Outcomes of multicystic peritoneal mesothelioma treatment with cytoreductive surgery and hyperthermic intraperitoneal chemotherapy.

BACKGROUND: Multicystic peritoneal mesothelioma (MCPM) is a rare neoplasm, generally considered a borderline malignancy, best treated by cytoreductive surgery (CRS) to remove macroscopic disease, combined with hyperthermic intraperitoneal chemotherapy (HIPEC). Owing to its rarity, little has been published on clinical presentation, clinical behaviour over time, or an optimal treatment approach. METHODS: A prospectively developed peritoneal malignancy database was interrogated for the years 2001-2018. Details on all patients with MCPM as a definitive diagnosis after CRS and HIPEC were analysed, including previous interventions, mode of presentation, surgical treatment, postoperative outcomes, and late follow-up information from abdominal CT and tumour markers. RESULTS: Some 40 patients with MCPM underwent CRS and HIPEC between 2001 and 2018. Of these, 32 presented with abdominal pain, distension or bloating, six patients presented with recurrence following previous surgery at the referring hospitals, and two had coincidental diagnoses during a surgical procedure. CRS involved peritonectomy in all 40 patients. Bowel resection was required in 18 patients, and seven had a temporary stoma. Thirty-eight patients were considered to have undergone a complete macroscopic tumour removal (completeness of cytoreduction CC0), and two had residual tumour nodules less than 2.5 mm in size, classified as CC1. Median duration of follow-up was 65 (range 48-79) months. There were no deaths during follow-up. The Kaplan-Meier-predicted recurrence-free interval was 115.4 months. CONCLUSION: MCPM is a rare peritoneal neoplasm with a heterogeneous pattern of presentation. CRS and HIPEC is an effective management option for this group of patients, with favourable long-term survival.

Clinical and surgical outcomes of patients with peritoneal mesothelioma discussed at a monthly national multidisciplinary team video-conference meeting.

BACKGROUND: Peritoneal mesothelioma (PM) is a rare primary neoplasm of the peritoneum with an increasing incidence worldwide. Cytoreductive surgery (CRS) with hyperthermic intraperitoneal chemotherapy (HIPEC) has shown promise as a treatment strategy. A national PM multidisciplinary team (national PM MDT) video-conference meeting was established in the UK and Ireland in March 2016, aiming to plan optimal treatment, record outcomes and provide evidence for the benefits of centralization. This article reports on the activities and outcomes of the first 2·5 years. METHODS: Between March 2016 and December 2018, patients with PM, referred to peritoneal malignancy centres in Basingstoke, Birmingham, Manchester and Dublin, were discussed by the national PM MDT via video-conference. The MDT was composed of surgeons, radiologists, specialist nurses and pathologists. Patients were considered for CRS and HIPEC if considered fit for surgery and if radiological imaging suggested that complete surgical cytoreduction could be achieved. Morbidity and mortality following surgery were analysed. Survival analysis following MDT discussion was conducted. RESULTS: A total of 155 patients (M : F ratio 0·96) with a mean(s.d.) age of 57(17) years were discussed. To date, 22 (14·2 per cent) have had CRS and HIPEC; the median Peritoneal Cancer Index for the surgical group was 17·0. Complete cytoreduction was achieved in 19 patients. Clavien-Dindo grade I-II complications occurred in 16 patients; there was no grade III-IV morbidity or 30-day in-hospital mortality. The median follow-up for the whole cohort was 18·7 months, and the 2-year survival rate from time of first review at the national PM MDT was 68·3 per cent. CONCLUSION: The centralized national PM MDT was effective at selecting patients suitable for CRS and HIPEC, reporting a good outcome from patient selection.

ANTECEDENTES: El mesotelioma peritoneal (peritoneal mesothelioma, PM) es una neoplasia primaria del peritoneo muy poco frecuente, con una incidencia creciente en todo el mundo. La cirugía citorreductora (cytoreductive surgery, CRS) con quimioterapia intraperitoneal hipertérmica (hyperthermic intraperitoneal chemotherapy, HIPEC) se ha mostrado prometedora como estrategia de tratamiento. En marzo de 2016, se organizó una reunión por videoconferencia del equipo multidisciplinar nacional de PM (national PM multi-Disciplinary Team, MDT) en el Reino Unido e Irlanda, con el objetivo de planificar un tratamiento óptimo, registrar los resultados y proporcionar evidencia de los beneficios de la centralización. Este manuscrito presenta las actividades y los resultados de los primeros 2,5 años. MÉTODOS: Entre marzo de 2016 y diciembre de 2018, 155 pacientes con PM, remitidos a centros de cirugía oncológica peritoneal en Basingstoke, Good Hope Hospital en Birmingham, Christie Hospital en Manchester y Mater Misericordiae en Dublín, fueron discutidos en el National PM MDT a través de una videoconferencia. El MDT estaba compuesto por cirujanos, radiólogos, enfermeras especializadas y patólogos. Los pacientes fueron considerados para CRS e HIPEC si se determinaba que eran aptos para la cirugía y si las imágenes radiológicas sugerían que se podía lograr una citorreducción quirúrgica completa. Se analizó la morbilidad y mortalidad después de la cirugía. Se realizó un análisis de supervivencia tras la discusión en el MDT. RESULTADOS: En total, se discutieron 155 pacientes (tasa varón/mujer 0,96) con una edad media de 57 ± 17 años. Hasta el momento, 22 (14,2%) habían sido sometidos a CRS y HIPEC y la mediana de PCI en el grupo quirúrgico fue de 17,0. La citorreducción completa se logró en 19 (86,4%), las complicaciones de Clavien-Dindo grado I/II ocurrieron en 16/22, sin morbilidad de grado III/IV, ni mortalidad a los 30 días. La mediana de seguimiento fue de 15,0 meses y la supervivencia a los 2 años desde el momento de la revisión en el National PM MDT fue del 66,7%. CONCLUSIÓN: El National PM MDT centralizado fue eficaz en la selección de pacientes adecuados para CRS e HIPEC, presentando un buen resultado a partir de dicha selección.

Stiff person syndrome: An unusual paraneoplastic neurological phenomenon in Carcinoid tumour

@#Stiff person syndrome is a rare neurologic disorder characterised by rigidity of the truncal and proximal limb muscles with intermittent superimposed spasms. It’s unique because it lacks similarity to any other neurologic disorder. Possibly tetanus is the closest related condition with both inhibiting central gamma-aminobutyric (GABA) systems. Stiff person syndrome is extremely rare with less than 20 cases reported from South Asia which has a population of nearly 2 billion. In its classic form, it is associated with the presence of high titres of glutamic acid decarboxylase (GAD) antibodies. Paraneoplastic stiff person syndrome comprising of around 5% of the patients has been reported with malignancies of the breast, colon, lung, thymus and Hodgkin’s lymphoma. Antibodies against amphiphysin and gephyrin are detected in paraneoplastic stiff person syndrome. We report a 58 year old Sri Lankan male with stiff person syndrome with a high GAD antibody titre and classical electromyographic changes, who was found to have an underlying carcinoid tumour. We postulate that stiff person syndrome was a paraneoplastic phenomenon secondary to the carcinoid in this case. Although neurological syndromes such as sensory neuropathy, limbic encephalitis and, myelopathy have been described as paraneoplastic features in carcinoid, we believe this is the first report of stiff person syndrome associated with carcinoid tumour.

Biological mesh is a safe and effective method of abdominal wall reconstruction in cytoreductive surgery for peritoneal malignancy.

BACKGROUND: Patients with peritoneal malignancy often have multiple laparotomies before referral for cytoreductive surgery (CRS). Some have substantial abdominal wall herniation and tumour infiltration of abdominal incisions. CRS involves complete macroscopic tumour removal and hyperthermic intraperitoneal chemotherapy (HIPEC). Abdominal wall reconstruction is problematic in these patients. The aim of this study was to establish immediate and long-term outcomes of abdominal wall reconstruction with biological mesh in a single centre. METHODS: A dedicated peritoneal malignancy database was searched for all patients who had biological mesh abdominal wall reconstruction between 2004 and 2015. Short- and long-term outcomes were reviewed. All patients had annual abdominal CT as routine peritoneal malignancy follow-up. RESULTS: Some 33 patients (22 women) with a mean age of 53·4 (range 19-82) years underwent abdominal wall reconstruction with biological mesh. The majority (23) had CRS for pseudomyxoma (19 low grade), six for colorectal peritoneal metastasis and four for appendiceal adenocarcinoma; 18 had undergone CRS and HIPEC previously. Twenty-five of the 33 patients had abdominal wall tumour involvement and eight had concurrent hernias. The mean duration of surgery was 486 (range 120-795) min and the mean mesh size used was 345 (50-654) cm2. Ten patients developed wound infections and four had a seroma. Two developed early enterocutaneous fistulas. Mean follow-up was 48 months. Five patients developed an incisional hernia. Four died from progressive malignancy. A further 15 patients had disease recurrence, but only one had isolated abdominal wall recurrence. CONCLUSION: Biological mesh was safe and effective for abdominal wall reconstruction in peritoneal malignancy. Postoperative wound infections were frequent but nevertheless incisional hernia rates were low with no instances of mesh-related bowel erosion or fistulation.

Impact of ovarian metastases on survival in patients treated with cytoreductive surgery and hyperthermic intraperitoneal chemotherapy for peritoneal malignancy originating from appendiceal and colorectal cancer.

AIM: Ovarian metastases from gastrointestinal tract malignancies have been considered an ominous finding with poor prognosis. The aim of this project was to determine the impact on survival, and potential cure, when cytoreductive surgery (CRS) and hyperthermic intraperitoneal chemotherapy (HIPEC) are combined to treat peritoneal malignancy in women with Krukenberg tumours. METHOD: A retrospective analysis of prospectively collected data between January 2010 and July 2015. Female patients undergoing complete CRS (macroscopic tumour removal) and HIPEC for pseudomyxoma peritonei (PMP) of appendiceal origin, or colorectal peritoneal metastases (CPM) were included. Survival was estimated using the Kaplan-Meier method and survival rates compared using the log-rank test. RESULTS: In total, 889 patients underwent surgery for peritoneal malignancy, of whom 551 were female. Of these, 504/551 (91%) underwent complete CRS and HIPEC. Overall, 405/504 (80%) had at least one involved ovary removed either during CRS and HIPEC or at their index prereferral operation. Three hundred and fifty-two patients (87%) had an appendiceal tumour and 53 (13%) had CPM. At a median follow up of 40 months, overall survival (OS) did not differ significantly between patients with or without ovarian involvement in women with a primary low-grade appendiceal tumour or CPM. In women with high-grade primary appendiceal pathology, OS was significantly lower in patients with ovarian metastases compared with those without ovarian involvement. CONCLUSION: Women with ovarian metastases from low-grade appendiceal tumours or colorectal cancer treated with CRS and HIPEC have similar survival rates to patients without ovarian metastases. Long-term survival and cure is feasible in patients amenable to complete tumour removal.

The modified Glasgow prognosis score predicts for overall and disease-free survival following cytoreductive surgery and HIPEC in patients with pseudomyxoma peritonei of appendiceal origin.

BACKGROUND: The modified Glasgow prognostic score (incorporating C-reactive protein and albumin) predicts survival in patients with gastro-intestinal tract cancer but has not been evaluated in patients with peritoneal malignancy. The aim was to evaluate the modified Glasgow score preoperatively in patients undergoing complete cytoreductive surgery (CCRS) with hyperthermic intraperitoneal chemotherapy (HIPEC) for pseudomyxoma peritonei (PMP) of appendiceal origin. METHODS: Prospectively collected data from patients with PMP of appendiceal origin following CCRS and HIPEC between January 2007 and December 2011 were analysed. The mGPS was calculated from preoperative C-reactive protein and albumin. Predicted overall survival (OS) and disease-free survival (DFS) for each mGPS score were calculated using the Kaplan-Meier model. In a separate analysis, a comparison was made between mGPS and Tumour Markers (TM). RESULTS: 260 patients were included in the study. The mGPS of 0, 1, and 2 were found in 111, 130, and 19 patients respectively. The median follow-up was 48 months. For mGPS-0, -1, and -2, the predicted OS was 82.2, 73.7, and 69.2 months and the DFS was 73.5, 62.9, and 54.4 months respectively. As mGPS increases, there is a reduction in long-term survival. There was no difference between mGPS and TM. CONCLUSION: Preoperative mGPS may be a cost effective prognostic tool for predicting OS and DFS in patients following complete CRS-HIPEC, and performs well compared to TM for predicting patients at high risk of recurrence.

Attitudes towards breast conservation in patients aged over 70 with breast cancer.

BACKGROUND: The majority of breast conserving surgery (BCS) is performed in younger women. There is little published information about the views of women aged over 70 regarding BCS. The aim of this study was to investigate the attitudes of this age group towards BCS, and factors which may influence their treatment decision-making. METHODS: A questionnaire was sent to all patients who were aged 70 or over at the time they had breast cancer surgery in NHS Lanarkshire between 1999 and 2013. This detailed surgical options and recommendations, timing of decision making, treatment expectations, psychological and cosmetic concerns and other factors which may have influenced any decision made e.g. travel for radiotherapy and potential side effects. RESULTS: Responses were received from 339 patients, 192 of whom had a mastectomy with the remaining undergoing BCS. In the mastectomy group 18 % (35) would have preferred to have BCS had it been an option, with 40 % (76) of group being happy to take neoadjuvant endocrine therapy to try and facilitate this. However, only 14 % (26) of patients would have considered neoadjuvant chemotherapy with the same aim. Almost half (82) of the mastectomy patients said that the risk of local recurrence following BCS was a factor which influenced their decision. CONCLUSION: BCS is something that patients aged over 70 are interested in considering in the same way as younger patients. More than a third of patients requiring mastectomy would be willing to take neoadjuvant endocrine therapy to attempt to downstage their tumour to facilitate BCS.

Cytoreductive surgery and hyperthermic intraperitoneal chemotherapy in 1000 patients with perforated appendiceal epithelial tumours.

PURPOSE: To report early and long term outcomes following cytoreductive surgery (CRS) and hyperthermic intraperitoneal chemotherapy (HIPEC) in 1000 patients with perforated appendiceal epithelial tumours, predominantly with pseudomyxoma peritonei (PMP). METHODS: Retrospective analysis of a prospective database of 1000 consecutive patients undergoing CRS and HIPEC for perforated appendiceal tumours between 1994 and 2014 in a UK National Peritoneal Malignancy unit. RESULTS: Overall 1000/1444 (69.2%) patients treated for peritoneal malignancy had appendiceal primary tumours. Of these 738/1000 (73.8%) underwent complete cytoreductive surgery (CCRS), 242 (24.2%) had maximal tumour debulking (MTD) and 20 (2%) had laparotomy and biopsies only. Treatment related 30-day mortality was 0.8% in CCRS and 1.7% in MTD group with major postoperative morbidity rates of 15.2% (CCRS) and 14.5% (MTD). Five- and 10-year overall survival was 87.4% and 70.3% in the 738 patients who had CCRS compared with 39.2% and 8.1% respectively in the MTD group. On multivariate analysis, significant predictors of reduced overall survival were male gender (p = 0.022), elevated CEA (p = 0.001), elevated CA125 (p = 0.001) and high tumour grade or adenocarcinoma (p = 0.001). CONCLUSIONS: Perforated epithelial appendiceal tumours are rare, though may be increasing in incidence and can present unexpectedly at elective or emergency abdominal surgery, often with PMP. CRS and HIPEC results in good long term outcomes in most patients.

Risk of definitive stoma after surgery for peritoneal malignancy in 958 patients: comparative study between complete cytoreductive surgery and maximal tumor debulking.

INTRODUCTION: Complete cytoreductive surgery (CRS) can achieve cure or long-term survival in selected patients with peritoneal malignancy. In selected patients, due to extensive disease, complete tumour removal is impossible and optimal strategy may be maximal tumour debulking (MTD). We analysed the stoma related outcome in a series of patients undergoing surgery in a National Peritoneal Malignancy Referral Centre. METHODS: All patients who underwent CRS, with or without, intra-operative hyperthermic intraperitoneal chemotherapy (HIPEC) between 1994 and 2012 were included. Data was collected prospectively in an institutional database and analysed retrospectively. RESULTS: CRS was performed in 958 patients (female: 595, male: 363) of whom 781 (81.5%) had a primary appendix tumour, 63 (6.6%) had a colorectal primary, 47 (4.9%) peritoneal mesothelioma, 38 (4%) an ovarian tumour and 29 patients (3%) other tumours. Complete CRS was achieved in 72% (693/958). Overall 352/958 (37%) had a stoma, which was permanent in 165/958 (17.2%). The median time interval from CRS to reversal of stoma was 4.4 months (range: 1.4-13.8). Stomas were created in 113/265 (42.6%) at MTD (permanent: n = 105 (93%), temporary: n = 8 (7%)), and 239/693 (34.5%) at complete CRS (permanent: n = 60 (25%), temporary: n = 179 (75%)) (p = 0.020). All temporary stomas in the 168/693 (24.4%) of patients who had complete CRS were subsequently reversed. CONCLUSION: To achieve complete CRS for peritoneal malignancy a stoma is often required and in a proportion this will be permanent. Overall over one third had a stoma at surgery with almost half subsequently reversed.

Pre-operative tumour marker status predicts recurrence and survival after complete cytoreduction and hyperthermic intraperitoneal chemotherapy for appendiceal Pseudomyxoma Peritonei: Analysis of 519 patients.

BACKGROUND: Cytoreductive surgery (CRS) combined with Hyperthermic Intraperitoneal Chemotherapy (HIPEC) is the optimal treatment for Pseudomyxoma Peritonei (PMP). Despite treatment, disease often recurs and may not be amenable to further CRS. Clinical experience suggests a spectrum of disease which may correlate with tumour marker levels. The aim of this study was to analyse the influence of markers on recurrence and survival. METHODS: The details of all patients undergoing surgery for PMP of appendiceal origin at a national centre for peritoneal malignancy were recorded in a dedicated prospective database. The data on all patients who had CRS and HIPEC between March 1994 and January 2012 was analysed and recurrence and survival correlated with pre-operative levels of CEA, CA-125 and CA19-9. RESULTS: Overall, 519 (69%) of 752 consecutive patients, underwent complete CRS and HIPEC. The median (range) age was 56 (20-82) years with 342/519 (66%) females. The mean overall (OS) and disease free survival (DFS) in the 131/519 patients who had normal preoperative tumour markers was 168 (128-207) and 125 (114-136) months respectively, significantly higher when compared with the 109/519 (21%) who had all three tumour markers elevated (OS of 65 (42-88) and DFS of 55 (41-70) months respectively) (P = 0.002). CONCLUSIONS: Elevated tumour markers predict an increased risk of recurrence and reduced survival after complete CRS. This may reflect cell biology in low grade tumours and is an independent prognostic feature. Further analysis may help to select patients for post-operative chemotherapy, second look procedures or stratification of follow up.

The degradation of p53 and its major E3 ligase Mdm2 is differentially dependent on the proteasomal ubiquitin receptor S5a.

p53 and its major E3 ligase Mdm2 are both ubiquitinated and targeted to the proteasome for degradation. Despite the importance of this in regulating the p53 pathway, little is known about the mechanisms of proteasomal recognition of ubiquitinated p53 and Mdm2. In this study, we show that knockdown of the proteasomal ubiquitin receptor S5a/PSMD4/Rpn10 inhibits p53 protein degradation and results in the accumulation of ubiquitinated p53. Overexpression of a dominant-negative deletion of S5a lacking its ubiquitin-interacting motifs (UIM)s, but which can be incorporated into the proteasome, also causes the stabilization of p53. Furthermore, small-interferring RNA (siRNA) rescue experiments confirm that the UIMs of S5a are required for the maintenance of low p53 levels. These observations indicate that S5a participates in the recognition of ubiquitinated p53 by the proteasome. In contrast, targeting S5a has no effect on the rate of degradation of Mdm2, indicating that proteasomal recognition of Mdm2 can be mediated by an S5a-independent pathway. S5a knockdown results in an increase in the transcriptional activity of p53. The selective stabilization of p53 and not Mdm2 provides a mechanism for p53 activation. Depletion of S5a causes a p53-dependent decrease in cell proliferation, demonstrating that p53 can have a dominant role in the response to targeting S5a. This study provides evidence for alternative pathways of proteasomal recognition of p53 and Mdm2. Differences in recognition by the proteasome could provide a means to modulate the relative stability of p53 and Mdm2 in response to cellular signals. In addition, they could be exploited for p53-activating therapies. This work shows that the degradation of proteins by the proteasome can be selectively dependent on S5a in human cells, and that this selectivity can extend to an E3 ubiquitin ligase and its substrate.

Peritoneal metastases from colorectal cancer: patient selection for cytoreductive surgery and hyperthermic intraperitoneal chemotherapy.

Cytoreductive surgery (CRS) in combination with hyperthermic intraperitoneal chemotherapy (HIPEC) is an established treatment modality for patients with pseudomyxoma peritonei. The majority of patients with pseudomyxoma who have complete tumour removal and HIPEC are cured. Over the last decade CRS for peritoneal metastasis of colorectal origin has emerged as an effective treatment strategy in carefully selected patients. Although convincing evidence is limited, available data shows promising results. The key to a successful outcome is appropriate selection of patients. In patients with extensive peritoneal disease, where complete cytoreduction is not achieved, surgical treatment may not be beneficial and might impair quality of life. In this paper we discuss the challenges of selecting patients with colorectal peritoneal metastases who are likely to benefit from CRS with HIPEC.

Sacral osteomyelitis following restorative rectal cancer resection: a delayed presentation of dormant pelvic sepsis.

Sacral osteomyelitis is a rare but potentially fatal complication of pelvic surgery. It is often diagnosed late due to the presence of vague, non-specific symptoms and a low index of suspicion. Previous literature has been limited to a number of case series of patients who have undergone ileoanal pouch formation following proctocolectomy and patients who have undergone pelvic floor reconstruction with mesh sacral colpopexy. Here, we present a patient presenting with sacral osteomyelitis 12 years following anterior resection for colorectal malignancy. We then review the previous literature and discuss the salient management points that may assist in the diagnosis and management of this uncommon complication.

p53 is activated in response to disruption of the pre-mRNA splicing machinery.

In this study, we show that interfering with the splicing machinery results in activation of the tumour-suppressor p53. The spliceosome was targeted by small interfering RNA-mediated knockdown of proteins associated with different small nuclear ribonucleoprotein complexes and by using the small-molecule splicing modulator TG003. These interventions cause: the accumulation of p53, an increase in p53 transcriptional activity and can result in p53-dependent G(1) cell cycle arrest. Mdm2 and MdmX are two key repressors of p53. We show that a decrease in MdmX protein level contributes to p53 activation in response to targeting the spliceosome. Interfering with the spliceosome also causes an increase in the rate of degradation of Mdm2. Alterations in splicing are linked with tumour development. There are frequently global changes in splicing in cancer. Our study suggests that p53 activation could participate in protection against potential tumour-promoting defects in the spliceosome. A number of known p53-activating agents affect the splicing machinery and this could contribute to their ability to upregulate p53. Preclinical studies indicate that tumours can be more sensitive than normal cells to small-molecule spliceosome inhibitors. Activation of p53 could influence the selective anti-tumour activity of this therapeutic approach.

Polymorphism of ZuBeCa3 microsatellite and its association with mammary tumor in dogs.

A study was conducted to explore the genetic polymorphism at ZuBeCa3 microsatellite and to estimate the association between microsatellite polymorphism and mammary cancer in dogs. Three genotypes namely AA, AB and BB were observed both in dogs affected by mammary cancer and in non-affected dogs. In the affected group, the frequency of genotype and allele was 0.63 for AA, 0.25 for AB and 0.12 for BB genotype, and 0.76 for A and 0.24 for B allele. In the nonaffected group, the frequency of AA, AB and BB genotype was 0.39, 0.38 and 0.23 and the frequency of A and B allele was 0.58 and 0.42, respectively. Histopathological observation classified the cancer-affected animals into three groups namely, malignant solid mammary carcinoma, malignant papillary adenocarcinoma and benign papillary adenoma in which the frequency of A allele was relatively more predominant in benign tumor group, which is more than 80%. Statistical tests showed significant differences (P < 0.01) of allelic distribution between tumor-affected and non-affected group, which reveals the effect of polymorphism on occurrences of mammary cancer in dogs. Besides, chi(2) test also reflected the significant effect of genotypes on occurrences of three groups of mammary cancer in dogs.

The determination of nicotinic acid in plasma by mixed-mode liquid chromatography-tandem mass spectrometry following ion exchange solid phase extraction.

An assay for nicotinic acid in plasma samples has been developed using ion exchange solid phase extraction in 96-well format followed by mixed-mode ion exchange/reversed-phase liquid chromatography with positive ion tandem mass spectrometry detection. The assay avoids the need for time-consuming derivatisation procedures or involatile ion-pair chromatography reagents. The assay is linear over the wide range 0.05-20 microg/mL, based on a 100 microL sample (correlation coefficient>0.99). The assay is accurate and precise (bias and coefficient of variation<18%) over this calibration range.

Genetic polymorphism of alpha-lactalbumin gene in riverine buffalo.

Alpha-lactalbumin (alpha-LA) is a major whey protein found in milk. Polymorphs of alpha-LA gene are reported to be significantly associated with milk production and constituent traits. Therefore, the present study was undertaken to detect polymorphism in alpha-LA at the genic level and to explore allelic variability at this locus. A total of 196 animals, belonging to four breeds of riverine buffalo viz. Bhadwari, Mehsana, Surti and Murrah were included under the present investigation. Two fragments i.e. 133 bp (Exon 1) and 159 bp (Exon 2) of alpha-LA gene were amplified by polymerase chain reaction and subsequently, single strand confirmation polymorphism (SSCP) study was carried out to identify different allelic pattern and genotypes of the animal included in the study. Both fragment of alpha-LA gene was found to be polymorphic in all the four breeds of riverine buffalo. Number of genotypes and allele varied breed to breed for both the fragments. In case of 133 bp fragment, four alleles A, B, C and D were found among different breeds of buffalo whereas in 159 bp fragment, five alleles namely A, B, C, D and E was found in different breeds. Nucleotide sequence data of different alleles showed the presence of both silent as well as functional mutation leading to variability in polypeptide chain.