical study.¹⁸

Methodology Source of Data

The present prospective study was undertaken in the Department of Pathology, JSS Medical College and Hospital, Mysuru.

Type of Study Descriptive study

Inclusion criteria

Histopathologically confirmed invasive duct carcinoma of breast were included in the study.

Exclusion criteria:

Breast carcinoma other than invasive duct carcinoma, diagnosed on core biopsy and patients having other known malignancy were excluded from the study.

Method of collection of Data

• Clinical details were collected from the patients through structured questionnaire.
• Venous blood samples were collected preoperatively; serum was separated and stored at temperature-80°c from patients with breast carcinoma undergoing surgery.
• Serum samples from normal women and benign breast tumor patients with age matched volunteers were used as control samples.
• Serum HGF was estimated using HGF ELISA kit (Thermofischer, Sandwich enzyme method)
• Gross and microscopic features of the mastectomy specimen were studied.

PROCEDURE:

Serum HGF assay by ELISA

The estimation of serum HGF was done by using HGF ELISA kit (Thermofischer, Co, Ltd.) using sandwich ELISA method with assay sensitivity of<10pg/ml. To Anti-HGF antibody precoated microplates wells, serum samples of the cases, controls and standards were pipetted. During the first incubation, the protein antigen bounds to the capture antibody. After washing, a detection antibody was added to the wells, and this antibody bounds to the immobilized protein captured during the first incubation. After removal of excess detection antibody, streptavidin peroxidase (HRP) conjugate was added which bounds to the detection antibody. After a third incubation and washing, to remove the excess HRP conjugate, a substrate solution was added and was converted by the enzyme to a detectable color signal. The intensity of this colored product was directly proportional to the concentration of antigen present in the original specimen. Values were read at 450nm OD. Standard curve is obtained by using a software from which the concentrations of the samples and standards are obtained.

Histopathological Examination:

Mastectomy specimens were fixed in 10% neutral buffered formalin within one hour of resection. They were examined grossly according to standard guidelines and noting all the important parameters, specimens were fixed for 24-48 hours. Sections were taken from representative sites, processed in automated tissue processor followed by paraffin embedding and staining with Haematoxylin & Eosin.

Sections were studied to establish final histopathological diagnosis.

Statistical analysis

The results were statistically analysed using SPSS version 22.

Statistical Methods

1 Descriptive statistics

2. Chi-square tests
3. Contingency table analysis using SPSS for Windows
4. P values <0.05 were considered statistically significant

The present study was undertaken from September 2016 to July 2018 in the Department of Pathology, J.S.S. Medical College and Hospital, Mysuru. A total number of 35 invasive ductal breast carcinoma as cases; 9 benign breast disease and 8 normal samples as controls were evaluated.

AGE AT THE ONSET OF DISEASE:                                                                                     

Total number of cases studied were 35, age range varied from 33-65years with a mean age of 45±15years and maximum number of cases were in the age group of 50-60 years accounting for 34.2% . (Graph 1)

Graph 1

Out of 35 cases, majority of cases were from rural area (68.57%) , rest being distributed in urban area(31.43%) (Graph 2)

Graph 2

RELATION WITH MENOPAUSE:

Out of 35 patients, 31 (88.57%) were in premenopausal period and the remaining 04(11.43%) in postmenopausal period. (Graph 3)

Graph 3

HISTORY OF BREAST FEEDING

All 35 cases were given positive history of breast feeding

LATERALITY OF BREAST CARCINOMA

Out of 35 cases, 25(71.42%) had tumour on left side and 10 (28.57%) had tumour on right side.(Graph 4)

Graph 4

ASSOCIATED MORPHOLOGICAL PARAMETERS:

 Lymphovascular invasion was observed in 62.8% of cases while necrosis desmoplasia , in-situ component, and perineural invasion were seen in 31.4 %,77.1%, 20% and 8.5% of cases respectively.(Graph 5) (Figure No 6, 7,8)

Graph 5

MODIFIED BLOOM RICHARDSONS SCORING

35 mastectomy specimens were graded based on Nottingham modification of the Scarff Bloom Richardson grading system.

Tubule formation:

A score of 3 was given for 77.14 %cases, score 1, 2 was given for 5.7% and 17.1% of cases respectively. (Graph 6)

Graph 6

Nuclear Pleomorphism:

Out of 35 cases, 21(60%) cases were given a score of 3(Marked pleomorphism) and 14(40%) cases with score 2(Moderate pleomorphism)

Graph 7

Mitotic activity:

Mitotic count of 6-9/HPF was present in 22 cases(62.8%), 0-5/HPF in 4 cases(11.4%) and >10/HPF in 9 cases(25.7%).

Graoh 8

HISTOPATHOLOGICAL GRADE OF THE TUMOUR:  

 Out of 35 cases 21(60%)were of grade 3 and rest 14(40%) were of grade 2.(Graph 9)(Figure No 6,7)

Graph 9

TNM STAGING OF THE TUMOUR:

Staging of the tumours was done using AJCC system of classification based on tumour (T), node (N) and Metastasis (M). Majority of cases (27)were of pT2 stage tumours being more than 2cm but less than 5cm in their greatest dimension, 5 cases belong to pT3 stage(tumor more than 5cm in greatest dimension), 2 cases belong to pT4 stage(tumor of any size with direct extension to skin/ chest wall) and one case pT1 stage(tumor less than 2cm in greatest dimension). (Graph 10 A)

Graph 10 A

pN stage of Tumour

Out of 35 cases, 8(22.85%) cases show no lymphnode metastasis(N0). 19(54.28%) cases showed lymphnode metastasis in 1-3 lymphnodes (N1), 5 (14.28%)cases showed lymphnode metastasis in 4-9 lymphnodes ( N2 )and 3 (8.57%)cases showed metastasis in>10 axillary lymphnodes(N3).(Figure-7)

Graph 10 B

SERUM HGF VALUES:

Serum HGF values in Invasive duct carcinoma was in the range of 936-2731pg/ml with a mean of 1386pg/ml.and other values as depicted in the Table No 1. The p value is <0.0001 which shows statistical significance in the present study.(Table No 1)

Table 1: SERUM HGF VALUES

Graph 11

ASSOCIATION OF SERUM HGF VALUES WITH KNOWN PROGNOSTIC FACTORS OF BREAST CARCINOMA

With age groups as shown in Table No.2 and showed no statistical significance (p –value-0.265).(Table 2)

Table 2: Association of different age groups with serum HGF

Menstrual status -Out of 35 patients, 31 (88.57%) were in premenopausal period and the remaining 04(11.43%) in postmenopausal period. No statistical significance seen when correlated with serum HGF levels(p-value-0.850).

Desmoplasia – It was present in 27(77.1%) cases with mean serum HGF value of 1859pg/ml and 8(22.8%) cases were devoid of desmoplasia with mean serum HGF of 1246pg/ml. There was a statistically significant correlation between desmoplasia and serum HGF levels. (p-value-0.004)(Table No 3)

Table 3: Association of Desmoplasia with serum HGF

Lymphovascular invasion- It was seen in 22(62.8%) cases with mean serum HGF level of 1370pg/ml and was absent in 13(37.1) cases with mean serum HGF level of 1412pg/ml. Serum HGF values when compared show no statistical significance(p-value-0.829)(Table No 4)

Table 4: Association of Lymphovascular invasion with serum HGF levels

Perineural invasion(PNI) – It was absent in maximum number of cases(91.4%) with a mean serum HGF of 1394pg/ml and only 3 cases showed PNI with a mean serum HGF value of 1302pg/ml. Serum HGF values when compared show no statistical significance ( p-value-0.787)(Table No 5)

Table 5: Association of Perineural invasion with serum HGF values

Modified Bloom Richardson score (MBR)

Tubule formation with serum HGF

Maximum cases(77.14%) were given a score of 3 with mean serum HGF level of 2015pg/ml and distribution of other cases are as shown in table no.7. Serum HGF values when compared among different groups show statistical significance (p-value -0.004).(Table No 6)

Table 6: Association of Tubule formation with serum HGF

 Nuclear pleomorphism with serum HGF

60% cases were given a score of 3 with mean serum HGF level of 1375pg/ml and 40% cases were given a score of 2 with mean serum HGF level of 1402pg/ml. Serum HGF values when compared among different groups show no statistical significant difference (p –value-0.887).(Table No 7)

Table 7: Association of Nuclear pleomorphism with serum HGF

Mitotic activity with serum HGF

Out of 35 cases, 22 cases were given a score of 2 with a mean serum HGF value of 1305pg/ml, 9 were cases given a score of 2(Mean serum HGF=1574) and 4 cases with score 1 and mean serum HGF of 1409pg/ml. Serum HGF values when compared among different groups show no statistical significant difference (p –value-0.476)(Table No 8)

Table 8: Association of Mitotic activity with serum HGF

Histologic grade with serum HGF

57.1% of the cases were of grade 3 with mean serum HGF level of 1375pg/ml and 42.8% were of grade 2 (Mean serum HGF=1402pg/ml). Serum HGF values compared among different grade show no statistical significance( p-value-0.887) (Table No 09)

Table 9: Association of Histologic grade with serum HGF

Tumor size of pTNM stage with serum HGF

Out of 35 cases maximum number of cases(77.14%) were of pT2 with a mean serum HGF of 1417pg/ml and other case distribution are as shown in table no 10.Serum HGF values compared among different tumor size show no statistical significant difference ( p-value-0.746) (Table No 10)

Table 10: Association of Tumor size of pTNM stage with serum HGF

Lymphnode( pN)metastasis with serum HGF

Majority of the cases(54.28%) showed lymphnode metastasis in 1-3 lymphnodes(N1). Distribution of other cases are as shown in Table No 11. Serum HGF values on correlation show no statistical significance(p-value-0.816)(Table No 11)

Table 11: Association of lymphnode( pN)metastasis with serum HGF

Figure 3: (Invasive Breast Carcinoma of No Special Type, Grade I): Tumour cells arranged in tubules, showing mild nuclear pleomorphism. (H&E, x100)

Figure 4: (Invasive Breast Carcinoma of No Special Type, Grade II): Tumour cells arranged in clusters cells, showing nuclei with moderate pleomorphism. (H&E, x100)

Figure 5: (Invasive Breast Carcinoma of No Special Type, Grade III): Tumour cells arranged in sheets, showing marked nuclear pleomorphism and frequent mitosis. (H&E, x100)

Figure 6: Desmoplasia – Invasive breast carcinoma-NST with areas of desmoplasia. (H&E, x40)

Figure 7: (Lymph node involvement): Tumour cells involving the axillary lymph node. (H&E, x 40)

Figure 8: (Lymphovascular invasion): Tumour cells are seen invading a blood vessel. (H&E, x100)

Figure 9 : Ductal Carcinoma In-Situ (DCIS) (H&E, x10)

Introduction

Breast cancer is the second most common cancer among in the world. In order to reduce the morbidity and mortality associated with breast cancer, there is a need to search for a biomarker to diagnose at early stage and to predict its prognosis.¹⁹ HGF known to be identical to scatter factor plays an important role in various stages of cancer progression including cell proliferation, movement, invasiveness, morphogenesis, and angiogenesis.²⁰Estimation of serum HGF in many other solid carcinomas has indicated to be a good prognostic marker. This study was conducted to estimate the serum levels of HGF in invasive ductal carcinoma of breast to know its significance.

AGE DISTRIBUTION:

In the present study, the age range of the patients with invasive breast carcinoma varied from 33years to 65 years with a mean age of 45.63±15 years. Mean age of other studies were variable and the results of present study was comparable with H A Attar et al.²¹ as shown in the Table No 14.

Table 14: Comparison Of Age Distribution With Other Studies

RELATION WITH MENOPAUSE:

Out of 35 patients, 31 (88.57%) were in premenopausal period and the remaining 04(11.43%) in postmenopausal period. This was comparable with the study done by H H Ahmed et al.²⁴, where maximum cases(72.73%) were in premenopausal period.

HISTOPATHOLOGICAL GRADE       

Grading was done according to Nottingham modification of the Scarff Bloom Richardson grading system. In present study out of 35 cases, 15 cases were of grade 2 (moderately differentiated)and 20 cases were of grade 3(poorly differentiated).Histological grade and its comparison with other studies as shown in Table No 15 .

Table 15: Comparison of histopathological grade with other studies

ASSOCIATION OF AGE WITH SERUM HGF AND COMPARISON WITH OTHER STUDIES:

In the present study the serum HGF values when compared among different age groups show no statistical significance (p-value-0.265) which was similar to H A Attar et al. ²¹(p-value-0.186) and SMS chen et al. 58(p-value-0.545) findings. The mechanisms by which serum HGF is released in different age groups are unclear but found to be regulated by many other factors like IL-1β, prostaglandin E2, heparin, bFGF, EGF and PDGF ²⁵

Serum HGF levels:

There was a significant increase in preoperative serum HGF levels of invasive ductal carcinoma patients as compared to benign breast disease and normal women. Serum HGF levels obtained among carcinoma patients was in the range of 936-2731pg/ml, mean value being 1386pg/ml as compared to benign breast diseases with a range of 726-1556pg/ml, mean value =944pg/ml and normal samples ranged 160-481pg/ml, mean value=337g/ml. The p value is <0.001 which shows statistical significance in the present study. These values were comparable with the following studies-

Table 16: Comparison of serum HGF values with other studies

HGF is thought to be a stromally derived paracrine growth factor in breast cancer, because human cultured breast cancer cells express the HGF receptor, c-Met, but not to produce HGF by themselves ²⁶.The increase in HGF could be explained by overstimulation of the cells that secrete HGF through autosecretion or mutation and aberrant regulation of the HGF-cMet signalling pathway or an imbalance between HGF activators and inhibitors²⁷ .In Study done by Tanaguchi et al., it was found that serum HGF levels was due to the presence of tumor and removal of the primary tumor clearly decreased the serum HGF level in primary breast cancer patients²². Several growth factors or cytokines including tumor necrosis factor alpha, interleukin 1 , and transforming growth factor 3 are known to be responsible for the production of HGF in stromal cells ²⁸This result was consistent with SMS Chen et al.²⁰and H H Ahmed et al.²⁴who detected that preoperative serum HGF levels were significantly elevated in the patients compared to those in control (p-value-0.026). Thus, the preoperative level of serum HGF may reflect the severity of invasive breast cancer and may be useful to pick up the higher risk patients for more aggressive treatment.

In present study LVI was seen in 22 cases (62.8%) with a mean serum HGF of 1370pg/ml and 1412pg/ml in cases with absent LVI without statistical significance (p-value=0.829). This conflicting result speaks to the complex nature of HGF and cMet interactions. The tumor microenvironment promotes the production of HGF variants that could disrupt or modify HGF/cMet function during tumor progression ²⁹.In present study PNI was absent in majority of cases (91.4%) with a mean serum HGF of 1394pg/ml and rest of the cases in which PNI was present had a mean serum HGF of 1302pg/ml. There was inverse relationship in serum HGF levels and showed no statistical significance (p-value-0.787). The possible existence of decoy cMet composed of the extracellurar domain of cMet released from cells has also been suggested³⁰; a soluble cMet was reported in multiple myeloma patients ³¹ As various fragmented forms of HGF and souble cMet exists in the sera of cancer patients, the identity of the HGF measured is probably different among ELISA and the correlation between serum HGF level and clinical features could be variable. ²⁹In present study desmoplasia was present in 77.1% cases with mean serum HGF value of 1859pg/ml and was absent in 22.8% cases with mean serum HGF of 1246pg/ml which was statistically significant (p-value-0.004). Fibroblasts and myofibroblasts are found abundant in the tumor stroma and secrete several tumor promoting chemokines and growth factors. HGF is a major component of the cancer-associated fibroblasts has been shown to promote tumorogenesis ³². In addition, fibroblasts (or recombinant HGF) promote survival of cancer cells and represent an important source of primary and acquired resistance to targeted therapy, including inhibitors of EGFR³³

In present study majority of the malignant cases (77.14%) had a score of 3 for tubule formation with a mean serum HGF level of 2015pg/ml. Serum HGF values when compared among different groups show statistical significance (p-value -0.004). In tumors, HGF disrupts adherens junctions and promotes cell scattering, dissociation and disruption of E-cadherin mediated breast cancer cell adhesion.³⁴ Following HGF stimulation, the CrkII and CrkL adapter proteins are recruited to Met-dependent signalling complexes. This appears to be the key to the breakdown of adherens junctions, the spreading of epithelial colonies and the formation of lamellipodia in response to HGF.³⁵

In present study 21 cases were given score of 3 with a mean serum HGF level of 1375pg/ml and 14 cases were of score 2 with a mean serum HGF level of 1402 pg/ml. Serum HGF values which were showing inverse relationship, when compared among different groups show no statistical significance (p-value-0.887).

In present study 22 cases had a score of 2 with a mean serum HGF of 1305 pg/ml. Serum HGF values when compared among different groups show no statistical significant difference (p –value-0.476).

It has been reported that patients with clinical infections theoretically cause an altered concentration of serum cytokine and may result in this discrepancy between serum HGF level and nuclear pleomorphism and mitotic activity^{. 20}

Histological grade when correlated with serum HGF levels, tumors of grade 2(43%) had a higher mean of 1402 pg/ml while grade 3 (57%) tumors had lower mean of 1375pg/ml and showed no statistical significance (p-value-0.887). This finding was similar to the study done by H A Attar et al.²¹ (p value-0.472) but in contrary SMS Chen et al.²⁰ had significant association. Differentiation of breast carcinoma cells represents a balance between a variety of physiologic and pathologically-relevant processes of serum HGF secretion and undergo an evolutionary adaptation to their microenvironment. cMet is a tyrosine kinase receptor that when activated by its ligand-HGF autophosphrylates and initiates an intracellular signalling cascade that involves many targets.

Majority of the cases (77.14%) were of pT2 stage with a mean serum HGF of 1417pg/ml while rest of the cases had mean value in the range of 966-1425 pg/ml. There was no statistical significance when tumor stage was compared to serum HGF levels (p-value-0.746). This was comparable with the study done by H A Attar et al. ²¹and Tanaguchi et al.²² but not with H H Ahmed et al.²⁴ Previous study has reported that irrespective of the tumor stage, elevated HGF levels were associated with disease recurrence and poorer survival rates enhancing the importance of estimating preoperative serum HGF levels in breast carcinoma. ²⁵

In present study axillary lymph nodes metastasis was found in 77.1% of cases with mean serum HGF level of 1403pg/ml and lymphnode metastasis was absent in 22.8% of cases with mean of 1357pg/ml. There was no statistical significant association between lymph node metastasis and serum HGF levels (p-value- 0.816). This finding was similar to studies conducted by other researchers.^21,24. It is important to estimate preoperative levels of serum HGF as H. Funakoshi et al.²⁵ had noted that node-negative patients with an elevated HGF had significantly poorer outcome than did node-positive patients with a low HGF. However, patients in more advanced TNM staging were shown to have higher serum HGF²⁰

HGF is a heterodimeric molecule composed of a 69-kDa α-chain and a 34-kDa β-chain which is synthesized and secreted as a biologically inactive precursor form and activated by serine proteases. The receptor for HGF was identified as a c-Met proto-oncogene. HGF known to be identical to scatter factor plays an important role in various stages of cancer progression including cell proliferation, movement, invasiveness, morphogenesis, and angiogenesis. The present study was a descriptive type of study conducted at JSS hospital, Mysuru. Preoperative serum HGF estimation was done in 35 cases of invasive ductal carcinoma using sandwich ELISA assay which were compared with benign breast disease and normal samples as controls. The study showed a significant increase in serum HGF levels in invasive ductal carcinoma as compared to benign breast disease and normal samples and there was statistical significance. This was comparable with various previous studies . The increase in HGF could be explained by overstimulation of the cells that secrete HGF through autosecretion or mutation and aberrant regulation of the HGF/cMet signalling pathway or an imbalance between HGF activators and inhibitors. Serum HGF levels were correlated with known prognostic factors. Statistical significant association was seen with factors like desmoplasia and tubule formation. Association of desmoplasia with increase in HGF levels may also be due to secretion of HGF by stromal components like fibroblasts and myofibroblasts that are found abundant in the tumor stroma and has been shown to promote tumorogenesis. In tumors, HGF disrupts adherens junctions and promotes cell scattering, dissociation and disruption of E-cadherin mediated breast cancer cell adhesion. Following HGF stimulation, the CrkII and CrkL adapter proteins are recruited to cMet-dependent signalling complexes. This appears to be the key to the breakdown of adherens junctions, the spreading of epithelial colonies and the formation of lamellipodia in response to HGF.

No statistical significance seen with respect to parameters like LVI, PNI, tumor size, lymphnode status which explains the complex interaction of HGF and cMet pathway. Some of the previous studies have shown association with the above parameters which may be due to that the tumor microenvironment promotes the production of HGF variants and could disrupt or modify HGF/cMet function during tumor progression. As various fragmented forms of HGF and soluble cMet exists in the sera of cancer patients, the identity of the HGF measured is probably different among ELISA and the correlation between serum HGF level and clinical features could be variable.

Thus the preoperative level of serum HGF has reflected the severity of invasive breast cancer in our study and is useful to pick up the higher risk patients for more aggressive treatment

1. Blobe GC, Schiemann WP, Lodish HF. Role of transforming growth factor β in human disease. New England Journal of Medicine. 2000 May 4;342(18):1350-8.
2. Kumar V, Abbas AK, Aster JC. The Breast. In: Lester SC, editor. Robbins and Cotran:Pathologic basis of disease. 9th ed. Philadelphia: Elsevier Saunders; 1999. p.1055-57.
3. Ahmed HH, Metwally FM, Mahdy ES, Shosha WG, Ramadan SS. Clinical value ofserum hepatocyte growth factor, B-cell lymphoma-2 and nitric oxide in primary breast cancer patients. Eur Rev Med Pharmacol Sci. 2012 Jul 1;16(7):958-65
4. Funakoshi H, Nakamura T. Hepatocyte growth factor: from diagnosis to clinical applications. Clinica chimica acta. 2003 Jan 1;327(1-2):1-23.
5. Pollard JW. Tumour-stromal interactions: Transforming growth factor-β isoforms and hepatocyte growth factor/scatter factor in mammary gland ductal morphogenesis. Breast Cancer Research. 2001 Aug;3(4):230.
6. Owusu B, Galemmo R, Janetka J, Klampfer L. Hepatocyte growth factor, a key tumorpromoting factor in the tumor microenvironment. Cancers. 2017 Apr 17;9(4):35.
7. Jiang WG. Hepatocyte growth factor and the hepatocyte growth factor receptor signalling complex as targets in cancer therapies. Current Oncology. 2007 Apr;14(2):66.
8. Brinkmann V, Foroutan H, Sachs M, Weidner KM, Birchmeier W. Hepatocyte growthfactor/scatter factor induces a variety of tissue-specific morphogenic programs In epithelial cells. The Journal of Cell Biology. 1995 Dec15;131(6):1573-86.
9. Pollack AL, Runyan RB, Mostov KE. Morphogenetic mechanisms of epithelial tubulogenesis: MDCK cell polarity is transiently rearranged without loss of cell–cell contact during scatter factor/hepatocyte growth factor-induced tubulogenesis. Developmental biology. 1998 Dec 1;204(1):64-79.
10. Rosário M, Birchmeier W. How to make tubes: signaling by the Met receptor tyrosine kinase. Trends in cell biology. 2003 Jun 1;13(6):328-35.
11. Zhang YW, Su Y, Volpert OV, Woude GF. Hepatocyte growth factor/scatter factor mediates angiogenesis through positive VEGF and negative thrombospondin regulation. Proceedings of the National Academy of Sciences. 2003 Oct28;100(22):12718-23.
12. Yamamoto K, Morishita R, Hayashi SI, Matsushita H, Nakagami H, Moriguchi A, Matsumoto K, Nakamura T, Kaneda Y, Ogihara T. Contribution of Bcl-2, but not Bcl-xL and Bax, to antiapoptotic actions of hepatocyte growth factor in hypoxia-conditioned human endothelial cells. Hypertension. 2001 May 1;37(5):1341-8.
13. Wang X, DeFrances MC, Dai Y, Pediaditakis P, Johnson C, Bell A, Michalopoulos GK, Zarnegar R. A mechanism of cell survival: sequestration of Fas by the HGF receptor Met. Molecular cell. 2002 Feb 1;9(2):411-21.
14. Elliott BE, Hung WL, Boag AH, Tuck AB. The role of hepatocyte growth factor (scatter factor) in epithelial mesenchymal transition and breast cancer. Canadian journal of physiology and pharmacology. 2002 Feb 1;80(2):91-102.
15. Toi M, Taniguchi T, Ueno T, Asano M, Funata N, Sekiguchi K, Iwanari H, Tominaga T. Significance of circulating hepatocyte growth factor level as a prognostic indicator in primary breast cancer. Clinical cancer research. 1998 Mar 1;4(3):659-64.
16. Matsumoto K, Nakamura T, Sakai K, Nakamura T. Hepatocyte growth factor and Met in tumor biology and therapeutic approach with NK4. Proteomics. 2008 Aug;8(16):3360-70.
17. Elliott BE, Hung WL, Boag AH, Tuck AB. The role of hepatocyte growth factor (scatter factor) in epithelial mesenchymal transition and breast cancer. Canadian journal of physiology and pharmacology. 2002 Feb 1;80(2):91-102.
18. Jiang WG. Hepatocyte growth factor and the hepatocyte growth factor receptor signalling complex as targets in cancer therapies. Current Oncology. 2007 Apr;14(2):66.
19. Stewart BW, Wild CP. World cancer report 2014. Self. 2018 Oct 18.
20. Sheen-Chen SM, Liu YW, Eng HL, Chou FF. Serum levels of hepatocyte growth factor in patients with breast cancer. Cancer Epidemiol Biomarkers Prev2005;14(3):715-7.
21. El-Attar HA, Sheta MI. Hepatocyte growth factor profile with breast cancer. Indian J PatholMicrobiol2011;54(3):509-13.
22. Taniguchi T, Toi M, Inada K, Imazawa T, Yamamoto Y, Tominaga T. Serum concentrations of hepatocyte growth factor in breast cancer patients. Clin Cancer Res 1995;1(9):1031-4.
23. Kucera R, Cerna M, Narsanska A, Svobodova S, Strakova M, Vrzalova J, Fuchsova R, Treskova I, Kydlicek T, Treska V, Pecen L. Growth factors and breast tumors, comparison of selected growth factors with traditional tumor markers. Anticancer research. 2011 Dec 1;31(12):4653-6.
24. Ahmed HH, Metwally FM, Mahdy ES, Shosha WG, Ramadan SS. Clinical value of serum hepatocyte growth factor, B-cell lymphoma-2 and nitric oxide in primary breast cancer patients. Eur Rev Med Pharmacol Sci. 2012 Jul 1;16(7):958-65.
25. Funakoshi H, Nakamura T. Hepatocyte growth factor: from diagnosis to clinical applications. Clinica chimica acta. 2003 Jan 1;327(1-2):1-23.
26. Kamalati TA, Thirunavukarasu BI, Wallace AN, Holder NI, Brooks RO, Nakamura TO, Stoker MI, Gherardi ER, Buluwela LA. Down-regulation of scatter factor in MRC 5 fibroblasts by epithelial-derived cells. A model for scatter factor modulation. Journal of cell science. 1992 Feb 1;101(2):323-32.
27. Matteucci E, Bendinelli P, Desiderio MA. Nuclear localization of active HGF receptor Met in aggressive MDA-MB231 breast carcinoma cells. Carcinogenesis. 2009 Apr 8;30(6):937-45.
28. Tamura M, Arakaki N, Tsubouchi H, Takada H, Daikuhara Y. Enhancement of human hepatocyte growth factor production by interleukin-1 alpha and-1 beta and tumor necrosis factor-alpha by fibroblasts in culture. Journal of Biological Chemistry. 1993 Apr 15;268(11):8140-5
29. Kim H, Youk J, Yang Y, Kim TY, Min A, Ham HS, Cho S, Lee KH, Keam B, Han SW, Oh DY. Prognostic implication of serum hepatocyte growth factor in stage II/III breast cancer patients who received neoadjuvant chemotherapy. Journal of cancer research and clinical oncology. 2016 Mar 1;142(3):707-14.
30. Deheuninck J, Foveau B, Goormachtigh G, Leroy C, Ji Z, Tulasne D, Fafeur V. Caspase cleavage of the MET receptor generates an HGF interfering fragment. Biochemical and biophysical research communications. 2008 Mar 14;367(3):573-7.
31. Wader KF, Fagerli UM, Holt RU, Børset M, Sundan A, Waage A. Soluble c‐Met in serum of patients with multiple myeloma: correlation with clinical parameters. European journal of haematology. 2011 Nov;87(5):394-9.
32. Kalluri R. The biology and function of fibroblasts in cancer. Nature Reviews Cancer. 2016 Sep;16(9):582.
33. Owusu B, Galemmo R, Janetka J, Klampfer L. Hepatocyte growth factor, a key tumor-promoting factor in the tumor microenvironment. Cancers. 2017 Apr 17;9(4):35.
34. Li G, Schaider H, Satyamoorthy K, Hanakawa Y, Hashimoto K, Herlyn M. Downregulation of E-cadherin and Desmoglein 1 by autocrine hepatocyte growth factor during melanoma development. Oncogene. 2001 Dec;20(56):8125.
35. Jiang WG, Martin TA, Parr C, Davies G, Matsumoto K, Nakamura T. Hepatocyte growth factor, its receptor, and their potential value in cancer therapies. Critical reviews in oncology/hematology. 2005 Jan 1;53(1):35-69